Koehler illumination

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In early microscopy, the light sources used to illuminate the object was derived from candles or lanterns with lenses to focus the light. This resulted in uneven and unpredictable illumination. One of the many contributions that August Köhler made to microscopy was to invent a system where light from a bulb could be collected and emitted as a uniform beam or cone of light.

For each magnification there is an optimum set-up. Getting this configuration right is frequently referred to as setting up Köhler illumination, but is more accurate to refer to it as optimising Köhler illumination.


Step 1

Picture 1: This shows an unfocussed image with a dark shadow in the corner.

Adjust the focus until your slide is sharp. You may notice that the edges are darker as in Picture 1, but more commonly the field condenser will be wide open, so the field view will be completely illuminated.

Step 2

Picture 2: Shrinking the field diaphragm reveals the blurry outline of the light beam.

Adjust the field diaphragm to make it smaller. This is the diaphragm near the bottom of the microscope, just above where light emerges. As you shrink it, the illuminated circular polygon will also shrink as seen in Picture 2.

Step 3

Picture 3: The disc of light becomes crisp as it is focussed.

Adjust the focus of the substage condenser. This is the knob under the microscope stage. You will notice that the edge of the illuminated circle will go from blurry to sharp and distinct (Picture 3).

Step 4

Picture 4: The disc of light is now centred.

Centre the condenser using the 2 screws which emerge from the rim of the condenser. These screws cause movement 90° to each other, so you will often need both to get the condenser centred (end result in Picture 4).

Step 5

Picture 5: The final image!

Once centred, open the field diaphragm so that the edges of the illuminated circle just match the field view (Picture 5).

Step 6

Adjust the condenser diaphragm to about 70-80% of the diameter. There are 2 ways to do this. In the first method, remove the eyepiece and peer down the barrel. You will see a circle of light. Adjust the diaphragm so that it occupies about 70-80% diameter. The second method is to just look down the microscope as normal. Open the diaphgram completely, then close it gradually, stopping when the image is the sharpest. Adjust the light intensity accordingly.

Finally, although there is an optimum setting for each magnification, in real life, the pragmatic compromise is to set your field diaphgram to suit the lowest objective that uses the condenser (usually x10) while keeping the condenser diaphragm at the optimum size for the highest magnification (x40).