Transcript Tutorial on Microscopy September 15, 2007 Why the need to study microscopy? • It is a tool complementary to molecular biology • It.

Tutorial on Microscopy
September 15, 2007
Why the need to study microscopy?
• It is a tool complementary to molecular biology
• It has become an indispensable tool for many
biologists and pathologists
• to check sterility of cultures
• to study the histology of biopsies
• to study the developmental program of organ
• to follow the movement of a protein from
the cytoplasm to the chloroplast
Students need to understand the microscope
• to get a sense of size,
• to get the best image possible,
• to learn how to enjoy using it,
• to use it for cool and fun purposes,
• to keep it in good shape,
• to be able to share the instrument with others.
MOTIC
ZEISS
Eye-piece or ocular
Revolving nose
with objectives
Eye-piece or ocular
Revolving nose
with objectives
Stage and its controls
Focussing knob
Eye-piece or ocular
Revolving nose
with objectives
Stage and its controls
Condenser
Light source
Focussing knob
Light and lenses are the two pieces of equipment
which are used to manipulate the light. Both are
inherent to the microscope you used; they cannot
be changed.
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
The condenser: a combination of lenses
http://micro.magnet.fsu.edu/primer/anatomy/condensers.html
The condenser: a combination of lenses
• The simplest condenser
• Role: to condense and
focus light onto the
specimen
Also called an iris
diaphragm
Allows more or less
light to enter the condenser
http://micro.magnet.fsu.edu/primer/anatomy/condensers.html
The condenser: a combination of lenses
• Will possess specific
characteristics (correction,
numerical aperture and
others) depending on
manufacturer specifications
http://www.zeiss.com/C1256B5E0047FF3F?Open
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
The objective: a combination of lenses
http://micro.magnet.fsu.edu/primer/
The objective: a combination of lenses
Motic
Zeiss
http://micro.magnet.fsu.edu/primer/
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
http://micro.magnet.fsu.edu/primer/
Achromat:
Good color correction – exactly for two wavelengths.
Field flatness in the image center, refocusing also covers
the peripheral areas. Designed for fields of view up to
18 mm diameter.
Versions for phase contrast. Budget-priced objectives.
Names: CP-Achromat (CP: Clinical Plan) and
Achrostigmatism.
http://www.zeiss.com/C1256B5E0047FF3F?Open
The effect of chromatic aberration
Rays of longer λ focus further away that those of shorter λ.
http://micro.magnet.fsu.edu/primer/
The effect of chromatic aberration
Rays of longer λ focus further away that those of shorter λ.
http://micro.magnet.fsu.edu/primer/
Motic
Plan and Epiplan:
Improved Achromat objectives with good image flatness
for fields of view with dia. 20 or even 23 mm. Therefore
ideal for photomicrography.
Zeiss
Achromat:
Good color correction – exactly for two wavelengths.
Field flatness in the image center, refocusing also covers
the peripheral areas. Designed for fields of view up to
18 mm diameter.
Versions for phase contrast. Budget-priced objectives.
Names: CP-Achromat (CP: Clinical Plan) and
Achrostigmatism.
http://www.zeiss.com/C1256B5E0047FF3F?Open
Field curvature: the sharpest focus of a lens is
on a curved surface rather than on a flat plane.
Plant microtechnique and microscopy. E. Ruzin
http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield
http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
Magnification / Numerical Aperture
http://micro.magnet.fsu.edu/primer/
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
Magnification / Numerical Aperture
http://micro.magnet.fsu.edu/primer/
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
http://micro.magnet.fsu.edu/primer/
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
∞ / 0.17
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
∞/∞ / 0.17
http://micro.magnet.fsu.edu/primer/
All the objectives mentioned here are members of the family
of ICS-Optics (ICS: Infinity Color-corrected System). These
objectives project their images to “infinity” first. Only the tube
lens produces an intermediate image – to be more precise, at
a distance of approx. 164.5 mm behind the tube lens. This
distance was chosen to comply with the classical tube length.
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
∞ / 0.17
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
∞/∞ / 0.17
http://micro.magnet.fsu.edu/primer/
A coverslip is
• part of the image-forming system,
• a lens element,
• its power has been taken into account by the manufacturer,
• its thickness and the making of the glass will affect the
deviation of the light.
Klosevych, 1989
The thickness of the coverslip and the refractive
index of the glass will have an effect of the light
path
1 thickness= 0.13 to 0.17 mm
Satisfactory for NA ≤ 0.4.
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
∞ / 0.17
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
∞/∞ / 0.17
Any questions?
http://micro.magnet.fsu.edu/primer/
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
http://micro.magnet.fsu.edu/primer/anatomy/oculars.html
Eyepieces are not just simple
lenses, but are corrected
optical systems consisting of
several lenses.
http://www.zeiss.com/C1256B5E0047FF3F?Open
1. Position of the intermediate
image (also for the reticle)
2. Limit of the field of view
(black edge of image)
3. Eye-piece optics
(Ramsden ocular)
4. Position of the eyepiece pupil
(pupil of the observer’s eye)
5. Focusing ring for the diopter
compensation
Eyepieces: magnifiers to view the intermediate
Image produced by the objective and the tube lens.
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
There will be specifications on these lenses
Motic
WFPL 10x / 20
Glasses symbol
WF for Wide Field of view
PL to match the objective correction
Magnification 10X
Field number which refers to
the diameter (in mm) of the fixed
diaphragm in the eyepiece.
The lenses: three types in your microscope
• The condenser
• The objective
• The eye-piece or ocular
There will be specifications on these lenses
Zeiss
PL 10x / 18
Glasses symbol
Designed for eyeglass wearers.
The exit pupil is at a considerable
distance from the eyepiece.
Final image
Intermediate
image
Specimen
What is the main purpose of the microscope?
• The condenser
• The objective
• The eye-piece or ocular
What is the main purpose of the microscope?
• The condenser
• The objective
• The eye-piece or ocular
• To condense and focus light onto the specimen
What is the main purpose of the microscope?
• The condenser
• The objective
• The eye-piece or ocular
• To condense and focus light onto the specimen
• To form a clear intermediate image
What is the main purpose of the microscope?
• The condenser
• The objective
• The eye-piece or ocular
• To condense and focus light onto the specimen
• To form an intermediate image
• To form the final image
Final image
Intermediate
image
Specimen
Any questions?
What is the main purpose of the microscope?
• Magnification: apparent increase in size
• Resolution: the minimum distance between
2 dots that can be discerned
Airy disc: defined as
the region enclosed
by the first minimum of
the Airy pattern and
contains 84 % of the
luminous energy.
http://micro.magnet.fsu.edu/primer/lightandcolor
Resolution: the minimum distance
between 2 dots that can be discerned
http://micro.magnet.fsu.edu/primer/
The smaller the diameter of the Airy disc produced by
a lens, the higher is the resolving power of that lens,
the better you can separate two distinct points.
http://micro.magnet.fsu.edu/primer/
Resolution = (0.61 λ) / Numerical Aperture
The larger the numerical aperture of a lens,
The smaller the Airy disc,
The smaller and better the resolution.
http://micro.magnet.fsu.edu/primer/
Airy disk sizes vary with changes in objective
numerical aperture and illumination wavelength.
Resolution = (0.61 λ) / Numerical Aperture
Resolution = (0.61 λ) / (n x sin θ)
λ: wavelength of light
n: refractive index of the medium in the object space
Θ: angular aperture
Resolution = (0.61 λ) / Numerical Aperture
Resolution = (0.61 λ) / (n x sin θ)
λ: wavelength of light
n: refractive index of the medium in the object space
Θ: angular aperture.
Objective
=θ
Specimen
Angular aperture is a measure of the number of the highly
diffracted image-forming light rays captured by the
objective
• expressed as the angle between the microscope optical
axis and the direction of the most oblique light rays
captured by the objective.
http://micro.magnet.fsu.edu/primer/
Resolution = (0.61 λ) / (n x sin θ)
In theory, θ cannot be superior to 90o
sin(θ) cannot be superior to 1.
In the best microscopes, θ is about 70o
with a sine of 0.94.
The other limitation for the objective is
the refractive index of the medium;
generally, it is air with n=1.
Resolution = (0.61 λ) / (n x sin θ)
If in air and maximum θ, then r = (0.61 λ) / 0.94
where 0.61 represents the degree to which image points can overlap
and still be recognized by an observer as separate points
• the lower the wavelength,
• the lower and better the resolution,
Resolution = (0.61 λ) / (n x sin θ)
If in air and maximum θ, then r = (0.61 λ) / 0.94
Monochromatic light better
Possibility to use coloured filters
If blue light, λ = 450 nm, r = 274.5 / 0.94 = 292 nm
If green light, λ = 550 nm, r = 335.5 / 0.94 = 356 nm
If UV light, λ = 250 nm, r = 152.5 / 0.94 = 162 nm
Because the λ of an e- is much shorter than a that of a
photon, resolution is much greater in an e- microscope
(0.2 nm).
• The shorter the λ,
• The better the resolution
Resolution = (0.61 λ) / (n x sin θ)
To further improve resolution, to visualize
minute specimens, one could use a medium with
a n higher than that of air.
Use of immersion lenses
allows more light to be
collected
More points to
form an image,
Better resolution
There is no diffraction
of light because of the
homogeneity of the
refractive indices.
http://micro.magnet.fsu.edu/primer/
Resolution = (0.61 λ) / (n x sin θ)
If maximum θ and blue light, then r = 274 / (n x 0.94)
If air (n = 1), r = 274 / 0.94 = 292 nm
If 50% glycerol, r = 274 / (1.4 x 0.94) = 208 nm
If generic imm. oil, r = 274 / ( 1.515 x 0.94) = 192 nm
If permount, r = 274 / (1.525 x 0.94) = 191 nm
If Canada balsam, r = 274 / (1.545 x 0.94) = 189 nm
• The larger the refractive index of the medium,
• The better the resolution.
• angular aperture
NA = n • sin(θ)
The larger the angular aperture,
The higher the numerical aperture,
The more light the lens can capture,
The more information the lens can transmit.
The larger the numerical aperture of a lens,
The smaller the Airy disc,
The better the resolution.
Any questions?
The objective: a combination of lenses
Motic
Zeiss
Plan
CP-Achromat
4x / 0.10
10x / 0.25
40x / 0.65
∞ / 0.17
5x / 0.12
10x / 0.25
40x / 0.65
100x / 1.25 oil
∞/∞ / 0.17
r = (0.61 λ) / NA
http://micro.magnet.fsu.edu/primer/
It would be a pity if the intermediate
image produced with such sophisticated
optics were to be impaired just before it
reaches the eye because of poor handling
of the microscope.
Koehler illumination
Koehler or Köhler illumination
• Switch on the microscope
• Check that the light is on
http://www.zeiss.com
Koehler or Köhler illumination
• Open wide the field diaphragm:
the spot of light should be at
its maximum diameter
http://www.zeiss.com
Koehler or Köhler illumination
• Open wide the iris
diaphragm of the
condenser. The small
light spot shows its
maximum brightness
http://www.zeiss.com
Koehler or Köhler illumination
• Place slide on stage
• Reduce brightness of light if need be
• Position the oculars so that you are
comfortable when looking through them
• Adjust your view by focussing the
diopter compensation ring
http://www.zeiss.com
Relaxed viewing is important
First, look into the distance with your eyes relaxed
and then into the eyepieces – without changing the
setting of your eyes. Only then should you set the
inter-pupillary distance of the eyepieces via the folding
bridge until you see only one circle instead of two.
Remember to use both your eyes for viewing.
Keep your distance
Microscopes for teaching labs are usually designed for eyeglass
wearers. Therefore, the exit pupil of the eyepiece is at a considerable
distance from the eyepiece. Users who do not wear eyeglasses should
also keep this distance to permit the entire light from the microscope
to find its way to the iris of the eye. If you slowly move your head to
and fro in front of the eyepieces, you will soon find the optimum,
relaxed posture allowing you to see the entire circle of the field of view.
Koehler or Köhler illumination
• Focus grossly on the specimen
http://www.zeiss.com
Koehler or Köhler illumination
• Close the field diaphragm
• Focus the condenser by
using the focussing knob
on the left of the microscope
http://www.zeiss.com
Koehler or Köhler illumination
When the condenser is
focussed, you will see the
sharp edges of the field
diaphragm
http://www.zeiss.com
Koehler or Köhler illumination
• Center the light with
the centering knobs
placed below the stage
You should now see a
centered image of the
specimen surrounded by
black
http://www.zeiss.com
Koehler or Köhler illumination
• Open now the field
diaphragm
• Do open just enough
to fill the field of view
Do not touch again the condenser knob,
because your condenser is now focussed
I was taught to set up Koehler illumination with
a focussed slide on the microscope stage but
with the specimen out of the field-of-view so that
its staining could not interfere with the light source.
I was also taught to use whenever possible a blue
filter so that a monochromatic light with a low λ
is obtained.
Koehler or Köhler illumination
• Remove gently one of the eye-pieces (us. the left).
• Look down the tube to see the back focal plane of
the objective
http://www.zeiss.com
Koehler or Köhler illumination
http://www.zeiss.com
Koehler or Köhler illumination
Three elements essential for optimal results:
• circular outline of the objective aperture,
• iris opening of the aperture diaphragm of the condenser,
• image of the light source.
http://www.zeiss.com
Koehler or Köhler illumination
Three elements essential for optimal results:
• circular outline of the objective aperture,
• iris opening of the aperture diaphragm of the condenser,
• image of the light source.
• Adjust the size of the light disc by swinging
the iris diaphragm of the condenser
http://www.zeiss.com
Koehler or Köhler illumination
• A compromise between resolution and contrast
9/10: best resolution
2/3: best contrast
For research purposes,
you should set-up Koehler each time you change objectives.
9/10 position of aperture diaphragm
Maximum resolution
2/3 position of aperture diaphragm
Maximum contrast
Maximum depth of detail
1/2 position of aperture diaphragm
Creation of artefacts
Klosevych, 1989
Microscopy and photomicrography. R.F. Smith, CRC Press
1
2
3
1: Aperture diaphragm = NAobj
lack of contrast and low visibility
2: Aperture diaphragm at 90%
better contrast &
excellent resolution
3: Aperture diaphragm at 50%
excessive diffraction &
creation of artefacts
Tips given by Zeiss:
• Relaxed viewing is important
• Keep your distance
• Exclusively for eyeglass wearers: a little test
• Avoid the use of force
• Protect your investment: the dust cover
• Please avoid “do-it-yourself” work on the microscope
Two invaluable web-sites to visit
when you want to learn more about microscopy:
http://micro.magnet.fsu.edu/primer
http://www.zeiss.com
(Microscopy from the beginning)