Transmitted Light

Adjustment of a transmitted light microscope for Köhler illumination is a logical and relatively easy process that should be practiced until mastered by all serious students of microscopy. Each time a microscope is turned on, it should be carefully inspected to ensure proper alignment of all optical components, and to guarantee that the lamp is centered and the condenser and field diaphragm are properly adjusted for Köhler illumination.

The microscope illustrated in Figure 1 uses an advanced external light source to provide illumination for the microscope. The lamp housing contains both a field diaphragm and collector lens, in addition to a tungsten-halogen bulb. A separate voltage regulator provides an adjustable direct-current voltage source for the lamp that varies between 5 and 12 volts. Modern microscopes usually contain a light source built into the base of the microscope, along with the collector lens and field diaphragm. Adjustment of microscopes with either internal or external light sources for Köhler illumination is similar in theory, however there are certain differences in the procedures that will be described below.

An important step in alignment of the microscope, whether it is for Köhler, Nelsonian, or any other strategy for illumination, is alignment of the light source (usually a tungsten-halogen bulb). Many modern microscopes now have pre-centered and pre-focused lamps, but older models still require the light source to be centered and focused by the user. We suggest consulting the owner's manual for instructions about how the lamp is to be centered and for other important information about the illumination source. The following procedures are recommended for aligning both external and internal lamps:

Internal Tungsten-Halogen Lamps

Figure 3 illustrates a typical view of the microscope light port when the filament is made visible using the procedures outlined above. The port on the left in Figure 3(a) contains a filament that is severely out of alignment and not positioned properly to focus an image that will completely fill the aperture diaphragm nor the back focal plane of the objective. Using translational adjustments on the lamp housing, the filament is brought into focus (Figure 3(b)) and then centered within the light port (Figure 3(c)). Visitors and students can practice aligning a "virtual" lamp filament using the interactive Java tutorial below:

Lamp Filament Alignment

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Use this tutorial to practice lamp filament alignment while monitoring the filament both in the microscope port and at the back focal plane of the objective.

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External Tungsten and Tungsten-Halogen Lamps

Alignment of the Condenser and Field Diaphragm

In some instances the edge of the partially closed aperture diaphragm is not concentric with the illuminated back focal plane of the objective. This is due either to a misalignment of the substage condenser or the objective, and the manufacturer's instruction manual should be consulted for the correct centering regime. Some microscopes are shipped with the aperture diaphragm in a fixed and centered position that is impossible to change in the laboratory. Darkfield and phase contrast condensers usually always will provide a centering mechanism due to the crucial aspect of this step in setting up microscopes for these illumination methods. The condenser aperture should be centered with a 40x or 60x objective to avoid having to re-center the diaphragm with each objective (lower power objectives will almost always fall within acceptable margins using this method).

Now that Köhler illumination has been established with the 10x objective, it must be borne in mind that changing to a higher power objective will require a re-alignment of both the field and aperture diaphragms. For example, if you switch to the 40x objective, you will have to close the field diaphragm somewhat and re-center it (looking at a smaller area of the specimen). Also, the condenser aperture diaphragm should be opened slightly (the 40x objective has a higher numerical aperture than does the 10x objective). Each time an objective is changed, both diaphragms must be adjusted according to the steps outlined above.

The conditions of Köhler illumination do not apply at magnifications using lower power objectives (5x and below). When preparing for low magnification microscopy, first align the microscope and adjust the optical pathway for proper Köhler illumination using the 10x objective. Most modern microscope condensers have a top lens that swings out of the light path for use with objectives of 5x magnification and below, as illustrated in Figure 8. Other condenser models have top lenses that can be unscrewed or have a rotating turret of condenser lenses for use with both low and high magnification objectives.

When the swing-out lens is removed from the optical pathway, condenser performance is radically changed and the aperture diaphragm no longer functions to control the numerical aperture of illuminating light rays. To avoid vignetting, the condenser aperture diaphragm should be opened to its widest setting, and specimen contrast is controlled with the field diaphragm, which now controls the numerical aperture of the illuminating light rays.

Contrast Adjustment at Low Magnifications

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At low magnifications, the field diaphragm is used to control numerical aperture, specimen contrast, and overall image quality. Use this tutorial to explore the effects of field diaphragm opening size on these variables.

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By properly adjusting the microscope for Köhler illumination, the specimen will be well-illuminated with even, glare-free light, giving good image resolution and contrast. For further information, consult our section on the theory of Köhler illumination and/or the basics of microscope illumination. Reference material concerning microscope illumination can also be found in our bibliography and in the section on web resources, which contains links to other microscopy web sites.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.