Köhler Illumination

The Algal Web: Methods

Setting up Köhler Illumination on your microscope:

1. Place a specimen on the stage and focus on it (your microscope must be at least roughly set up to do this).
2. Reduce the aperture of the field iris diaphragm (located nearest the light source) so that you can see its edges.
3. Bring the edges of the field iris into focus by focusing on the condenser. Both the specimen and the iris should now be in focus.
4. Center the field iris using the two condenser-centering knobs.
5. Adjust the condenser iris (immediately below the condenser) to reduce glare around the edge of the field iris.
6. Open the field iris so the edges lie just beyond the field of view. The condenser iris can be adjusted slightly to optimize image contrast.
7. Do not use the condenser iris to adjust light intensity. Use the adjustable lamp power supply (which will change the color rendition),
or (for color photography) with neutral density filters placed between the lamp and the field iris.