Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Curr Protoc. 2022 Aug;2(8):e517. doi: 10.1002/cpz1.517.
Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is completely internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since 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. This overview will review the history, optical theory, and hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons. © 2022 Wiley Periodicals LLC.
全内反射荧光（TIRF）显微镜（TIRFM）是一种优雅的光学技术，可用于在极薄的轴向区域（“光学切片”）激发荧光团。该方法基于这样的原理，即当激发光在透明固体（例如盖玻片）与其与液体的界面处完全内反射时，在固液界面处会产生称为消逝波的电磁场，并且其频率与激发光相同。由于消逝波的强度随距离固体表面的距离呈指数衰减，因此只有距离固体几纳米内的荧光分子才能被有效地激发。本综述将回顾 TIRFM 的历史、光学理论和硬件配置。此外，它还将提供在神经元的质膜上研究受体的实验细节和方法学考虑。© 2022 Wiley Periodicals LLC.
