Imaging biological structures with fluorescence photoactivation localization microscopy.
Imaging biological structures with fluorescence photoactivation localization microscopy.
复制标题
DOI：
10.1038/nprot.2008.246
复制
发表时间：
2009
期刊：
影响因子：
14.8
通讯作者：
Hess, Samuel T.
中科院分区：
文献类型：
--
作者：
Gould, Travis J.;Verkhusha, Vladislav V.;Hess, Samuel T.
荧光光活化定位显微镜(FPALM)以亚衍射极限分辨率成像生物结构。通过重复的活化、读出和漂白循环，可以精确地定位大量的光活化探针，以获得有效分辨率为数十纳米的标记分子的地图(图像)。FPALM已被应用于多种生物成像应用，包括固定细胞和活细胞中的膜、细胞骨架和胞浆蛋白。分子运动是可以量化的。FPALM还可以应用于非生物样品，这些样品可以用光活化探针标记。重点是细胞成像，我们在这里描述了传统的宽视场荧光显微镜适用于FPALM，并介绍了成功获得和分析FPALM图像的一步一步的程序。该协议的基本原理也可能适用于使用类似成像技术的用户，这些成像技术应用光致可激活探测器的局部化来实现超分辨率。一旦完成设置对准，可以在大约1-30分钟内获得数据采集，并在大约0.5-4小时内进行分析。
Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1–30 min and analyzed in approximately 0.5–4 h.
登录
查看更多内容
影响因子：
48
作者：
Gould, Travis J.;Gunewardene, Mudalige S.;Gudheti, Manasa V.;Verkhusha, Vladislav V.;Yin, Shu-Rong;Gosse, Julie A.;Hess, Samuel T.
通讯作者：
Hess, Samuel T.
影响因子：
56.9
作者：
Bates, Mark;Huang, Bo;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.0401638101
发表时间：
2004-04-27
影响因子：
11.1
作者：
Gordon, MP;Ha, T;Selvin, PR
通讯作者：
Selvin, PR
影响因子：
46.9
作者：
Chudakov, DM;Belousov, VV;Lukyanov, KA
通讯作者：
Lukyanov, KA
影响因子：
3.4
作者：
Ando, Ryoko;Flors, Cristina;Miyawaki, Atsushi
通讯作者：
Miyawaki, Atsushi