Kohler Illumination

Setting up Kohler illumination on Zeiss Photomicroscope III

I was given a demonstration today on how to set up Kohler illumination on the Zeiss Photomicroscope III – as the chap says most researchers (including me) do not know how the get the most out of their microscopes. This process is the microscope equivalent of telescope collimation and, like telescope collimination, makes a big difference to the quality and contrast in the images seen.

The following are my notes from the session:

1. There are two lens holders just below condenser. One has lens and the other a diffuser. Rotate the diffuser out of the light path but leave the lens in the light path.
2. Ensure at this stage that the field diaphragm on the base just above the light is fully open.
3. Focus on sample (any slide) using 10-20x objective – do not use higher power – using the knob right at the BACK. Turning this knob leads to the stage moving up and down.
4. Stop the field diaphragm down until you can see the octagon shape of its edge easily.
5. Focus the condenser using the FRONT knob so that this octagon is in focus. Turning this knob leads to the condenser going up and down independent of the stage.
6. Turn the centring knobs on the condenser to centralise the octagon in the field of view. These two knobs are located at back left and back right on the condenser and work in a similar fashion to centring knobs on finder scopes on telescopes.
7. Open up the field diaphragm until the octagon edge is only just visible at edge of field of view.
8. The process is now complete and even high quality illumination should be achieved.