Köhler Illumination

A microscope has two interleaved sets of planes that are imaged onto each other. The field set runs through the field diaphragmLoading..., the specimen, the intermediate image and the camera sensor. The aperture set runs through the lamp filament, the condenser aperture diaphragm and the objective's back focal plane.

August Köhler's arrangement, published in 1893, puts the lamp in the aperture set. Each point of the filament then sends a parallel beam through the whole specimen instead of being imaged onto part of it, so the filament's structure never appears in the image and the field is evenly lit. It also separates the two controls: the field diaphragm sets how much of the specimen is lit, and the aperture diaphragm sets the angle of the light.

Focus on the specimen. Close the field diaphragmLoading... until its edge appears as a polygon in the field. Raise or lower the condenser until that edge is sharp; a thin color fringe is normal. Center the polygon with the condenser centering screws. Open the diaphragm until the edge just disappears beyond the field the camera sees.

Then set the condenser aperture diaphragm. Look at the objective's back focal plane through a Bertrand lens or with an eyepiece removed, and close the aperture to about 70–80% of the objective's numerical apertureLoading.... Repeat for each objective; at low magnification, often 4× and below, swing out the condenser top lens if your condenser has one.

The eye adapts to uneven light and hardly notices it; a camera records it exactly, and analysis software treats every brightness difference as information. An off-center or defocused condenser produces a brightness gradient across the field, which in brightfield reads as an optical densityLoading... gradient, a slope of apparent stain.

Köhler alignment removes the avoidable part of that unevenness at the source. The rest, the vignettingLoading... that comes from the optics themselves, is handled afterwards by flat-field correctionLoading.... Doing them in that order matters: a flat field captured with misaligned illumination corrects an error that changes the next time someone touches the condenser.