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

Tutorial on MicroscopySeptember 15, 2007

Why the need to study microscopyWhy 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 microscopeStudents 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 nosewith objectives

Eye-piece or ocular

Revolving nosewith objectivesStage and its controls

Focussing knob

Eye-piece or ocular

Revolving nosewith objectivesStage

Condenser

Light source

Focussing knob

and its controls

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



http://micro.magnet.fsu.edu/primer/anatomy/condensers.html

The condenser: a combination of lenses

http://micro.magnet.fsu.edu/primer/anatomy/condensers.html

• The simplest condenser• Role: to condense and

focus light onto the specimen

Allows more or less light to enter the condenser


Also called an iris diaphragm


• Will possess specific characteristics (correction, numerical aperture and others) depending on manufacturer specifications

http://www.zeiss.com/C1256B5E0047FF3F?Open



http://micro.magnet.fsu.edu/primer/

The objective: a combination of lenses



Motic Zeiss



Motic Zeiss

Plan CP-Achromat

Achromat:Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also coversthe 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.


The effect of chromatic aberration

Rays of longer λ focus further away that those of shorter λ.


The effect of chromatic aberration





Achromat:Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also coversthe 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.


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

Motic

Plant microtechnique and microscopy. E. Ruzin

Field curvature: the sharpest focus of a lens is on a curved surface rather than on a flat plane.

http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

Magnification / Numerical Aperture



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil

Magnification / Numerical Aperture



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil

∞ / 0.17 ∞ / - ∞ / 0.17

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.



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil

∞ / 0.17 ∞ / - ∞ / 0.17

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.

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.

Klosevych, 1989



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil

∞ / 0.17 ∞ / - ∞ / 0.17

Any questions?



http://micro.magnet.fsu.edu/primer/anatomy/oculars.html

Eyepieces are not just simple lenses, but are corrected optical systems consisting of several lenses.

Eyepieces: magnifiers to view the intermediate Image produced by the objective and the tube lens.


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



There will be specifications on these lenses

Motic

WFPL 10x / 20 Glasses symbol

WF for Wide Field of viewPL to match the objective correctionMagnification 10XField number which refers to the diameter (in mm) of the fixed diaphragm in the eyepiece.



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.

Specimen

Intermediate image

Final image

What is the main purpose of the microscope?



• To condense and focus light onto the specimen



• To condense and focus light onto the specimen• To form a clear intermediate image



• To condense and focus light onto the specimen• To form an intermediate image• To form the final image


Specimen

Intermediate image

Final image

Any questions?

• Magnification: apparent increase in size• Resolution: the minimum distance between

2 dots that can be discerned


http://micro.magnet.fsu.edu/primer/lightandcolor

Airy disc: defined as the region enclosed by the first minimum of the Airy pattern and contains 84 % of the luminous energy.


Resolution: the minimum distance between 2 dots that can be discerned

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.


The larger the numerical aperture of a lens, The smaller the Airy disc, The smaller and better the resolution.


Resolution = (0.61 λ) / Numerical Aperture


n: refractive index of the medium in the object space Θ: angular aperture

Resolution = (0.61 λ) / (n x sin θ)

λ: wavelength of light

Airy disk sizes vary with changes in objective numerical aperture and illumination wavelength.


n: refractive index of the medium in the object space Θ: angular aperture.


λ: wavelength of light

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.

= θ


Specimen

Objective

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.


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,

If in air and maximum θ, then r = (0.61 λ) / 0.94


If in air and maximum θ, then r = (0.61 λ) / 0.94

Monochromatic light betterPossibility 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 nmIf 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


To further improve resolution, to visualize minute specimens, one could use a medium with a n higher than that of air.

Use of immersion lensesallows more light to be collected

More points to form an image,

There is no diffraction of light because of thehomogeneity of the refractive indices.

Better resolution



If maximum θ and blue light, then r = 274 / (n x 0.94)

If air (n = 1), r = 274 / 0.94 = 292 nmIf 50% glycerol, r = 274 / (1.4 x 0.94) = 208 nmIf generic imm. oil, r = 274 / ( 1.515 x 0.94) = 192 nmIf permount, r = 274 / (1.525 x 0.94) = 191 nmIf 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

The larger the angular aperture, The higher the numerical aperture,The more light the lens can capture,The more information the lens can transmit.

NA = n • sin(θ)

The larger the numerical aperture of a lens, The smaller the Airy disc, The better the resolution.

Any questions?



Motic Zeiss

Plan CP-Achromat

4x / 0.1010x / 0.2540x / 0.65

5x / 0.1210x / 0.2540x / 0.65100x / 1.25 oil

∞ / 0.17 ∞ / - ∞ / 0.17

r = (0.61 λ) / NA

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


• Open wide the field diaphragm: the spot of light should be at its maximum diameter




• Open wide the iris diaphragm of the condenser. The small light spot shows its maximum brightness



• 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

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.

Relaxed viewing is important

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.



• Focus grossly on the specimen



• Close the field diaphragm• Focus the condenser by using the focussing knob on the left of the microscope



When the condenser is focussed, you will see the sharp edges of the field diaphragm



• Center the light with the centering knobsplaced below the stage

You should now see a centered image of the specimen surrounded by black


• 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 λis obtained.



• Remove gently one of the eye-pieces (us. the left). • Look down the tube to see the back focal plane of the objective





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

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.


• A compromise between resolution and contrast9/10: best resolution2/3: best contrast

For research purposes, you should set-up Koehler each time you change objectives.

9/10 position of aperture diaphragm

Maximum resolution


Maximum contrastMaximum depth of detail


Creation of artefacts

Klosevych, 1989

2

3

1: Aperture diaphragm = NAobj

lack of contrast and low visibility2: Aperture diaphragm at 90% better contrast & excellent resolution3: Aperture diaphragm at 50% excessive diffraction & creation of artefacts

1

Microscopy and photomicrography. R.F. Smith, CRC Press

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

http://micro.magnet.fsu.edu/primer

Two invaluable web-sites to visit when you want to learn more about microscopy:

http://www.zeiss.com(Microscopy from the beginning)

http://micro.magnet.fsu.edu/primer

http://www.zeiss.com/

Documents

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