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Total internal reflection fluorescence (TIRF) microscopy (TIRFM), also known as evanescent wave or evanescent field microscopy, allows selective excitation of fluorophores (e.g., GFP, membrane dyes, fluorochromes) in an aqueous environment remarkably close to a solid surface (within r100 nm), at a high refractive.
TIRFM allows the excitation of fluorophores in an extremely thin axial region ('optical section'). The TIRFM technique is based on the principle that when excitation light is completely reflected internally at the solid-liquid interface, an electromagnetic field, called the evanescent wave, will be generated in the liquid phase with the same frequency as the excitation light. As the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited.
Figure 1. The physical basis of TIRF
TIRFM produces wide-field images with extremely low background fluorescence, which makes TRIF a powerful tool for a wide range of cell biological characterizations:
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