Toomre Derek
Cold Spring Harb Protoc. 2012 Apr 1;2012(4):439-46. doi: 10.1101/pdb.ip068676.
Live cell fluorescent microscopy is important in elucidating dynamic cellular processes such as cell signaling, membrane trafficking, and cytoskeleton remodeling. Often, transient intermediate states are revealed only when imaged and quantitated at the single-molecule, vesicle, or organelle level. Such insight depends on the spatiotemporal resolution and sensitivity of a given microscopy method. Confocal microscopes optically section the cell and improve image contrast and axial resolution (>600 nm) compared with conventional epifluorescence microscopes. Another approach, which can selectively excite fluorophores in an even thinner optical plane (<100 nm) is total internal reflection fluorescence microscopy (TIRFM). The key principle of TIRFM is that a thin, exponentially decaying, evanescent field of excitation can be generated at the interface of two mediums of different refractive index (RI) (e.g., the glass coverslip and the biological specimen); as such, TIRFM is ill-suited to deep imaging of cells or tissue. However, for processes near the lower cell cortex, the sensitivity of TIRFM is exquisite. The recent availability of a very high numerical-aperture (NA) objective lens (>1.45) and turnkey TIRFM systems by all the major microscopy manufacturers has made TIRFM increasingly accessible and attractive to biologists, especially when performed in a quantitative manner and complemented with orthogonal genetic and molecular manipulations. This article discusses sample preparation for TIRFM, acquisition of time-lapse movies, and quantitative analysis. It also gives examples of imaging cytoskeleton dynamics and exo- and endocytosis using TIRFM.
活细胞荧光显微镜在阐明细胞信号传导、膜运输和细胞骨架重塑等动态细胞过程中非常重要。通常，只有在单分子、囊泡或细胞器水平进行成像和定量时，才会揭示短暂的中间状态。这种见解取决于特定显微镜方法的时空分辨率和灵敏度。与传统落射荧光显微镜相比，共聚焦显微镜对细胞进行光学切片，提高了图像对比度和轴向分辨率（>600 nm）。另一种方法是全内反射荧光显微镜（TIRFM），它可以在更薄的光学平面（<100 nm）中选择性地激发荧光团。TIRFM的关键原理是，在两种不同折射率（RI）的介质（如玻璃盖玻片和生物样本）的界面处，可以产生一个薄的、指数衰减的倏逝激发场；因此，TIRFM不适用于细胞或组织的深度成像。然而，对于靠近细胞皮质下层的过程，TIRFM的灵敏度非常高。所有主要显微镜制造商最近都推出了非常高数值孔径（NA）的物镜（>1.45）和交钥匙TIRFM系统，这使得TIRFM对生物学家来说越来越容易获得且具有吸引力，特别是当以定量方式进行并辅以正交的遗传和分子操作时。本文讨论了TIRFM的样品制备、延时电影的采集和定量分析。它还给出了使用TIRFM成像细胞骨架动力学以及胞吐和胞吞作用的示例。
