How to set up Koehler Illumination

How to set up Koehler illumination

Aligning your microscope for Koehler illumination is easy and will give you clearer images with much better contrast, so you can see (and capture) all the important details.

So how do you accomplish this task? Excellent question – I’m glad you asked.

It’s pretty simple to set up and it should only take a minute or two before you’re using your microscope to its full potential.

Optical components

Most modern microscopes are capable of using Koehler illumination or are at least capable of being retrofitted as long as certain criteria can be met.

To set up for Koehler, head for the (usually neglected) bottom half of your microscope because that’s where you’ll find all the components you’ll need to adjust – two diaphragms and your condenser.

You’ll find the field diaphragm at the top of your illuminator (it’s usually controlled by a little lever on the side or front).

You’ll find the aperture diaphragm at the bottom of the condenser (also usually controlled by a small level on the side).

The field diaphragm controls the area of the circle of light illuminating your specimen, while the aperture diaphragm controls the angular aperture of the cone of light from the condenser.

When your microscope is aligned with Kohler illumination properly, the condenser is at the correct height relative to the sample, the field diaphragm is adjusted to eliminate reflections and glare, and the aperture is moved to an optimal position to provide contrast without producing shading artifacts. 

It can be frustrating adjusting your controls each time the objective is changed – especially for critical work. But it quickly becomes second nature.

6 steps to Koehler illumination

1. Place your sample slide on the stage and focus on it using a 4x or 10x objective.
2. Completely open the condenser aperture diaphragm and completely close the field iris diaphragm. (Be sure you’ve correctly identified the diaphragms, or you won’t really be setting up Kohler illumination).
3. Now bring the small circle on your slide into sharp focus using the focus knobs on the side of your microscope. Try to make the edge as sharp as you can – the edges of the lit area will look like an octagon.
4. Once you have a sharp focus, use the condenser centring controls (often little screws on the side of the condenser) to move the illuminated area around until you have it exactly in the middle of your field of view.
5. Open the field diaphragm until its shadow just disappears from the field of view. Now you have a perfectly aligned microscope for Kohler illumination!
6. The last step is to use the aperture diaphragm under the condenser to find the setting that provides the best contrast and resolution for your sample.

Using this method, your images will have much greater clarity and contrast, with little or no glare. And if you’re into microscopy, you’ll be able to meet journal quality standards – in less than a minute of your time!