Modern microscopists have developed a wide spectrum of useful techniques designed to aid in contrast enhancement and provide better observation and photomicrography of specimens. This section of the Microscopy Primer describes many of these techniques in detail.

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 - Invented by Dr. Robert Hoffman, this contrast-enhancing technique serves to increase specimen visibility and contrast, especially for unstained and living specimens.

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 - 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.

Polarized Light Microscopy - Although much neglected and undervalued as an investigative tool, polarized light microscopy provides all the benefits of brightfield microscopy and yet offers a wealth of information, which is simply not available with any other optical microscopy technique. The technique exploits optical properties of anisotropy to reveal detailed information about the structure and composition of materials, which are invaluable for identification and diagnostic purposes.

Rheinberg Illumination - First described around the turn of the century by British microscopist Julius Rheinberg, this technique provides beautiful high-contrast colored images of unstained specimens.

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.

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 - 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.

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