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Fundamentals of Stereomicroscopy

Considering the wide range of accessories currently available for stereomicroscope systems, this class of microscopes is extremely useful in a multitude of applications. Stands and illuminating bases for a variety of contrast enhancement techniques are available from all of the manufacturers, and can be adapted to virtually any working situation. There are a wide choice of objectives and eyepieces, enhanced with attachment lenses and coaxial illuminators that are fitted to the microscope as an intermediate tube. Working distances can range from 3-5 centimeters to as much as 20 centimeters in some models, allowing for a considerable amount of working room between the objective and specimen.

Introduction to Stereomicroscopy - Stereomicroscopes have characteristics that are valuable in situations where three-dimensional observation and perception of depth and contrast is critical to the interpretation of specimen structure. These instruments are also essential when micromanipulation of the specimen is required in a large and comfortable working space. The wide field of view and variable magnification displayed by stereomicroscopes is also useful for construction of miniature industrial assemblies, or for biological research that requires careful manipulation of delicate and sensitive living organisms.

Illumination for Stereomicroscopy

Reflected (Episcopic) Illumination - Stereomicroscopes are often utilized to examine specimens under both reflected (episcopic) and transmitted (diascopic) illumination schemes, employing a variety of light sources and configurations, which are strategically positioned in the appropriate locations. In many circumstances, reflected and transmitted light sources are combined to take advantage of particular specimen characteristics in a manner that most effectively reveals the features of interest. This review focuses on the wide variety of techniques and equipment currently in use to illuminate a multitude of specimens observed with reflected light techniques.

Oblique Illumination - Specimens that are nearly transparent and colorless may be almost invisible when viewed in the stereomicroscope using traditional transmitted (diascopic) brightfield illumination techniques. However, if the illumination is directed so that it originates from a single azimuth and strikes the specimen from an oblique angle, details in the specimen may be revealed with much greater contrast and visual clarity than when the light is allowed to pass directly through specimen features along the optical axis of the microscope.

Darkfield Illumination - 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. The principal elements of darkfield illumination are the same for both stereomicroscopes and more conventional compound microscopes, which often are equipped with complex multi-lens condenser systems or condensers having specialized internal mirrors containing reflecting surfaces oriented at specific geometries.

Fluorescence Illumination - The application of stereomicroscopes for GFP observation is now so prevalent that stereo fluorescence illuminators are more frequently referred to as GFP illuminators, even though they can be utilized for many other applications in both the life sciences and the electronics manufacturing industry. Large specimens, such as larvae, nematodes, Zebrafish, oocytes, and mature insects can be easily selected and manipulated when they are labeled with GFP and illuminated by fluorescence techniques. The fluorescence illumination reveals which organisms are producing the fluorescent protein and the stereoscopic vision coupled to a large field of view and ample working distance enables observers to conduct experiments with forceps, pipettes, or micromanipulators. Other, more conventional, specimens are also easily observed and recorded using stereomicroscopes with fluorescence illumination.

Stereomicroscopy Digital Image Galleries

Fluorescence Stereomicroscopy Image Gallery - The application of stereomicroscopes for GFP observation is now so prevalent that stereo fluorescence illuminators are more frequently referred to as GFP illuminators, even though they can be utilized for many other applications in both the life sciences and industry. Large specimens, such as larvae, nematodes, zebrafish, oocytes, and mature insects can be easily selected and manipulated when they are labeled with GFP and illuminated by fluorescence techiques. This technique is also applicable to traditional fluorescence specimens, such as stained thin sections, cell culture mounts, and autofluorescence in plant tissues. Visit the gallery to observe the wide variety of specimens imaged using this novel new technique.

Interactive Java and Flash Tutorials

Oblique Coherent Contrast Illumination - Transparent specimens, which are almost invisible when viewed in the stereomicroscope using traditional brightfield illumination techniques, often assume a pseudo three-dimensional appearance with stark contrast when viewed in oblique illumination. This interactive Java tutorial explores variations in specimen contrast produced by Nikon's Oblique Coherent Contrast illumination system designed to optimize contrast in transmitted stereoscopic microscopy.

Toroidal Mirrors - The C-BD Diascopic Brightfield/Darkfield stand introduced with the Nikon SMZ1500 uses a seven-sided toroidal mirror to substantially reduce stray light. Use this interactive Java tutorial to explore how mirror shape affects the amount of light entering the objective in darkfield stereoscopic microscopy.

Stereomicroscopy Fluorescence - The illuminator for epi-fluorescence on a stereomicroscope functions in a manner that is similar to those employed on more complex compound microscopes. Typically, the fluorescence illuminator consists of a xenon or mercury arc lamp contained in an external lamphouse that is attached to the microscope via an intermediate tube (or vertical illuminator) positioned between the microscope zoom body and observation tubes. This interactive tutorial explores internal optical pathways of the Nikon SMZ1500 stereomicroscope equipped with an epi-illumination intermediate tube and lamphouse.

Focus and Alignment of Mercury and Xenon Arc Lamps - Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.

The SMZ-1500 Stereoscopic Microscope - Many stereoscopic microscopes feature the ability to perform a continuous magnification change by means of a zoom lens system placed between the objective and the eyepieces. Explore zoom magnification, focus, and illumination intensity in stereoscopic microscopes with this interactive Flash tutorial.

SMZ-1500 Oblique Coherent Contrast Illumination - Featuring new objectives with substantially higher numerical apertures and an industry-leading zoom ratio of 15x, the Nikon SMZ1500 establishes a new standard in stereomicroscopy. This interactive Flash tutorial explores the light path in Nikon's proprietary Oblique Coherent Contrast illumination system designed to optimize contrast in transmitted stereoscopic microscopy.

SMZ1500 Optical Pathways - Stereoscopic microscopes have separate optical pathways for each eyepiece, a system which is designed to produce a beautiful three-dimensional view of complex specimens at relatively low magnifications. In addition, these microscopes provide a large field and a larger working distance than compound microscopes.

Toroidal Mirrors - The C-BD Diascopic Brightfield/Darkfield stand introduced with the Nikon SMZ1500 uses a seven-sided toroidal mirror to substantially reduce stray light. Use this interactive Flash tutorial to explore how mirror shape affects the amount of light entering the objective in darkfield stereoscopic microscopy.

Contributing Authors

Paul E. Nothnagle - Avimo Precision Instruments, 78 Schuyler Baldwin Drive, Fairport, New York, 14450.

William Chambers - Industrial Microscope Division, Nikon Instruments Inc., Melville, New York 11747.

Matthew J. Parry-Hill, Thomas J. Fellers, 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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