The microscope objective is the heart of every confocal fluorescence microscope. It serves both to focus the excitation light onto the specimen and to collect the emitted fluorescence. The optical performance of the microscope as a whole is determined to a large degree by the quality of the objective, because the tolerances for all the other optical elements (such as relay and projection lenses, dichroic mirrors, etc.) are largely relaxed because of the smaller optical angles. Every objective consists of a number of optical elements (as many as 10-12 for the top-range objectives), all carefully positioned with respect to one another, of different refractive index material, and specific radius of curvature. Over years of use, the optical performance of a microscope objective may gradually decrease because of wear on the optical coatings and possible misalignment of the optical elements resulting from thermal effects or shock. To ensure optimal performance, the quality of the objective should be checked routinely. However, elaborate experiments, such as the measurement of the point spread function at various field positions and at different wavelengths, are required to check the quality of the objective. As a result, the microscope objective in practice is often not much more than a black box to its user.
This section summarizes some of the main issues related to microscope objectives. Some of the important information relating to a given objective's specifics is denoted on its side. Most of these indications have been standardized. Figure 2.1 shows two illustrations of typical microscope objectives. One is an oil-immersion Plan-Neofluar microscope objective with a NA of 1.3 and a 100x magnification, the other a water-immersion C-Apochromat microscope objective with correction collar, a NA of 1.2 and a 40x magnification. O
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