Köhler Illumination - Optimal Alignment of the Light Microscope

                              Rev 11/06/2012

 

Based on principles developed by August Köhler, 1893.

 

The basic procedure:

  1. Place a slide on the stage of the microscope and focus on the specimen. This step is most important, a specimen must be in focus to the eye of the observer.

  2. Decrease the size of the field diaphragm located nearest the light source (the field iris) so that you can see its edges. Opening and closing the field diaphragm does not alter the brightness of the image but merely controls the width of the light beam being transmitted to the condenser and restricts the light to the part of the specimen which is actually being observed.

  3. Bring the edges of the field iris into focus by raising or lowering the sub-stage condenser. Both the specimen and the edges of the iris diaphragm should be in sharp focus.

  4. Center the image of the field iris using the condenser-centering knobs.

  5. Open the centered and focused field diaphragm (field iris) so that the edges are just outside the field of view.

  6. Adjust the condenser iris, using the sliding lever, to increase or decrease image contrast. The optimum opening depends on the specimen. Do not use the condenser iris to control light intensity. Adjusting the condenser iris affects the angle of the light being transmitted to the specimen. Resolution and contrast increase as the condenser iris is opened.

  7. Adjust light intensity with the light power supply or with neutral density filters.

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