Lecture 1 The Principles of Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” Purdue University Department of Basic Medical Sciences, School.
Download ReportTranscript Lecture 1 The Principles of Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” Purdue University Department of Basic Medical Sciences, School.
Slide 1
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 2
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 3
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 4
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 5
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 6
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 7
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 8
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 9
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 10
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 11
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 12
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 13
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 14
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 15
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 16
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 17
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 18
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 19
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 20
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 21
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 22
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 23
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 24
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 25
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 26
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 27
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 2
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 3
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 4
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 5
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 6
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 7
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 8
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 9
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 10
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 11
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 12
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 13
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 14
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 15
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 16
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 17
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 18
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 19
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 20
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 21
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 22
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 23
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 24
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 25
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 26
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
•
•
•
•
•
•
•
•
Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
Slide 3 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to Lecture 1
•
•
•
•
•
Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
Slide 4 t:/PowerPoint/confoc/lect1nu.ppt
Microscopes
•
•
•
•
Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
Slide 5 t:/PowerPoint/confoc/lect1nu.ppt
Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
Slide 6 t:/PowerPoint/confoc/lect1nu.ppt
Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
Slide 8 t:/PowerPoint/confoc/lect1nu.ppt
Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
Slide 9 t:/PowerPoint/confoc/lect1nu.ppt
Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
Slide 10 t:/PowerPoint/confoc/lect1nu.ppt
Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
Slide 11 t:/PowerPoint/confoc/lect1nu.ppt
Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
Slide 12 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
Slide 13 t:/PowerPoint/confoc/lect1nu.ppt
Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
Slide 15 t:/PowerPoint/confoc/lect1nu.ppt
Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
Slide 16 t:/PowerPoint/confoc/lect1nu.ppt
Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
Slide 18 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
Slide 19 t:/PowerPoint/confoc/lect1nu.ppt
Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
Slide 22 t:/PowerPoint/confoc/lect1nu.ppt
Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
Slide 23 t:/PowerPoint/confoc/lect1nu.ppt
The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 25 t:/PowerPoint/confoc/lect1nu.ppt
Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt
Slide 27
Lecture 1
The Principles of Microscopy
BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
Professor of Immunopharmacology
Director, Purdue University Cytometry Laboratories
These slides are intended for use in a lecture series. Copies of the graphics are distributed and
students encouraged to take their notes on these graphics. The intent is to have the student
NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All
material copyright J.Paul Robinson unless stated. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
Slide 1 t:/PowerPoint/confoc/lect1nu.ppt
Purdue University Cytometry Laboratories
UPDATED October 27, 1998
Evaluation
• End of term quiz - 100% grade
Purdue University Cytometry Laboratories
Slide 2 t:/PowerPoint/confoc/lect1nu.ppt
Introduction to the Course
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Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
Purdue University Cytometry Laboratories
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Introduction to Lecture 1
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Purdue University Cytometry Laboratories
Early Microscope
Modern Microscopes
Magnification
Nature of Light
Optical Designs
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Microscopes
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Upright
Inverted
Köhler Illumination
Fluorescence Illumination
Purdue University Cytometry Laboratories
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Earliest Microscopes
• 1590 - Hans & Zacharias Janssen of Middleburg, Holland
manufactured the first compound microscope
• 1673 Antioni Van Leeuwenhoek created a “simple” microscope that
could magnify to about 275x, and published drawings of
microorganisms in 1683
Purdue University Cytometry Laboratories
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Early Microscopes (Hooke)
Purdue University Cytometry Laboratories
Slide 7 t:/PowerPoint/confoc/lect1nu.ppt
Secondary Microscopes
• In 1827 Giovanni Battista Amici, built high quality microscopes and
introduced the first matched achromatic microscope in 1827. He
recognized the importance of coveralls thickness and developed the
concept of “water immersion”
• Carl Zeiss and Ernst Abbe developed oil immersion systems by
developing oils that matched the refractive index of glass. Dr Otto
Schott formulated glass lenses that color-corrected objectives and
produced the first “apochromatic” objectives in 1886.
Purdue University Cytometry Laboratories
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Modern Microscopes
• Early 20th Century Professor Köhler
developed the method of illumination still
called “Köhler Illumination”
• Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
Purdue University Cytometry Laboratories
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Köhler
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
Purdue University Cytometry Laboratories
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Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Purdue University Cytometry Laboratories
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Some Principles
• Rule of thumb is is not to exceed 1,000
times the NA of the objective
• Modern microscopes magnify both in the
objective and the ocular and thus are called
“compound microscopes”
Purdue University Cytometry Laboratories
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Magnification
• An object can be focussed generally no
closer than 250 mm from the eye
(depending upon how old you are!)
• this is considered to be the normal viewing
distance for 1x magnification
• Young people may be able to focus as close
as 125 mm so they can magnify as much as
2x because the image covers a larger part of
the retina
Purdue University Cytometry Laboratories
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Magnification
1000mm
35 mm slide
24x36 mm
1000 mm
M = 36 mm = 28
The projected image is 28 times larger than we would
see it at 250 mm from our eyes.
If we used a 10x magnifier we would have a magnification
of 280x, but we would reduce the field of view by the same
factor of 10x.
Purdue University Cytometry Laboratories
Slide 14 t:/PowerPoint/confoc/lect1nu.ppt
Basic Microscopy
• Bright field illumination does not reveal
differences in brightness between structural
details - i.e. no contrast
• Structural details emerge via phase
differences and by staining of components
• The edge effects (diffraction, refraction,
reflection) produce contrast and detail
Purdue University Cytometry Laboratories
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Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending
upon the color absorbed. Thus the selective absorption of white light
produces colored light.
• Refraction
– Direction change of a ray of light passing from one transparent medium to
another with different optical density. A ray from less to more dense
medium is bent perpendicular to the surface, with greater deviation for
shorter wavelengths
• Diffraction
– Light rays bend around edges - new wavefronts are generated at sharp
edges - the smaller the aperture the lower the definition
• Dispersion
– Separation of light into its constituent wavelengths when entering a
transparent medium - the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow
Purdue University Cytometry Laboratories
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Control
Absorption
No blue/green light
red filter
Purdue University Cytometry Laboratories
Slide 17 t:/PowerPoint/confoc/lect1nu.ppt
Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
Purdue University Cytometry Laboratories
blue
blue
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Refraction
Short wavelengths are “bent”
more than long wavelengths
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Purdue University Cytometry Laboratories
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Refraction
He sees the
fish here….
But it is really here!!
Purdue University Cytometry Laboratories
Slide 20 t:/PowerPoint/confoc/lect1nu.ppt
Upright Scope
Purdue University Cytometry Laboratories
Slide 21 t:/PowerPoint/confoc/lect1nu.ppt
Inverted Microscope
Purdue University Cytometry Laboratories
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Microscope Basics
• Originally conformed to the German DIN
standard
• Standard required the following
– real image formed at a tube length of 160mm
– the parfocal distance set to 45 mm
– object to image distance set at 195 mm
• Currently we use the ISO standard
Purdue University Cytometry Laboratories
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The Conventional Microscope
Mechanical
tube length
= 160 mm
Object to
Image
Distance
= 195 mm
Focal length
of objective
= 45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Purdue University Cytometry Laboratories
Slide 24 t:/PowerPoint/confoc/lect1nu.ppt
Conventional Finite Optics
with Telan system
Ocular
Intermediate Image
195 mm
160 mm
Telan Optics
Other optics
Objective
45 mm
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
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Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
The main advantage of infinity
corrected lens systems is the
relative insensitivity to
additional optics within the
tube length. Secondly one can
focus by moving the objective
and not the specimen (stage)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
Purdue University Cytometry Laboratories
Slide 26 t:/PowerPoint/confoc/lect1nu.ppt
Summary Lecture 1
• Upright and inverted microscopes
• Köhler illumination
• Refraction, Absorption, dispersion,
diffraction
• Magnification
• Optical Designs - 160 mm and Infinity
optics
Purdue University Cytometry Laboratories
Slide 27 t:/PowerPoint/confoc/lect1nu.ppt