Total internal reflection fluorescence (TIRF) microscopy represents a method of exciting and visualizing fluorophores present in the near-membrane region of live or fixed cells grown on coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high-refractive medium (e.g., glass) into a low-refractive medium (e.g., cell, water). The evanescent field produced by total internally reflected light excites the fluorescent molecules at the cell-substrate interface and is accompanied by minimal exposure of the remaining cell volume. This technique provides high-contrast fluorescence images, with very low background and virtually no out-of-focus light, ideal for visualization and spectroscopy of single-molecule fluorescence near a surface. This unit presents, in a concise manner, the principle of operation, instrument diversity, and TIRF microscopy applications for the study of biological samples.