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Introduction to microscopy
Introduction to microscopy

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6 Fluorescence microscopes

Although it is not used routinely in histology laboratories, fluorescence microscopes are used extensively in research. They are particularly valuable for viewing live cells that have been labelled with fluorescent markers. For example it is possible to transfect cells with fluorescent versions of their endogenous proteins so that the cellular location of a particular protein can be identified. They are also used in a technique analagous to immunohistochemistry, called immunofluorescence, in which cells are stained with antibodies that are either directly coupled to fluorescent tags, or the primary antibodies are detected with a fluorescent secondary antibody. In all cases the cells or tissues are illuminated with a high intensity light of a suitable wavelength to excite the fluorescent tag. The emitted light is collected by the objective and viewed through the eyepiece as normal, but usually additional filters are placed in the path of the emitted light so that only light of the correct wavelength, forming the image is observed. The usual arrangement of a fluorescence microscope is using incident illumination, generating a dark field image (Figure 11).

Figure 11 A fluorescence micrograph - dark field image. The image shows astrocytes in tissue culture that have been stained with three fluoresceent reagents. The cell nuclei are blue, the microfilaments of the cytoskeleton are red, and the contact points that the cell makes with the substratum (focal adhesions) are in green. (Courtesy of Mrs Christine Lancashire)