title: An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cellular actin filaments.
abstract: Single-molecule speckle (SiMS) microscopy has been a powerful method to analyze actin dynamics in live cells by tracking single molecule of fluorescently labeled actin. Recently we developed a new SiMS method, which is easy-to-use for inexperienced researchers and achieves high spatiotemporal resolution. In this method, actin labeled with fluorescent DyLight dye on lysines is employed as a probe. Electroporation-mediated delivery of DyLight-actin (DL-actin) into cells enables to label cells with 100% efficiency at the optimal density. DL-actin labels cellular actin filaments including formin-based structures with improved photostability and brightness compared to green fluorescent protein-actin. These favorable properties of DL-actin extend time window of the SiMS analysis. Furthermore, the new SiMS method enables nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm. With these advantages, our new SiMS microscopy method will help researchers to investigate various actin remodeling processes. In this chapter, we introduce the methods for preparation of DL-actin probes, electroporation to deliver DL-actin, the SiMS imaging and data analysis.
authors: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.
authors: Sawako
authors: S
authors: Yamashiro
authors: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.
authors: Hiroaki
authors: H
authors: Mizuno
authors: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.
authors: Naoki
authors: N
authors: Watanabe
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008853:Microscopy / Q000379:methods*; D036103:Nanotechnology; D011817:Rabbits; D013997:Time Factors; D014981:Xenopus
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Actin; Computer-assisted tracking of speckles; Electroporation; Nanometer-scale displacement analysis; Single-molecule speckle microscopy