Koehler Illumination

Koehler Illumination achieves a homogeneously illuminated object plane imaged with optimal contrast and resolution. This illumination approach was first introduced in 1893 by August Koehler and manufactured by the leading microscope manufacturer Carl Zeiss Jena as a method of providing the ideal specimen illumination with optimal contrast and structural resolution.

Key Learnings:

• Koehler Illumination aligns the light source for homogenous illumination and contrast. It uses a collector lens system, field stop diaphragm function, and condenser aperture stop diaphragm.
  
• The appropriate use of the adjustable aperture stop diaphragm is significant to achieve suitable resolution, optimal contrast, and depth of field.
• Experienced microscopists can adjust the condenser aperture stop diaphragm accurately by observing the image without the need to view the diaphragm in the objective's back focal plane.

Before looking in detail at how to establish Koehler Illumination, let us have a look at the Condenser Aperture Stop Diaphragm Function. The angle of the light cone produced by the condenser corresponds to the actual illumination aperture of the condenser in transmitted light. It is modulated by the diameter of the aperture stop diaphragm. A fully open condenser aperture stop diaphragm will allow for the maximum possible numerical aperture of the given condenser. The appropriate use of the adjustable aperture stop diaphragm is of significant importance to achieve suitable resolution, optimal contrast and depth of field.

The tutorial initializes with a focused sample image on the left-hand side of the tutorial. Along with an animation of the condenser aperture stop diaphragm size adjacent to the specimen as it appears when viewing the objective’s back focal plane through a Bertrand lens or by removing one of the eyepieces and peering into the observation tube. In order to operate the tutorial, use the Aperture Diameter slider to adjust the aperture stop diameter, and thus modulate the contrast level of the image.

Since then, manufacturers have outfitted the modern microscope with the necessary hardware for Koehler Illumination: A so-called collector lens system, a field stop iris diaphragm and condensers housing an aperture stop diaphragm. Usually, the collector lens system is built into the base of the microscope. The collector lens system will project an enlarged and focused image of the light source (e.g. halogen lamp filament, LED-array) into the plane of the aperture diaphragm of a properly height positioned condenser. The correct height of the condenser is achieved when the image of the field stop appears with maximum sharpness in the focused object plane. By doing so, the light source can never become focused into the specimen plane (instead the light source image is projected into the objective’s back focal plane). As a result, the focused object plane is illuminated very homogenously. The proper adjustment of the field stop diameter will reduce the amount of contrast- diminishing straylight. Closing or opening the condenser aperture diaphragm controls the angle of the light rays emerging from the condenser. By doing so, the optimum ratio between maximum resolution and strongest contrast can be individually set.

Now, this interactive tutorial explores how to establish Koehler illumination on a transmitted light microscope.

The pre-condition for a homogeneous Koehler Illumination is the alignment of the light source (e.g. halogen lamp filament images) and the appropriate use of diffusing disks (if available). Otherwise the image background will not have the optimal illumination homogeneity.
Please be aware that many modern light sources can no longer be aligned by the microscopist (factory pre- aligned light sources and fixed diffusing disks in the illuminating beam path).
In this tutorial, the correct alignment of a halogen lamp in transmitted light brightfield is explained. Typically, this procedure is required after changing the halogen lamp bulb.
  

First, set your microscope for transmitted light brightfield and align Koehler Illumination. Use the brightfield position of the condenser modulator disk (“H” or “BF”), preferably work with a non- Ph objective. The image of the light source can best be seen employing a 40x dry objective. Removing one eyepiece, allows to inspect the full light source image in the objective´s back focal plane. Make yourself familiar with the mechanical alignment options of your lamp.
Switch on the light source. Set the lamp intensity to a convenient brightness level. Remove all diffusing disks from the beam path. Focus an empty area of a slide. Remove one eyepiece. With a 40x objective magnification the image of the light source can be seen in the objectives back focal plane if Koehler Illumination was carried out beforehand. A halogen lamp house usually has a concave mirror to produce a second image of the light source for better light efficiency. Both halogen lamp filament images are mechanically adjustable in x/y (position) and z (focus).