Coordinate-based colocalization analysis of single-molecule localization microscopy data
Coordinate-based colocalization analysis of single-molecule localization microscopy data
复制标题
DOI：
10.1007/s00418-011-0880-5
复制
发表时间：
2012-01-01
影响因子：
2.3
通讯作者：
Heilemann, Mike
中科院分区：
文献类型：
--
作者：
Malkusch, Sebastian;Endesfelder, Ulrike;Heilemann, Mike
不同标记的生物分子的共定位是荧光显微镜中一个有价值的工具，可以提供生物分子相互作用的信息。随着超分辨率显微镜的出现，共定位分析越来越接近分子分辨率，弥补了与荧光共振能量转移等其他技术的差距。在这些新的显微技术中，基于单分子定位的超分辨率方法提供了单分子坐标的优势，而不是强度信息，可用于共定位分析。这需要适应现有的定位显微镜数据的数学算法。本文介绍了一种适用于单分子超分辨率数据的基于坐标的共定位分析算法。此外，我们提出了一个同时双色成像的实验配置，以及一种校正光学像差的鲁棒方法，精度为几纳米。我们证明了我们的方法对细胞结构和两种结合肌动蛋白丝的蛋白质的潜力。
Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.