2015年
An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cellular actin filaments
BIOPHYSICAL METHODS IN CELL BIOLOGY
- ,
- ,
- 巻
- 125
- 号
- 開始ページ
- 43
- 終了ページ
- 59
- 記述言語
- 英語
- 掲載種別
- 論文集(書籍)内論文
- DOI
- 10.1016/bs.mcb.2014.10.013
- 出版者・発行元
- ELSEVIER ACADEMIC PRESS INC
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.
- リンク情報
- ID情報
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- DOI : 10.1016/bs.mcb.2014.10.013
- ISSN : 0091-679X
- PubMed ID : 25640423
- Web of Science ID : WOS:000370488400005