Total internal reflection fluorescence (TIRF) microscopy.
Total internal reflection fluorescence (TIRF) microscopy.
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DOI：
10.1002/0471142956.cy1218s50
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发表时间：
2009-10-01
影响因子：
--
通讯作者：
Fish, Kenneth N
中科院分区：
文献类型：
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作者：
Fish, Kenneth N
全内反射荧光（TIRF）显微镜（TIRFM）是一种优雅的光学技术，其提供在极薄的轴向区域（“光学切片”）中的荧光团的激发。该方法基于这样的原理：当激发光在透明固体（例如，盖玻片），在固体-液体界面处的液体中产生电磁场，称为渐逝波，并且其频率与激发光相同。由于倏逝波的强度随着与固体表面的距离呈指数衰减，因此仅在固体的几百纳米内的荧光分子被有效地激发。本单元将简要回顾历史，光学理论，和不同的硬件配置中使用的TIRFM。此外，它将提供实验的细节和方法的考虑，研究受体在质膜上的神经元。
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 totally 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 unit will briefly review the history, optical theory, and different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.