Most commonly, one eyepiece is removed (Figure 5) giving full view of the objectives back focal plane (seen as a bright circle). When closing the condenser aperture diaphragm, its sharp image will appear in the objectives back focal plane. As a rule-of-thumb, for visual microscopy, the condenser aperture stop is closed by 20% - 25% of the back focal plane diameter (75% - 80% of the back focal plane diameter is still open). For digital imaging, the condenser aperture stop is opened more to achieve a better resolution.

202258 — It shows how to achieve Kohler on some different microscopes, and even something approaching Kohler but without a field iris.

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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.

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Opticalmicroscope

If the image is totally dark, open the field stop diaphragm slightly and center it with the condenser centering screws. This is rarely necessary, especially if the condenser was removed from the microscope.

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.

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Microscope

The objective lens forms a real intermediate image which is then greatly magnified by the eyepiece. The objective lens and eyepiece are maintained at a fixed distance and focusing is achieved by moving the whole assembly up and down in relation to the sample. High magnification requires very bright illumination of the sample and a condensor lens is usually placed between the light source and the sample stage to focus light onto the sample.

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.

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Criticalillumination

When the condenser aperture diaphragm is closed too far, diffraction artifacts will cause visible fringes. Opposite, when opening the condenser aperture too wide, a significant loss of contrast is the result. The correct setting of the aperture diaphragm will vary from specimen to specimen, and the experienced microscopist will soon learn to accurately adjust the condenser aperture diaphragm by observing the image without having to view the condenser aperture diaphragm in the back focal plane of the objective.

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.

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Please note, that in some microscopes, the upper condenser height position can be limited mechanically. Make sure that the condenser can move high enough to produce a sharp field stop diaphragm within the sample plane. Take care not to move the condenser to high. Otherwise, the slide will be catapulted out of the specimen holder, which can easily damage the objective front lens.

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The simplest light microscope consists of an objective lens and an eyepiece. Microscope objectives and eyepieces usually consist complex lens systems of two or more lenses to correct for lens aberrations.

Switch on the light source. Use a transmitted light sample for brightfield (e.g. cytological smear, thin stained tissue section). Without a sample, Koehler Illumination cannot be aligned. To practice Koehler Illumination start with an objective magnification of 10x or 20x. This allows You to see the field stop diaphragm image with best clarity. Start with fully open condenser aperture diaphragm.

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).

Light microscopy uses electromagnetic radiation in the ultraviolet or visible wavelength range to obtain a magnified image of an object. The resolution of the imaging is limited by the minimum focus of the radiation due to diffraction. For light microscopy the diffraction limit is approximately 1 µm (10-6 m). Imaging objects smaller than 1 µm is possible using electrons instead of light. High-resolution electron microscopy can image features as small as approximately 1 Å (10-10 m). Microscopes

Differential interference contrast microscopy

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As a result, its image will be concentric with the field of view.   In some routine microscopes tools may be needed (e.g. SW 1.5 hex keys). Open the centered field stop diaphragm, until its image has just disappeared. Please do not fully open the field stop diaphragm, as otherwise straylight will be produced. This will reduce the image contrast. By looking at the sample, close the condenser aperture diaphragm until You have reached the best compromise between maximum resolution (condenser aperture fully open) and strongest contrast (condenser aperture diaphragm fully closed).

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.

The second method is to close the condenser aperture diaphragm to the position, where the visual image brightness will drop significantly and suddenly. Both methods will give similar results.

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The above tutorial workflow describes the Koehler Illumination alignment for transmitted brightfield illumination with higher objective magnifications (10x and higher). If the condenser contains phase ring stops, DIC- prisms, other masks, or filters, rotate the condenser turret to the free brightfield position. The best specimens to practice Koehler illumination alignment are well-stained brightfield samples, such as very thin cytological/hematological smears or tissue sections (8 micrometer or less). Please note, that Koehler Illumination is done for the objective magnifications of 10x – 100 (150)x and must be repeated for each objective in transmitted light. For the lower objective magnifications (1x – 5x), the Koehler Illumination is first aligned for the 10x objective. Afterwards the condenser front lens is swung out (achromatic aplanatic condenser) or a low-power illumination lens is used below the condenser (abbe condenser). The sample is in focus and the condenser diaphragm is fully opened. The already centered field stop diaphragm will now act as aperture diaphragm, which is used for contrast/ resolution modulation as usually.

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