Illumination of the specimen is the most important variable in achieving high- quality images in microscopy and critical photomicrography. The Köhler illumination technique was first introduced in 1893 by August Köhler as a method of providing the optimum specimen illumination.
This technique is recommended by all manufacturers of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope’s full potential.
Step by Step guide
- Switch on the microscope.
- Start with a 10X objective
- Place the sample on the stage and clamp it with the specimen holder. Make sure the coverslip is facing the objective lens.
- Focus the sample
- Close the field diaphragm
- Focus the image of the field diaphragm into the image plane by adjusting the condenser height, using the condenser focusing knob. You should see crisp edges of the field diaphragm.
- Centre the image of the field diaphragm using the centering screws in the condenser
- Open the field diaphragm just until you cannot see its edges any longer. The idea is to illuminate the imaged area, but no more.
- Adjust the aperture diaphragm on condenser to optimise contrast. Remove one eyepiece, close the aperture diaphragm until the bright area viewed in the eyepiece is about 66%-75% of the total area.
- Congratulations! You have achieved Köhler illumination.
Be careful when adjusting the condenser aperture diaphragm. Closing the condenser aperture diaphragm reduces resolution. To maximize both contrast and resolution, close the diaphragm just to the point where the image begins to get dark and no further. This position is especially important when using DIC (Differential Interference Contrast) optics.
For critical work, it is necessary to touch up the alignment if objectives are switched, or even for large variations in focus plane in the sample.
N.B. – Köhler illumination is a prerequisite to any other transmitted light technique, e.g. DIC!