How to adjust a microscope for Köhler Illumination

Köhler illumination is a microscope alignment technique, most often used for transmitted light, that ensures images are evenly illuminated and at maximum resolution. The microscope should be adjusted for Köhler illumination before transmitted light imaging, phase contrast and especially differential interference contrast (DIC), which is highly sensitive to incorrect alignment.

Alignment Procedure

Focus on the specimen using a low power lens like a 10x air. Fully close the field diaphragm and adjust the condenser focus so the image of the iris is in sharp focus. Centre the image of the iris using the two centring screws. Open the field diaphragm until it is just outside the field of view. Remove an eyepiece and look into the empty opening. Adjust the iris aperture until it is about 70% open and then replace the eyepiece. The microscope is now adjusted for Köhler illumination.