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Darkfield Microscopy

Darkfield microscopy is a specialized illumination technique that capitalizes on oblique illumination to enhance contrast in specimens that are not imaged well under normal brightfield illumination conditions. After the zeroth order (direct) light has been blocked by an opaque stop in the substage condenser, light passing through the specimen from oblique angles at all azimuths is diffracted, refracted, and reflected into the microscope objective to form a bright image of the specimen superimposed onto a dark background.

Transmitted Darkfield Illumination - Transmitted darkfield illumination can be used to increase the visibility of specimens lacking sufficient contrast for satisfactory observation and imaging by ordinary brightfield microscopy techniques. This section discusses various aspects of darkfield illumination, including theory of the technique, condenser design for transmitted darkfield illumination (at both low and high magnifications), microscope configuration parameters, and suggestions for choosing suitable candidates for observation.

Reflected Darkfield Illumination - Darkfield illumination with reflected light enables visualization of grain boundaries, surface defects, and other features that are difficult or impossible to detect with brightfield illumination. The technique relies on an opaque occluding disk, which is placed in the path of the light traveling through the vertical illuminator so that only the peripheral rays of light reach the deflecting mirror. These rays are reflected by the mirror and pass through a hollow collar surrounding the objective to illuminate the specimen at highly oblique angles.

Darkfield Illumination for Stereomicroscopy - Darkfield observation in stereomicroscopy requires a specialized stand containing a reflection mirror and light-shielding plate to direct an inverted hollow cone of illumination towards the specimen at oblique angles. A number of aftermarket products are currently available for retrofitting stereomicroscopes with transmitted darkfield illumination. In addition, many of the microscope manufacturers offer illumination accessories that can be conveniently utilized to achieve darkfield conditions for their stereo systems. The principal elements of darkfield illumination are the same for both stereomicroscopes and more conventional compound microscopes.

Darkfield Microscope Configuration - A step-by-step guide to configuration of transmitted light microscopes for use with both low and high magnification darkfield condensers is provided in this review. Careful attention should always be given to microscope alignment and configuration, irrespective of whether the illumination mode is brightfield, darkfield, phase contrast or some other contrast enhancement technique. Time spent in this endeavor will be repaid in excellent performance of the microscope both for routine observation and critical digital imaging or photomicrography.

Troubleshooting Darkfield Microscopy - There are numerous common problems associated with darkfield microscopy and photomicrography or digital imaging. These range from insufficient illumination and condenser mis-alignment to using a field stop of incorrect size. Most darkfield illumination problems are associated with the substage condenser, and this should be the first suspect when things do not work properly. This section addresses some of the more common problems encountered with darkfield microscopy, along with suggested remedies.

Darkfield Photomicrograph Gallery - The Molecular Expressions gallery of darkfield illumination photomicrography and digital imaging contains a wide spectrum of images captured under a variety of conditions and utilizing many different specimens. Included in this unique gallery are specimens ranging from simple diatoms to fossilized dinosaur bones, insects, Moon rocks, and integrated circuits.

Darkfield Microscopy Interactive Java Tutorials - Explore various aspects of darkfield microscopy theory and practice using these tutorials, which are designed to complement text pages by enabling visitors to use a web browser to simulate configuration and operation of a microscope under darkfield illumination. Both the theory and practice of darkfield microscopy are addressed by the tutorials.

Contributing Authors

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

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


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