Field Diaphragm

A transmitted-light microscope has two adjustable irises, and they are easy to confuse. The field diaphragm, near the lamp, sets how much of the specimen is illuminated; with Köhler illuminationLoading... its edge is focused in the specimen plane. The condenser aperture diaphragm, under the stage, sets the angle of the illumination, which trades contrast against resolution.

Neither one is a brightness control, although both change brightness. Using the aperture diaphragm to dim the image changes contrast and resolution; using the field diaphragm cuts off the field. Brightness belongs to the neutral density filterLoading... and the exposure time.

Close it until the edge is visible, focus and center it with the condenser, then open it until the edge just clears the field. The field that matters is the camera's, which is often a different size and shape from what the eyepieces show, and usually rectangular. Set the diaphragm while watching the live camera image, not through the eyepieces.

Repeat for each objective, because the field of view changes with magnification. A diaphragm opened for a 10× objective is far too wide for a 40×.

Opened well past the field, the diaphragm lights specimen and glass that the camera does not image. That light still scatters inside the specimen, the coverslip and the objective, and some of it reaches the sensor as a diffuse glare added to every pixel.

In brightfield the glare does most damage to the darkest areas: it adds light to strongly stained regions, so their measured optical densityLoading... is compressed toward the background and strong staining reads weaker than it is. Closed too far, the problem is the opposite and obvious: dark, cut-off corners that look like severe vignettingLoading....