Proper use of the Compound Microscope  
     Kohler Illumination & Staining-3

The best resolution occurs when all elements of the microscope are in perfect alignment and the iris diaphragms are properly adjusted to the best aperture. On simple microscopes you may not be able to alter the alignment of the different parts, but on these Leitz microscopes it is possible to align and focus the condenser to achieve "Kohler Illumination".

Because we will be using a lot of thick hand-sections in this class, it is vital that you learn how to achieve Kohler illumination. Otherwise, you will not be able to analyze your specimens.

1] Place a commercially prepared slide on the stage.

2] Make sure the swinging lens is in the light path (facing up) and focus on the specimen 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 knurled focusing ring to see its effect. My German friends have told me that the correct way to focus the second ocular is to make it more negative so it is out of focus, then rotate it in a positive direction until it is focused.

5] Having the oculars focused will improve image quality and will decrease eye strain. Once this is done it need not be changed during a given session. However, it is a good habit to do this at the beginning of each lab. It is best done at 10X because there is less chance for errors at this magnification compared to 4 X.

6] Make sure the aperture iris is completely open [rotated all the way counter-clockwise].

7] Reduce the field of illumination by rotating the knurled ring on the field diaphragm completely clockwise. Be gentle with the field diaphragm. It should close without any effort.

8] You should see a small circle of light. If you are lucky, it will be in the center of the field. However, it will most likely be off-center and out of focus. Let us know if you can't find it!!

9] Use the vertical condenser adjusting knob to make the circle as small as possible by gently rotating it. This moves the condenser up and down. Do this carefully so that the circle of light is not pushed laterally. 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. This is a little hard to do so don't be too worried if you have some red or blue in the halo.

10] Expand the field diaphragm by rotating its knurled ring counter-clockwise, until the light touches one edge of the field. If the light is perfectly centered it should touch the entire circumference of the field. This is unlikely.

11] Center the circle of light by using the two small adjustable knobs on the front 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 just enough to extend beyond the field of view.

12] Repeat this with the 20 or 40 X objective. For critical work this should be done for each objective. This is especially important for taking photographs and for examining minute, translucent specimens like fungi and algae. For our labs, it will be good to do this for the 10X objective at the start of each session. You need not do this for 4X and 40X. However, if you are having some problems resolving details, check to be sure that you have the condenser aligned and focused.

It may be difficult to do this with the 100 X objective. However, if you achieve proper alignment with the 40 X objective, the 100 X will be similar.

13] When working at 20 - 100 X it is important to adjust the condenser aperture iris. This is especially important for translucent structures. Closing this iris increases contrast. Thus something fuzzy becomes smooth and something faint becomes dark. It is usually possible to close the iris and judge its effects subjectively. However, there is a "tried & true" procedure which you should know.

14] Remove one of the oculars and look directly down the tube at the light field. Close the iris so that it occludes 1/4 - 1/3 of the area. This should give the best contrast. 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 also increases depth of focus up to a point. Thus, more areas of a three dimensional specimen will be in focus If it is closed to much, a flat indistinct image results.
The example shows part of a diatom frustule. There is little detail when the iris is wide open (top). When it is fully closed (middle) the contrast is increased but there are aberrations which make the small holes appear larger than they are in actuality. The outline of the small holes is also indistinct. When the iris is closed 25 - 30 % there is improved contrast and less aberration.

15] 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.

As part of the first lab, we will be using different stains to study their effects an fresh specimens. Experiment with the aperture iris as you study these. Fresh sections are usually too thick for detailed examination at high magnification, but the aperture iris can be used to great effect with this type of material.

While these procedures may seem tedious, they will become routine as you progress. If you look carefully you can see that Professor Kohler is smiling!