光开关动力学和标记密度对超分辨率荧光成像的影响。

The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging.

机构信息

Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, 97074 Würzburg, Germany.

出版信息

J Biotechnol. 2010 Sep 15;149(4):260-6. doi: 10.1016/j.jbiotec.2010.02.010. Epub 2010 Feb 20.

Abstract

Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution.

摘要

基于荧光团可逆光开关的超分辨率荧光成像方法,随后进行定位,目前已发展成为细胞成像的有前途的工具。由于这些方法中的大多数都依赖于将大多数荧光团转移到非荧光的暗态,并对分离的荧光荧光团进行精确定位,因此必须仔细控制光可开关荧光团的光物理性质。可实现的分辨率和分辨结构特征的能力不仅取决于荧光团的亮度,还取决于标记密度以及非荧光暗态的稳定性或寿命。在这里,我们讨论了荧光团的关-开比如何影响分辨率。我们将实验数据与理论模拟进行了比较,并提出了一种定制光开关特性以实现最佳光学分辨率的策略。

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