Shivanandan A, Deschout H, Scarselli M, Radenovic A
Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland.
Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland.
FEBS Lett. 2014 Oct 1;588(19):3595-602. doi: 10.1016/j.febslet.2014.06.014. Epub 2014 Jun 10.
Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo-activable fluorescent proteins (PA-FPs) are used as labels, we focus specifically on Photo-Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co-localization analysis.
单分子定位显微镜（SMLM）在空间分辨率上比传统荧光显微镜提高了一个数量级，有潜力成为定量生物学实验的一种非常有用的工具。它已经在从分子生物学到神经科学等生物学的各个领域用于此目的。在这篇综述文章中，我们简要回顾了SMLM在定量生物学中的应用，以及所涉及的挑战和一些已提出的解决方案。由于其在标记特异性方面的优势，以及当使用光激活荧光蛋白（PA-FPs）作为标记时光闪烁导致的相对较低的过计数，我们特别关注光激活定位显微镜（PALM），尽管所提出的观点可能总体上适用于SMLM。此外，我们关注以下三种定量测量：单分子计数、蛋白质空间分布异质性分析和共定位分析。
