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Oblique or Anaxial Illumination
Introduction to Oblique Illumination - Achieving conditions necessary for oblique illumination, which has been employed to enhance specimen visibility since the dawn of microscopy, can be accomplished by a variety of techniques with a simple transmitted optical microscope. Perhaps the easiest methods are to offset a partially closed condenser iris diaphragm or the image of the light source. In former years, some microscopes were equipped with a condenser having a decenterable aperture iris diaphragm. The device was engineered to allow the entire iris to move off-center in a horizontal plane so that closing the circular diaphragm opening would result in moving the zeroth order to the periphery of the objective rear focal plane. In advanced models, the entire diaphragm was rotatable around the axis of the microscope so that oblique light could be directed toward the specimen from any azimuth to achieve the best desired effect for a given specimen.
In practice, utilizing a partially closed condenser iris diaphragm technique to achieve oblique lighting is plagued with problems similar to those hampering brightfield observation. The results are a general loss of resolution and superimposition of diffraction rings, which surround and confuse the interpretation of minute specimen detail. In addition, Becke lines and other undesirable optical effects originating from regions of the specimen that are not in exact focus complicate the image. To circumvent many of these problems, a combination of oblique illumination at large condenser numerical aperture, coupled to video contrast enhancement, has been utilized as an effective method to generate optical thin sections that strongly resemble those observed with differential interference contrast (DIC) microscopy.
Oblique Illumination in Stereomicroscopy - 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.
Interactive Java Tutorials
Oblique Illumination Light Pathways - Achieving conditions necessary for oblique illumination can be accomplished by a variety of techniques with a simple transmitted light optical microscope. The easiest methods are to offset a partially closed condenser iris diaphragm, insert an opaque sector stop near the condenser aperture, or de-center the image of the light source. Regardless of the mechanism utilized to establish oblique illumination, the conditions required for image formation remain the same. This interactive tutorial explores changes in microscope light paths and demonstrates events at the objective rear focal plane as illumination progresses from axial to highly oblique.
Contrast Enhancement Technique MTF Curves - The utilization of contrast enhancement techniques in optical microscopy affects the response when relative modulation is calculated as a function of specimen spatial frequency. This interactive tutorial explores the effects of popular contrast modes on image contrast and the modulation transfer function of the modified microscope.
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 stereomicroscopy.
Refractive Index Determination by Oblique Illumination - Oblique illumination is sometimes utilized as an alternative to the Becke line test to determine whether the refractive index of a specimen is higher or lower than that of the surrounding medium. This interactive tutorial explores how variations in the refractive index of a specimen and its surrounding medium alter visibility in the microscope when utilizing oblique illumination techniques.
Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
William Chambers - Industrial Microscope Division, Nikon Instruments Inc., Melville, New York 11747.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
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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