How to Align Köhler Illumination in 6 Simple Steps

By Lauren Alvarenga - 3 February, 2022

Comparison of image quality when aperture stop is too wide, optimally set, and too small during Köhler illumination alignment

Bright, even illumination from a microscope isn’t always as simple as turning on the light source. Since microscopes are sophisticated optical systems, they need care and adjustment to ensure that they are producing optimal quality images. One of the easiest optical alignments is Köhler illumination for transmitted light brightfield images.

The alignment process for Köhler illumination is the same for upright and inverted microscopes, but the locations of the condenser and field and aperture stops vary from microscope to microscope. Be sure to check your microscope manual if you’re having trouble locating these components.

Pro tip: Make sure your optics are clean before you start this process; a dirty objective can make Köhler alignment more difficult than it needs to be.

1. Change to a low magnification objective

Changing to a low magnification objective on your microscope, such as 10X, gives you a wide field of view and a long depth of focus.

Blurry image when objective is set to the lowest magnification objective in the Köhler illumination process

2. Focus on your sample

To ensure the proper position of the apertures in the optical path, focus on the sample as the starting point for the remainder of the optical adjustments.

Focused image of the sample in the Köhler illumination process

3. Close the field stop

Once the sample is in focus, close the field stop completely. If it is unfocused, adjust the condenser’s height until the blades of the field stop are as sharp as possible, matching the focus of the sample.

Illustration showing how to close the field stop for Köhler illumination

4. Center the field stop

Next, adjust the field stop’s position so that it is in the center of your microscope image. Use the two knobs on the front of the condenser to adjust the XY position of the field stop.

To check the center position, open the field stop until you see the edges of the octagon reach the edge of the field of view. Adjust as needed.

Illustration showing how to center the field stop for Köhler illumination

5. Adjust the aperture stop

Once you have an image, adjust your aperture stop to control the contrast and resolution. You can use a Bertrand lens or similar optic to visualize the back focal plane. You should fill it about 75% of the field of view.

Image quality when the aperture stop is too small for Köhler illumination

Comparison of image quality when aperture stop is too wide (left), optimally set (center), and too small (right).

6. Eliminate the field stop from the FOV

Finally, open the field stop so that it is just outside of the field of view. Avoid opening it all the way as this will degrade your image.

And that’s it! Now you’ll have an even, bright field of illumination for high-quality microscopy images!

If you’re interested in delving deeper into Köhler illumination, check out our Microscope Resource Center primer, which explores the technique in detail.

Discovery Blog