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Total internal reflection fluorescent microscopy

This review discusses applications of fluorescence microscopy using totally internally reflected excitation light. When totally internally reflected in a transparent solid at its interface with liquid, the excitation light beam penetrates only a short distance into the liquid. This surface electromagnetic field, called the ‘evanescent wave’, can selectively excite fluorescent molecules in the liquid near the interface. Total internal reflection fluorescence (TIRF) has been used to examine the cell/substrate contact regions of primary cultured rat myotubes with acetylcholine receptors labelled by fluorescent α‐bungarotoxin and human skin fibroblasts labelled with a membrane‐incorporated fluorescent lipid. TIRF examination of cell/substrate contacts dramatically reduces background from cell autofluorescence and debris. TIRF has also been combined with fluorescence photobleaching recovery and correlation spectroscopy to measure the chemical kinetic binding rates and surface diffusion constant of fluorescent labelled serum protein binding (at equilibrium) to a surface.