In the quest for getting best performance from the microscope, I’ve read several descriptions on the setting up of Köhler illumination. – Some of which leave much to be desired regards clarity of information. I’ve described below, a collective from these descriptions, which I believe to be the correct method, and that which I’m currently using.
As always, if any experienced microscopists can see any errors with this method, I’d be very grateful for good advice.
The routine is as suggested for transmitted light (brightfield illumination) with today’s typical compound microscope, having an “Abbe” sub-stage condenser with built-in iris diaphragm, on board illumination, and a separate field iris diaphragm located just above the lamp housing.
Some sub-stage condensers are fitted with a swing out top lens assembly. This lens usually needs to be in the light path at all times except when using very low power objectives of x4. (But to be certain, always refer to manufacturer’s instructions for the specific microscope).
Other types of sub-stage condenser may be fitted with a swing out bottom lens, (sometimes referred to as a “bulls-eye” lens). In this case, the swing out lens is usually removed from the light path at all times other than when using low power x4 objectives. (But again, always refer to manufacturers instructions for the specific microscope).
1 – Noting the above, ensure that any swing out lens is either in or out of the light path, according to the specific requirements of the microscope concerned.
2 – Place a suitable pre-prepared specimen slide (with cover slip) on the microscope’s stage. A thin, translucent specimen should be used for this purpose. Adjust the lamp intensity to a comfortable brightness, and using the x10 objective, bring the specimen into sharp focus.
3 – Close the field iris diaphragm so that only a small circle of light is allowed through.
4 – Without altering focus on the specimen slide, slowly rack the sub-stage condenser assembly up/down (see note below) until the field iris diaphragm blades are sharply in focus, superimposed onto the specimen on the slide. (You may also need to open/close the field iris diaphragm slightly whilst viewing, to get a better feel for the shape of the blades). There is a critical point during this focussing of the field iris blades, where red fringing occurs on one side of the focus point, and blue fringing occurs on the other side. Slow and careful condenser height adjustment is therefore needed in order to attain the optimal point of focus between the two Note
– It is as well to ensure prior to this, that the sub-stage condenser’s top limit of travel does not allow it to crash into the bottom of the microscope slide, but leaves just the tiniest amount of air space between.
5 – Centre the now sharp image of the field iris diaphragm blades within the field of view, by using the two condenser centring screws.
6 – Open the field iris diaphragm until the blades just go out of field of view. (If properly centred, the blades will all exit the field of view at the same time).
7 – Remove the eyepiece, (or in the case of a binocular microscope, one of the eyepieces), and look down the tube from about 6 inches away. You will see a sharply defined ring of light, which is passing through the iris of the sub-stage condenser. Adjust the width of the sub-stage condenser diaphragm until it opens just more than the entire field of view, and then close it back in so that only about three quarters of the maximum field of view can be seen. Note
– Some sources advise setting the condenser iris such that four fifths of the field of view can be seen, but a setting of three quarters is claimed to be the best compromise in providing an even field of illumination, and optimal contrast.
8 – Replace the eyepiece. Basic Köhler set-up is now complete.
In theory, steps 3 to 8 should be repeated with each subsequent change of objective lens. In practice, repeating only steps 7 & 8 should suffice.