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Optical Pathways in the Transmitted Light Microscope

The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. Illumination of the specimen is the most important controllable variable in achieving high-quality images in microscopy, critical photomicrography, and digital imaging. When properly adjusted, light from the condenser will fill the rear focal plane of the objective with image-forming light by projecting a cone of light to illuminate the field of view. The condenser aperture diaphragm is responsible for controlling the angle of the illuminating light cone and, consequently, the numerical aperture of the condenser. It is the major variable that enables the right balance of resolution and contrast in the microscope. This interactive tutorial explores the function of the field and condenser aperture diaphragms of a transmitted light microscope.

The tutorial initializes with a cut-away diagram of a typical upright transmitted light microscope showing the important internal components. In order to operate the tutorial, use the Illumination Intensity slider to increase the lamp output to approximately 80 to 90 percent of the maximum (enough to see the illumination beam). Next, use the Field Diaphragm and Aperture Diaphragm sliders to open and close these iris diaphragms and observe how they affect the illumination.

The optical train and numerous other components of a modern microscope are presented in the tutorial cut-away diagram. The condenser containing the aperture diaphragm and the luminous-field diaphragm normally contained in the stand base are the critical elements in achieving Köhler illumination. Image forming light rays passed through the specimen are captured by the microscope objective and directed either into the eyepieces or to a beamsplitter into the camera port. Throughout the optical train of the microscope, illumination is directed and focused through a series of diaphragms and lenses as it travels from the light source to illuminate the specimen and then into the eyepieces or camera attachment. Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the condenser and reaching the specimen from all azimuths. Because the light source is not focused at the level of the specimen, illumination at the specimen level is essentially grainless and extended (in effect, producing a uniform field of illumination), and does not suffer deterioration from dust and imperfections on the glass surfaces of the condenser. The setting of the condenser's aperture diaphragm, along with the numerical aperture of the objective, determines the realized numerical aperture of the entire microscope system. The luminous-field diaphragm determines which portion and size of the specimen is illuminated. The aperture diaphragm of the condenser is imaged on the pupil of the objective and regulates the illumination of this pupil. Because the light source is imaged into the aperture diaphragm, this component becomes the accessible de-facto illumination source.

Contributing Authors

Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.

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