利用干涉光电激活定位显微镜 (iPALM) 对 F-肌动蛋白丝进行三维超分辨显微观察。

IF 0.5 Q4 EDUCATION & EDUCATIONAL RESEARCH
Paginas de Educacion Pub Date : 2016-12-01 DOI:10.3791/54774
Yilin Wang, Pakorn Kanchanawong
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引用次数: 0

摘要

荧光显微镜可直接观察细胞内的特定生物分子。然而,传统荧光显微镜的空间分辨率受衍射限制,图像平面内约为 200 纳米,沿光轴方向大于 500 纳米。因此,长期以来荧光显微镜在观察细胞内超微结构特征方面受到严重限制。近年来超分辨显微镜方法的发展克服了这一限制。尤其是可光电开关荧光团的出现,使基于定位的超分辨显微技术得以实现,其分辨能力接近分子长度尺度。在此,我们介绍了一种基于单分子定位显微镜和多相干涉仪的三维超分辨显微镜方法,即干涉光激活定位显微镜(iPALM)的应用。这种方法可在所有三个维度上提供近乎各向同性的 20 纳米分辨率。本文介绍了观察丝状肌动蛋白细胞骨架的方法,包括标本制备和 iPALM 仪器的操作。这些方法也可用于研究细胞中的其他超微结构特征。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

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Paginas de Educacion
Paginas de Educacion EDUCATION & EDUCATIONAL RESEARCH-
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