Total internal reflection fluorescence microscopy (TIRF)

About this technique

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Total internal reflection fluorescence (TIRF) microscopy is an imaging technique that overcomes the limited axial (or depth) resolution of conventional fluorescence microscopy. It is most commonly used for fluorescence microscopy of the cell membranes of live cells. It  exploits the formation of an electromagnetic field, or evanescent wave, that is generated when laser light undergoes TIR at the interface between a glass cover slip (or similar) and an aqueous or cellular sample. The evanescent wave excites fluorescent molecules in the sample, but only to a depth of approximately 100 nm, thereby giving outstanding axial resolution.

Traditional wide-field fluorescent signals from membrane molecules tend to be dwarfed by cytoplasmic fluorescence, TIRF microscopy is widely used to distinguish biological processes that occur at cell surfaces from those that occur within the cytoplasm. Such processes include adhesion of cells to surfaces, cellular secretion processes, and binding of proteins to receptors at cell surfaces. TIRF is particularly valuable for examining dynamic and single molecule events (like endocytosis and exocytosis, and protein trafficking) occurring on or near the cell membrane of living cells.