Koehler illumination is a process that provides optimum contrast and resolution by focusing and centering the light path, thereby spreading the light evenly over the field of view. To allow a microscope to be set up for Koehler illumination, it must have two adjustable iris diaphragms: The aperture diaphragm (aperture stop, AS) at the condenser and the field diaphragm (field stop, FS) nearer to the lamp. The AS diaphragm controls the angular aperture of the light cone from the condenser, while the FS diaphragm controls the area of the circle of light illuminating the specimen.
Light from the primary light source is collected by the collector lens and forms an image at the AS (aperture stop) position. This image acts as a secondary light source, reproducing the image at the objective's exit pupil position and casting telecentric (parallel light) illumination on the specimen surface. This is generally known as "Koehler illumination" and has two main features. One is its brightness and uniformity; the other is that it allows both AS and FS (field stop) settings to be changed independently.
The effect of FS
FS settings adjust the field of view at the extreme periphery and prevent extraneous reflected light from interference with the image forming light. This effectively eliminates flares from the whole image.
The effect of AS
The AS is effective when adjusting the angular aperture of the illuminating light cone or changing the image contrast. Generally, the best contrast is obtained by diapharagming the objective pupil diameter to about 80%. However, when using objectives of over 100x magnification, better contrast images are obtained by diapharagming the objective pupil diameter to less than 50%.

(A) Focusing the condenser:
1. First place a stained specimen on the stage and focus using a 10x objective.
2. Now locate the FS diaphragm control, located near the light outlet in the base of the microscope. Close this diaphragm right down while looking down the microscope - you will see a dark circle encroaching on the image.
3. Locate the focus control on the condenser, which is usually a knob on either or both sides of the condenser. By rotating the knob while observing the specimen you will be able to focus the condenser so that the edge of the dark circle (the blades of the iris) appears sharp. Do not move the main focus controls during this procedure.

(B) Centring the condenser:
Find the controls for centring the condenser. These are generally two thin knobs projecting at angles from the front of the condenser carrier. While observing the specimen, rotate either or both controls to bring the bright circle to the centre of the field of view. This process is made easier in the final stages by opening the diaphragm almost to the edge of the field of view so that the image of the edges of the blades can be aligned with the edge of the field of view. Once the condenser has been focused and centred in this way, the diaphragm can be opened so that it is just outside the field of view. The condenser will remain centred when different objectives are selected, but the field iris diaphragm will have to be adjusted to just outside the field of view at different magnifications.

(C) Adjusting the aperture iris:
This important step is often neglected, leading to either suboptimal resolution and/or poor contrast.
1. First locate the control for the aperture iris, which may be a ring around the condenser. If necessary, remove the condenser to locate the ring and check its action.
2. With the condenser in place, focused and centred, remove an eyepiece and look down the tube while turning the iris control. You will see a dark circle encroaching on the image at the bottom of the tube (the back focal plane).
3. This process can be made easier by slipping a centering telescope (used for setting up phase contrast) into the tube in place of the eyepiece. For most specimens, the iris should be closed down so that it occupies the outer 20% or so of the field. This increases the contrast, making observation easier. Although some specimens may need variation on the 20%, beware of closing the iris too much as resolution will be drastically reduced. A more accurate way of adjusting the aperture iris is to note the numerical aperture (NA) on the objective, then set the NA on the condenser to 20% less. For example, with a 40x objective with an aperture of 0.65, set the graduation on the condenser to 20% less, approx. 0.5.
4. It can be seen that unlike the field iris setting, the aperture iris should be reset for each objective to match the NA of the condenser to 80% of the NA of the objective.