The primary advantage of Köhler illumination is the extremely even illumination of the sample. This reduces image artifacts and provides high sample contrast. Even illumination of the sample is also critical for advanced illumination techniques such as phase contrast and differential interference contrast microscopy.
1) Turn on the illuminator.
2) Open the field iris fully.
3) Open the condenser (aperture) iris fully.
4) Select a low power, dry objective lens (10 X or 20 X).
5) Put a specimen slide on the stage. A well-stained specimen is best.
6) Adjust the binocular tubes for your interpupillary distance. You should see one big circle of light.
7 ) Focus on the specimen.
8) Adjust the eyepieces and / or binocular tube to correct for your vision.
9) While observing the specimen, close the field iris until its edge appears. This is the hardest step for beginners since the field iris may be hard to find if it is very much misaligned. Watch for the movement of the iris or its shadow as you close and open it.
10) Focus the field iris using the condenser focus knob. Try for a thin purple fringe at the edge of the iris. If you can’t focus on the field iris, check the condenser for flip- in lenses and find the configuration that lets you focus on the field iris. If the iris moves out of the field of view as it is focused, bring it back into the field of view with the condenser centering screws.
11) Center the field iris with the condenser centering screws.
12) Open the field iris just to the edge of the field of view.
13) Pull out an eyepiece
14) While looking down the empty tube close the condenser iris to cover 10% to 50% of the full bright circle.
15) Put back the eyepiece.
Both specimen and field iris should be in focus together.
Tip:When changing often between objectives, align the field iris for the lowest magnification and the condenser iris for the highest.