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Adjusting Koehler Illumination

Found in: Techniques

Koehler illumination ensures that the specimen receives a bright uniform light. Only those areas actually seen are illuminated.

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Koehler illumination ensures that the specimen receives a bright uniform light. Only those areas actually seen are illuminated.A uniform, bright light source of the correct color is very important for obtaining high quality microscopic images. One problem is that the lamp is not able to produce a uniform light, because the filament of the lamp is brighter than its surrounding. One solution is to place a frosted glass plate above the light source as a diffuser. This reduces the light intensity and changes the color of the light, however.

Koehler illumination was developed by August Köhler (1866-1948). This illumination principle greatly enhances the quality of the microscopic images (especially photographs). The illumination principle offers the following advantages:

  • It illuminates the specimen uniformly without the need of a diffuser.
  • It only illuminates the part of the specimen which is actually observed (at a higher magnifications a smaller section of the specimen). This reduces the heating of the specimen.
  • It reduces internal reflections. This improves the contrast in photomicrographs.

The Koehler illumination must be adjusted before observation:

  1. Rotate a low power objective (eg. 4x or 10x) into position. This will increase the field of view.
  2. Insert a slide with a specimen and focus it.
  3. Adjust the field iris diaphragm (the diaphragm of the light source) in such a way that its edges become visible. The field of view is reduced this way, only a small round part of the specimen is visible.
  4. Raise or lower the condenser (not the stage!) and bring the edges of the field iris diaphragm (not the condenser aperture diaphragm) into focus. The focus of the specimen is not changed. Now both the edge of the iris diaphragm and and the specimen should be in focus. If the height of the condenser is not properly adjusted, then dust of the lamp will come into focus and disturb the image.
  5. There are two condenser centering screws/knobs at the side of the condenser. Turn these knobs to bring the field into the center of view.
  6. Now you can open the field diaphragm and start regular microscopic observation.
  7. When doing photographic work, open the field diaphragm only as far as necessary. Opening it further will increase internal light reflections and result in a lower contrast. You need to observe the edges of the field diaphragm through the camera viewfinder. It may also be necessary to refocus the specimen when looking through the camera.
koehler

The Koehler diaphragm is centered and in focus. The adjustment is correct.

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The Koehler diaphragm is centered but out of focus. Raise or lower the condenser to focus the diaphragm.

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The Koehler diaphragm is centered but out of focus. Raise or lower the condenser to focus the diaphragm.

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