全内反射荧光显微镜利用全内反射产生的隐逝波激发样品，从而使样品表面数百纳米内的荧光基团受到激发，产生荧光信号。因只有靠近样品表面区域产生荧光信号，全内反射显微镜技术极大的改善了图像的信噪比，从而可以观察到样品表面细节信号甚至单分子的活动情况。

Total internal reflection fluorescence (TIRF) microscopy, also known as evanescent wave/field microscopy, is a technique that uses a special mode of sample illumination to exclusively excite fluorophores that are within approximately 100 nm of the cover glass/sample interface. This mode of illumination is based on an evanescent field which is produced when light rays are totally internally reflected at the cover glass/substrate interface. The evanescent field does not propagate deeper into the sample; therefore, images are not contaminated with fluorescence from out-of-focus planes. As a result, TIRF microscopy provides a means of imaging fluorophores near the cover glass/substrate interface with a high signal-to-noise ratio, thus making it the current gold standard technique for studying events that occur at the adherent plasma membrane of cells.