Zeiss Photomicroscope

Photomicroscope Tutorial

Because the light microscope will be one of the most important tools we use, it is important to know how to get the best results from it. We will be using a Zeiss microscope for photomicrography in this class. However, the instructions for correct alignment of the condenser will be applicable to other microscopes with adjustable condensers.

The diagram of a typical compound microscope is given below. Light is provided by a built in bulb which is reflected through the field diaphragm, the condenser aperture iris, the condenser, the specimen, the objective, the tube, ocular into your Eye or a Camera.

Light Source

Mirror

Field Diaphragm (Iris)

Condenser Iris

Condenser Lenses

Specimen

Objective Lens

Microscope Tube (Body)

Ocular Lens

Eye or Camera

Light Path in a Typical Microscope

The Light Path in the Zeiss Photomicroscope is a little different.

Light travels through the Objective to a Mirror which reflects it towards the back of the microscope.

It passes through a Beam Splitter which directs some light towards the Cassette holding the Film.

Part of the Beam is split again.

Some light is projected onto the Sensor for the Light Meter.

The rest is directed towards the Oculars.

The Light Meter Sensor and the Film Cassette constitute the "Camera" for this microscope.

1] Locate the parts of the microscope by referring to the figures above & below.

There are various control knobs on the microscope which affect the light path. In addition, there are knobs for coarse and fine focus, as well as knobs to move the stage. There are also some buttons which insert filters into the light path. Refer to the figure below.

2] Locate the coarse and fine focusing knobs, and the knobs which control the mechanical stage. The latter are on the right side of the microscope as it faces you. Also locate the Condenser Focusing Knob which is also on the right side of the microscope.

3] The Beam Path Control Knob is used to direct the light path. When the knob is pushed fully into the scope, all of the light goes to the oculars. When it is pulled out 2 stops (to the black line) half of the light goes to the oculars and the rest goes to the "camera". The photographic reticule is also inserted into the ocular field of view.

The condenser aligns and focuses light on the specimen.
It focuses light just like a magnifying glass can focus sunlight to produce a hot spot. It has a knob on the right side which raises and lowers the condenser for focusing.

You will find a knob which controls a swung in or out lens on the left side of the scope. This is similar to your classroom scopes. This swinging lens is left out for low-power illumination [i.e. 4X], and swung into the light path for objectives of 10X or greater magnification.

There are two small knobs on the rear of the condenser, set at 45^o which are used to center it.

Finally there is a lever which controls the aperture iris in the condenser.

3] Locate all of the controls for the condenser.

The light source is housed in the base of the microscope. It passes through the field diaphragm which contains an iris. Rotating a knurled ring which is concentric with it controls the size of the field diaphragm. The field diaphragm controls the area of illumination. The proper area varies with magnification. At low magnification you need a wide area, but for high power you should reduce the area of illumination. The best results are obtained when the area of illumination extends just beyond the field of view. This is very critical for photomicrography but is less critical for routine work.

4] Locate the field diaphragm and its knurled ring.

The magnification of an image is controlled by the objectives which are housed in a rotating nosepiece, and the oculars.

There is a separate projection lens inside the scope for photography.

To change objectives you rotate the nosepiece counterclockwise, starting with the 4X objective.

Notice that we have 2.5, 4 or 6.3, 16, 25, 40 & 100X objectives.

Once one of the lenses is focused a specimen, the others should also be in focus when they are swung into place. This property is referred to by the term parafocal.

However, in actual practice some focusing adjustment is required when you switch from one objective to another.
Do so with care!!!

Specific Instructions for our Photomicroscope

When you swing in the 16X objective you will need to rotate the focusing knob CLOCKWISE (towards the rear wall).

If you then switch to the 25X objective, you need to rotate the focusing knob COUNTERCLOCKWISE.

When you go from the 25X to the 40X objective CLOCKWISE focusing adjustments are required.

We will not use the 100X objective in this course. This is an oil immersion objective. If you must use this and similar objectives, be sure that the specimen was in focus at 40X before switching to 100X. Place a drop of oil over the area to be studied before the lens is swung into position. Never focus down on the specimen with an oil immersion lens. Always change the focus so that the objective is traveling away from the slide.

Cryosections are a little thicker than commercial slides. Consequently, greater care must be taken when changing objectives with these. When in doubt, play it safe and ask for help until you get acquainted with the material that you are studying.

This microscope has a built in set of ancillary
magnification lenses. This is called the "Optivar".

The Optivar is located on the right side of the scope, just above the nosepiece which holds the objectives.

It is normally set at 1.25. You can rotate it to 1.6 or 2.0.

Thus, you can increase magnification without changing objectives. This is very useful.

The PH spot on the Optivar is used to align Phase Rings for Phase Contrast microscopy. We will deal with this later.

Kohler Illumination

The best resolution occurs when all elements of the microscope are in perfect alignment and the iris diaphragms are properly adjusted. On simple microscopes you may not be able to alter the alignment of the different parts, but on the photomicroscope it is possible to align and focus the condenser to achieve "Kohler Illumination". August Kohler was a German microscopist who discovered the benefits of proper illumination for light microscopy, and this procedure retains his name. This is critical for photomicrography.

Procedure

Examine the Stage of the photomicroscope.

a] Gently pull back the lever which is used to hold a slide in place on the stage.

b] Place your slide on the stage & release the lever.

c] Locate the knobs which move the stage in X or Y directions. These are on one side of the stage.

d] Grasping the indicated area in the figure below can rotate this stage. This allows you to compose the best orientation for your pictures without handling the slide manually.

1] Place a commercially prepared slide on the stage.

2] Make sure the swinging lens is in the light path and focus using the 10X objective.

3] Use only one eye [right eye with right ocular or left eye with left ocular] and focus the specimen with the coarse/fine-focusing knob.

4] Use the knurled ring below the other ocular to focus it while looking through it with your other eye. You may not need to change the focus. However, experiment by rotating the focusing ring to see its effect.

5] Make sure the condenser iris is completely open [rotated all the way counter-clockwise].6] Reduce the field of illumination by rotating the knurled ring on the field diaphragm completely clockwise. 7] You should see a small circle of light.

8] Use the condenser focusing knob to make the circle as small as possible by gently rotating it. This moves the condenser up and down. As you focus the field diaphragm you will notice that its halo turns from blue to red and red to blue. The best focus occurs when you adjust the condenser so that the halo is just between red and blue.

9] Expand the field diaphragm by rotating its knurled ring counter-clockwise, until the circle of light touches one edge of the field.

10] Center the light by using the two small adjustable knobs on the rear of the condenser. When you are satisfied, expand the field so that the light fills it completely. However, do not fully open the field diaphragm. Open it enough to extend just beyond the field of view.

11] For photography this should be done for each objective and for each Optivar setting.

Kohler illumination can’t be achieved for some low power objectives. I will discuss this later.

12] Adjust the Condenser aperture iris by removing one ocular and looking directly down the tube at the light field with a small telescope. Close the iris so that it occludes 1/4 of the area. This should give the best contrast. This has its greatest effect at higher magnifications (25 - 100X).

13] Examine a specimen before and after adjusting the aperture iris. This should be done for each objective for critical viewing. In practice, you can experiment with this while viewing a specimen and adjust it without removing the ocular. Closing the aperture iris increases contrast and depth of field up to a point. If it is closed too much, a flat indistinct image results. Closure also diminishes the color of the specimen.

14] Experiment with the aperture iris while viewing a prepared slide. Once you have achieved what you think gives the best image quality, remove one of the oculars and see how much of the field is occluded.While these procedures may seem tedious, they will become routine as you progress and they are essential if you want to get good photographic results.

Typical films

Color Slides

Ektachrome 100

Fujichrome 100

Ektachrome 200 or 400

Slide film with greater sensitivities (800, 1600) can be used fluorescence microscopy.

I haven't used Print Film very much. However, Fuji Color or Kodak ASA 100 films should be adequate. You can scan the prints and the negatives which can be an advantage. Print films are more forgiving with bad exposures but they may not have the best resolution.
It is essential to use the daylight blue filter located in the base of the microscope when you use daylight films in the microscope!!!!!
You can buy Tungsten light-balanced Ektachrome film but it is more expensive and does not yield better results, in general.
Use ASA 100 Black & White film as well. Use the built in green filter for B & W photos.

Removing the Film Magazine (FM) from the Scope
(Film Magazine = Film Cassette)

Locate the FM on the Right Side of the Scope

Brace the microscope body with your left hand

Press in on the Film Magazine

Rotate it Counterclockwise

The Red Line on the FM should be Opposite the Red Dot on the Mounting Bracket

The FM can be removed with a little Jiggling

Rewinding Exposed Film

Place the Rewind Crank in the hole of the FM

Turn Clockwise until there is no tension

You should hear and feel the film leader disengage from the spindle

Opening the Film Magazine

Rotate the Film Magazine until you see a Silver Button in a slot near its base.

Hold with BOTH HANDS, apply slight Pressure.

Rotate until the Silver Button is out of the groove.

Place the FM on the counter and Remove the Outer Housing.

Remove the Film Holder & place it on the counter.

Removing Film

Place the Film Holder in your Hand to Cushion the Cover of the Film Holder

Find the Silver, Slotted Button near the top of the Film Cassette

Press the Slotted Button

The Film Holder Cover will be released

Remove the Film Cassette

Placing a Film Magazine in the Scope

The Camera Port MUST NEVER be left Open!

Be sure that a film Magazine, even an empty one, is placed into the photomicroscope.

Lock the Cover of the Film Holder by pressing it so that it engages the silver, slotted button and snaps in place.

Reassemble the Film Magazine

Insert it into the Scope

Match the Red Line with the Red Dot

Brace the Scope

Press on the Film Magazine

Rotate it Clockwise

The Red Line should be Aligned with a Black Dot.

Inserting a New Roll of Film

Remove the Film Holder from the Film Magazine (See Above)

Press The Silver, slotted Button

The Cover Opens

Insert the New Roll of Film

It will only go in ONE Way

Do NOT Force it

Drag the Film across the front of the Film Holder

Crease the end of the Film at the 2nd Sprocket Hole

LOOP THE FILM UNDER the far spindle

Insert the end of the film in the slit on the spindle so that a sprocket hole is caught in the small hook (in the slit)

Manually roll the spindle clockwise until the film is clearly secure and will not fall out of the slit

Check to see that the two sprockets of the film holder are aligned and engaged by the sprocket holes in the film

Close the Film Cover

Reassemble the Film Magazine

Insert the Film Magazine (as above)

Preparing to take the First Exposure

The Film Advance Button (Yellow Light) on the Exposure Meter should be Blinking

This happens at the end of a roll &

When a new roll of film is added

Press the Blinking Yellow Button

You should hear the film advance

Press the button again

The film should advance

However, sometimes it does not’t

Do Not Panic!!!!!!!!!!!!!!!!!!!!!!!!!!

By pressing the yellow button you are advancing film that was exposed to light while you were loading it into the film holder.

If the yellow button only works once, the next 2-3 frames will probably not be any good

If the yellow button works twice, the next 1-2 frames will probably not be good

Place a ANY slide on the stage, and focus on it, be sure that light is directed to the camera and click off the first 1-3 exposures by pressing the A button on the exposure meter.

The Next Exposure will be OK!!!

Your First Exposure

1] Pull out the Beam Path Control Knob 2 clicks so that the black circle is just visible. The photographic reticule should be visible.

2] The reticule has a circle at he center and four lines emanating from the corners of the rectangle.

3] Focus on a Specimen.

4] Move the specimen out of the frame.

5] Completely defocus both oculars by rotating them fully counterclockwise.

6] Using one eye at a time - Rotate the oculars clockwise until you can see that each line is actually composed of two lines. (See Below) You have now focused the oculars for your eyes!!

7] This is a critical step!!!! If you do not focus the oculars this way your photos will all be out of focus!!!!!

8] Bring the specimen into the field.

9] Compose the picture by adjusting the magnification and orientation of the subject.

10] Check your Kohler Illumination!!!!!

11] Use the circle in the reticule to center the condenser.

12] Expand the light just beyond the field of view.

13] Adjust the Condenser Iris

Filters

Make sure you have the correct filters in place.

Check on the right side of the base to be sure that the filter with the yellow line is pushed into the scope.

This is the Daylight Blue Filter & it MUST ALWAYS be inserted for Color Photos with outdoor films. For Unstained Specimens, or those stained with phloroglucinol, place a second blue filter over the Field Diaphragm.

For specimens stained with Tol. Blue or another blue dye, omit the second blue filter, but leave the Daylight Blue Filter in place.

For Polarizing photos, place a polarizer on top of the Field Diaphragm and rotate it to get the desired degree of polarization..

For Black & White photos, insert the built-in green filter on the right-hand base of the scope and remove all other filters. You can use a polarizer for B & W photos.

The other built-in filters are neutral density filters. You will not use these.

Low Magnification, Field Diaphragm & Filters

Swing out the high power condenser lens for magnifications with the 2X & 4X objectives.

The 6.3X objective is on the border line between low and high magnification & photos can be taken with or without the filters below.

Place the Sintered Blue Filter over the Field Diaphragm.
This will diffuse the light and make it more uniform.

If you are photographing specimens which have stained Blue, place a piece of fine lens paper over the field diaphragm.

In both cases you may need to defocus the Condenser.
Otherwise the image of the filters may appear in your photos!!!!!!!

Open the Field Diaphragm and the Condenser Iris completely!

Use Maximum Illumination!!!

It may be impossible to get good low magnification pictures of cryosections because they are relatively thick.

Now he tells me!!!

Furthermore, capturing colors is greatly hampered by the thickness of cryosections.

Consequently, I often use Crossed Polarizers for low mag. photos. These lack some details but can be effective nonetheless!!!

If you use crossed polarizers, turn the illumination to maximum and open the Field Diaphragm and the Condenser Iris completely!

Adjusting The Light Meter

It has Two Dials, one Meter and a set of Buttons.

The Left Knob adjusts the light intensity.

The Meter gives a "read out" of the light intensity.

The Right Knob sets the film speed (sensitivity)

The Buttons control the shutter.

Make sure that the Left Knob is rotated fully counterclockwise.

Push in the Green Button. This turns the lamp ON.

Rotate the Left Knob until the Meter reads 5-6.

Adjust the illumination as necessary.

You will need greater illumination for high magnifications.

Use bright illumination to focus on the specimen.

Reduce the illumination to 6 to take a picture.

This is an old light meter

It gives proper exposures if they are of long duration

Short exposures are unreliable.

Thus, the light must be reduced to get a good photo.

The Film Speed is set with the Right Knob

We typically use Fujichrome or Ektachrome Slide Film with a speed/sensitivity of ASA 100 (DIN 21)

ASA = American Rating (Orange Numbers)

DIN = European Rating (White Numbers)

If you rotate the small central knob which sticks out, the reading will change by 3 DIN numbers which corresponds with corresponding changes in the ASA rating.

A greater ASA or DIN signifies a MORE SENSITIVE film which requires a shorter exposure.

The wider (outer) ring which is flush with the front panel changes the sensitivity by one DIN number on either side of indicator.

DO NOT TRY TO TURN THIS TO MOVE MORE THAN 1 DIN UNIT - IT WILL BREAK!!!!

Trial and Error has taught me that you get the best result with DIN 27 -30 for ASA 100 films.

To play it safe, Take one exposure at DIN 27 & Another at DIN 30.

This is due to the age of the light meter!!!

Check to see that you have the correct film sensitivity selected.
Press the A Button

You will hear

the shutter open (Oh happy day!)

the shutter close &

the film transported

You may notice that the yellow (Film Advance) button flickers when the film is transported.

It will stay ON when the roll is finished.

Top

Lock the Cover of the Film Holder by pressing it so that it engages the silver, slotted button and snaps in place.
Reassemble the Film Magazine Insert it into the Scope Match the Red Line with the Red Dot Brace the Scope Press on the Film Magazine Rotate it Clockwise The Red Line should be Aligned with a Black Dot.