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From the theory of image formation developed by Ernst Abbe in 1872 until his death in 1905, there was a period of great advances in light microscopy. The use of apochromatic microscope objectives and oil-immersion objectives resulted in high-quality light microscopes that reached the theoretical limit of lateral resolution of approximately 200 nm (0.2 ¼m). The next important step was to improve image contrast. This chapter reviews several methods and techniques to provide contrast. We define contrast as ratios of or differences of light intensities between different areas (or pixels) in the optical plane, such as a difference in intensity between different points of a specimen, or between a specimen and the background: Contrast=I max −I min I max +I min . Contrast in an image is determined by several parameters: signal strength (number of detected photons), the dynamic range of the signal (lowest to highest level of signal), optical aberrations of the optical system, and the number of picture elements per unit area (pixels). The technical advances that resulted in greatly improved contrast can be divided into two groups: nonoptical techniques, as exemplified by fluorescence microscopy and the development of epi-fluorescence microscopy; and optical techniques, such as phase contrast microscopy and differential interference microscopy. The techniques of fluorescence microscopy provide contrast as well as high specificity and sensitivity. Phase contrast and differential contrast microscopy permit the microscopic observation of live, unstained cells in tissue culture. Another important technique is video-enhanced contrast microscopy. These groups of techniques that provide and enhance image contrast have resulted in advances in cell biology, neurobiology, and developmental biology as well as diagnostic techniques in clinical medicine. Note that two types of microscopy can be combined, called correlative microscopy, to further minimize artifacts and the false interpretation of images. 5.1 Nonoptical Techniques In the previous sections we discussed the use of stains and dyes as well as the great specificity that is possible with the use of immunocytochemical methods. The autofluorescence of organelles, cells, and tissues was known for a long time.
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