Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy.
Ultrahigh resolution imaging of biomolecules by fluorescence photoactivation localization microscopy.
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DOI：
10.1007/978-1-59745-483-4_32
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Mason MD
中科院分区：
文献类型：
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作者：
Hess ST;Gould TJ;Gunewardene M;Bewersdorf J;Mason MD
衍射限制了可以通过普通光学显微镜成像的生物结构。然而，最近开发的技术正在打破衍射造成的限制，并允许在分子长度尺度上对生物样品进行成像。荧光光活化定位显微镜（FPALM）和相关方法现在可以在固定和活细胞中成像分子分布，测量分辨率优于30 nm。基于单个光活化分子的定位，FPALM使用活化、定位和光漂白的重复循环，结合高灵敏度荧光成像，来识别和定位样品中的大量分子。详细讨论了这种显微镜的构造和使用的程序和陷阱。胞质蛋白、膜蛋白和其他结构的最终图像，以及采集过程中的结果示例。希望这些细节可以用于对各种生物样品进行FPALM，以显着推进对生物系统的理解。
Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Final images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, in order to significantly advance the understanding of biological systems.