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Specialized Microscopy Techniques

Modern microscopists and optical engineers have developed a wide spectrum of useful techniques designed to aid in contrast enhancement, provide better observation, and assist in the collection of photomicrographs and digital images of a wide variety of specimens. This section of the Molecular Expressions Microscopy Primer describes many of these techniques in detail.

Contrast Enhancing Techniques

Contrast in Optical Microscopy - With the assistance of Dr. Robert Hoffman, we review the problems of contrast enhancement with both amplitude and phase specimens and review techniques that have been developed to assist with specimen contrast.

Darkfield Microscopy - Oblique illumination can be used to increase the visibility of specimens lacking in sufficient contrast that are difficult to observe with standard brightfield microscopy. This section discusses various aspects of the theory and practice of condenser design and other important concepts in both transmitted and reflected light darkfield microscopy.

Differential Interference Contrast - An excellent mechanism for rendering contrast in transparent specimens, differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk. The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast. As the gradient of optical path difference grows steeper, image contrast is dramatically increased.

Hoffman Modulation Contrast - Hoffman modulation contrast is an oblique illumination technique that enhances contrast in both stained and unstained specimens by detection of optical phase gradients. This section includes discussions of transmitted and reflected light applications using Hoffman modulation contrast and links to interactive Java tutorials designed to aid in understanding the technique. Also included are virtual microscopes and an image gallery of photomicrographs made using modulation contrast either alone or in combination with other illumination mechanisms.

Oblique or Anaxial 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.

Phase Contrast - A large spectrum of living biological specimens are virtually transparent when observed in the optical microscope under brightfield illumination. To improve visibility and contrast in such specimens, microscopists often reduce the opening size of the substage condenser iris diaphragm, but this maneuver is accompanied by a serious loss of resolution and the introduction of diffraction artifacts. Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by Zeiss laboratories into a commercial microscope several years later. This technique provides an excellent method of improving contrast in unstained biological specimens without significant loss in resolution, and is widely utilized to examine dynamic events in living cells.

Comparison of Phase Contrast and DIC Microscopy - Phase contrast and differential interference contrast (DIC) microscopy are complementary techniques capable of producing high contrast images of transparent biological phases that do not ordinarily affect the amplitude of visible light waves passing though the specimen. The most fundamental distinction between differential interference contrast and phase contrast microscopy is the optical basis upon which images are formed. Phase contrast yields image intensity values as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. The situation is quite distinct for differential interference contrast, where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for introducing contrast into specimen images.

Polarized Light Microscopy - The polarized light microscope is designed to observe and photograph specimens that are visible primarily due to their optically anisotropic character. In order to accomplish this task, the microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer. Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to other techniques such as darkfield and brightfield illumination, differential interference contrast, phase contrast, Hoffman modulation contrast, and fluorescence.

Rheinberg Illumination - Rheinberg illumination, a form of optical staining, was initially demonstrated by the British microscopist Julius Rheinberg to the Royal Microscopical Society and the Quekett Club (England) over a hundred years ago. This technique is a striking variation of low to medium power darkfield illumination using colored gelatin or glass filters to provide rich color to both the specimen and background. In Rheinberg illumination, the oblique light rays traversing a brightfield condenser pass through an annular filter of one or more colors, while the central rays of light pass through another spot-shaped filter fitted into the circular opening of the annular-shaped filter. The objective is used at full aperture.

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.

Deconvolution in Optical Microscopy - Deconvolution is a computationally intensive image processing technique that is being increasingly utilized for improving the contrast and resolution of digital images captured in the microscope. The foundations are based upon a suite of methods that are designed to remove or reverse the blurring present in microscope images induced by the limited aperture of the objective. Practically any image acquired on a digital fluorescence microscope can be deconvolved, and several new applications are being developed that apply deconvolution techniques to transmitted light images collected under a variety of contrast enhancing strategies. One of the most suitable subjects for improvement by deconvolution are three-dimensional montages constructed from a series of optical sections.

Live-Cell Imaging - An increasing number of investigations are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. As such, live-cell imaging has become a requisite analytical tool in most cell biology laboratories, as well as a routine methodology that is practiced in the wide ranging fields of neurobiology, developmental biology, pharmacology, and many of the other related biomedical research disciplines. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated in the presence of synthetic fluorophores and/or fluorescent proteins.

Introduction to Confocal Microscopy - Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest.

Principles and Applications of Interferometry - The foundation for interferometry (often referred to as microinterferometry) dates back to the nineteenth century with the introduction of the first interference microscope, which was based on the principles of the Jamin interferometer. Since that period, a number of commercial interference microscopes, both with transmitted and reflected light capabilities, have been produced by a number of manufacturers. Primarily designed to yield quantitative data from interference images, these microscopes utilize various technologies to determine parameters such as refractive index, birefringence, and thickness for a wide spectrum of materials.

  • Two Beam Interferometry - A two-beam interferometer functions by dividing originally coherent light into two beams of equal intensity, directing one beam onto the reference mirror and the other onto the specimen, and measuring the optical path difference (the difference in optical distances) between the resulting two reflected light waves.

  • Multiple-Beam Interferometry - The technique of multiple-beam interferometry is based upon situating two surfaces of high reflectivity in close proximity and using a lens to converge beams which have undergone multiple-reflection between the surfaces.

Near-Field Scanning Optical Microscopy - For ultra-high optical resolution, near-field scanning optical microscopy (NSOM) is currently the photonic instrument of choice. Near-field imaging occurs when a sub-micron optical probe is positioned a very short distance from the sample and light is transmitted through a small aperture at the tip of this probe. The near-field is defined as the region above a surface with dimensions less than a single wavelength of the light incident on the surface. Within the near-field region evanescent light is not diffraction limited and nanometer spatial resolution is possible. This phenomenon enables non-diffraction limited imaging and spectroscopy of a sample that is simply not possible with conventional optical imaging techniques.

Fluorescence Microscopy Techniques

Fluorescence Microscopy - Used primarily with episcopic illumination, fluorescence microscopy is rapidly becoming a standard tool in the fields of genetics, embryology, and cell biology.

Fluorescence and Differential Interference Contrast Combination Microscopy - Fluorescence microscopy can be combined with contrast enhancing techniques such as differential interference contrast (DIC) illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC then, without relocating the specimen, switching the microscope to fluorescence mode.

Fluorescence and Phase Contrast Combination Microscopy - To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.

Olympus FluoView Laser Scanning Confocal Microscopy - The new Olympus FluoViewTM FV1000 is the latest in point-scanning, point-detection, confocal laser scanning microscopes designed for today's intensive and demanding biological research investigations. Excellent resolution, bright and crisp optics, and high efficiency of excitation, coupled to an intuitive user interface and affordability are key characteristics of this state-of-the-art optical microscopy system.

Interactive Java Tutorials - A gallery of interactive Java applets designed to aid students in understanding difficult concepts in specialized microscopy techniques.

Contributing Authors

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

Robert Hoffman - Modulation Optics, Inc., 100 Forest Drive, Greenvale, New York 11548.

Tatsuro Otaki - Optical Design Department, Instruments Company, Nikon Corporation, 1-6-3 Nishi-Ohi, Shinagawa-ku, Tokyo, 140-8601, Japan.

Philip C. Robinson - Department of Ceramic Technology, Staffordshire Polytechnic, College Road, Stroke-on-Trent, ST4 2DE, United Kingdom.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

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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