An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cellular actin filaments

Sawako Yamashiro, Hiroaki Mizuno, Naoki Watanabe

    Research output: Contribution to journalArticlepeer-review

    8 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)43-59
    Number of pages17
    JournalMethods in Cell Biology
    Volume125
    DOIs
    Publication statusPublished - 2015 Jan 1

    Keywords

    • Actin
    • Computer-assisted tracking of speckles
    • Electroporation
    • Nanometer-scale displacement analysis
    • Single-molecule speckle microscopy

    ASJC Scopus subject areas

    • Cell Biology

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