Transcript Lect2nu BMS 524 Confocal Microscopy

Lecture 2
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 otherwise stated, however, the material may be
freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose.
The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures
in these lecture notes are taken from this text.
UPDATED October 27, 1998
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Review
• Microscope Basics
• Magnification
• Optical systems
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Microscope Components
•
•
•
•
•
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Fluorescence Microscope
Numerical Aperture
Refractive Index
Aberrations
Objectives
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Definitions
• Refractive Index
• Aberrations
• Fluorescence
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Fluorescent Microscope
Arc Lamp
Excitation Diaphragm
Excitation Filter
EPI-Illumination
Ocular
Objective
Emission Filter
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Construction of Filters
Dielectric filter
components
“glue”
Single Optical
filter
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Anti-Reflection Coatings
Coatings are often magnesium fluoride
Optical Filter
Multiple
Elements
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Standard Band Pass Filters
630 nm BandPass Filter
White Light Source
Transmitted Light
620 -640 nm Light
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Standard Long Pass Filters
520 nm Long Pass Filter
Light Source
Transmitted Light
>520 nm Light
Standard Short Pass Filters
575 nm Short Pass Filter
Light Source
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Transmitted Light
<575 nm Light
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Optical Filters
510 LP dichroic Mirror
Dichroic Filter/Mirror at 45 deg
Light Source
Transmitted Light
Reflected light
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Filter Properties
Light Transmission
100
Notch
50
Bandpass
%T
0
Wavelength
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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.
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Light striking a surface
Incident Beam
i
Transmitted
(refracted)Beam
t
r
Reflected Beam
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Properties of thin Lenses
f
f
p
q
1
p
Resolution (R) = 0.61 x
(lateral)
(Rayleigh criterion)
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+
1
q
l
NA
=
1
f
q
Magnification =
p
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Microscope Objectives
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Objectives
PLAN-APO-40X 1.30 N.A. 160/0.22
Flat field Apochromat Magnification Numerical Tube Coverglass
Aperture Length Thickness
Factor
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Objectives
Limit for smallest
resolvable distance d
between 2 points is
(Rayleigh criterion):
d = 1.22l

This defines a “resel” or “resolution element”
Thus high NUMERICAL APERTURE is
critical for high magnification
In a medium of refractive index n the
wavelength gets shorter: lln
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Numerical Aperture
• The wider the angle the lens is capable of
receiving light at, the greater its resolving
power
• The higher the NA, the shorter the working
distance
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Numerical Aperture
• Resolving power is directly related to numerical aperture.
• The higher the NA the greater the resolution
• Resolving power:
The ability of an objective to resolve two distinct lines very close
together
NA =  sin m
– (n=the lowest refractive index between the object and first
objective element) (hopefully 1)
– m is 1/2 the angular aperture of the objective
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Numerical Aperture
• For a narrow light beam (i.e. closed illumination aperture diaphragm)
the finest resolution is (at the brightest point of the visible spectrum i.e.
530 nm)….
l
=
NA
.00053
1.00 = 0.53 mm
• With a cone of light filling the entire aperture the theoretical resolution
is…..
l
2 x NA
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=
.00053
2 x 1.00 = 0.265 mm
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Object Resolution
• Example: 40 x 1.3 N.A. objective
l
2 x NA
.00053
2 x 1.3 = 0.20 mm
=
40 x 0.65 N.A. objective
l
2 x NA
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=
.00053
2 x .65 = 0.405 mm
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Microscope Objectives
60 1.4 NA
PlanApo
Oil
Microscope
Objective
Stage
Coverslip
Specimen
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Refractive Index
Objective
n = 1.52
n = 1.52
n=1.52
n=1.52
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Oil
n = 1.5
n = 1.0
Air
n = 1.52
Coverslip
Specimen
Wat er
n=1.33
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Sources of Aberrations
• Monochromatic Aberrations
–
–
–
–
–
Spherical aberration
Coma
Astigmatism
Flatness of field
Distortion
• Chromatic Aberrations
– Longitudinal aberration
– Lateral aberration
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Monochromatic Aberration
– Spherical aberration
F1
F2
F1
Corrected lens
Generated by nonspherical wavefronts produced by the objective, and increased tube length,
or inserted objects such as coverslips, immersion oil, etc. Essentially, it is desirable only to
use the center part of a lens to avoid this problem.
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Monochromatic Aberrations
– Coma
Fig 12 p117
From:”Handbook of Biological Confocal Microscopy”
J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
The figure is not reproduced in this presentation because
we do not have permission to place this figure onto a
public site.
Note: For class use
Figure is under box
Coma is when a streaking radial distortion occurs for object points away from the optical axis. It
should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in
confocal systems.
From:Handbook of Biological Confocal Microscopy
J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
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Monochromatic Aberrations
–Astigmatism
Fig 13 p118
From:”Handbook of Biological Confocal Microscopy”
J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed
The figure is not reproduced in this presentation because
we do not have permission to place this figure onto a
public site.
Note: For class use
Figure is under box
If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated.
Fig 13 p118
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From:Handbook of Biological Confocal Microscopy
J.B.Pawley, Plenum Press, NY, 1995, 2nd Ed.
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• Monochromatic Aberrations
– Flatness of Field
– Distortion
Lenses are spherical and since points of a flat image are
focused onto a spherical dish, the central and peripheral
zones will not be in focus. Complex Achromat and
PLANAPOCHROMAT lenses partially solve this problem
but at reduced transmission.
DISTORTION occurs for objects components out of axis.
Most objectives correct to reduce distortion to less than
2% of the radial distance from the axis.
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Useful Facts
• The intensity of light collected decreases as
the square of the magnification
• The intensity of light increases as the square
of the numerical aperture
Thus when possible, use low magnification
and high NA objectives.
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Fluorescence Microscopes
• Cannot view fluorescence emission in a single
optical plane
• Generally use light sources of much lower flux
than confocal systems
• Are much cheaper than confocal systems
• Give high quality photographic images (actual
photographs) whereas confocal systems are
restricted to small resolution images
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Summary Lecture 2
•
•
•
•
•
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Properties of optical filters
Objectives
Numerical Aperture
Refractive Index/Refraction
Aberrations
Fluorescence Microscope - introduction
Purdue University Cytometry Laboratories
Purdue
University
Cytometry
Laboratories
Purdue
University
Cytometry
Laboratories
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