A practical guide to multicolor live-cell single-molecule imaging

Yanagawa, Masataka; Yoda, Toshiki; Sugioka, Ryosuke; Inoue, Asuka

doi:10.51094/jxiv.5935

##article.authors##

Yanagawa, Masataka Graduate School of Science, Kyoto University https://researchmap.jp/masataka_yanagawa
Yoda, Toshiki Graduate School of Pharmaceutical Sciences, Tohoku University
Sugioka, Ryosuke Graduate School of Pharmaceutical Sciences, Kyoto University
Inoue, Asuka Graduate School of Pharmaceutical Sciences, Kyoto University

DOI:

https://doi.org/10.51094/jxiv.5935

キーワード:

全反射蛍光顕微鏡、光学系セットアップ、 1分子イメージング、 1分子追跡解析、変分ベイズ法ー隠れマルコフモデル、 smDA、蛍光標識、 FiBiT、 Gタンパク質共役型受容体 (GPCR)

抄録

Single-molecule imaging enables quantitative analysis of molecular dynamics on cell membranes, providing information on molecular diffusion, oligomer size, and membrane association/dissociation dynamics. Multicolor single-molecule imaging with orthogonal labeling further allows investigation of molecular interactions. Successful implementation of multicolor single-molecule imaging, however, requires careful optimization of optical design, sample preparation, and data processing. This chapter describes methods for multicolor single-molecule imaging and analysis. First, we describe the construction of a total internal reflection fluorescence microscopy (TIRFM) system equipped with rotating TIRF illumination, with detailed illustrations. We then present sample preparation methods, focusing on orthogonal molecular labeling techniques and optimization of expression levels for single-molecule detection. Finally, we introduce the Auto Imaging System (AIS), Auto Analysis System (AAS), and an updated version of the single-molecule Dynamics Analyzer (smDA, https://github.com/yanagawamasataka5z-oss/smDA-Igor), software tools for single-molecule imaging and analysis. Together, these tools enable researchers to establish a robust platform for multicolor single-molecule imaging and quantitative analysis of membrane protein dynamics.

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