Bright, even illumination from a microscope isn’t always as easy as turning on the light source. Since microscopes are complex optical systems, they need care and adjustment to ensure that they are producing top-quality images. Köhler illumination for transmitted light brightfield images is one of the easiest optical alignments.
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 are different on each microscope. Refer to your microscope manual if you’re having trouble locating these components.
Pro tip: Your optics must be 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.
2. Focus on your sample
For proper position of the apertures in the optical path, focus on the sample as the starting point for the remainder of the optical adjustments.
3. Close the field stop
Once the sample is in focus, close the field stop completely. If it isn’t focused, adjust the condenser’s height until the blades of the field stop are as sharp as possible, matching the focus of the sample.
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.
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.
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!
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