TY - JOUR
T1 - An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cellular actin filaments
AU - Yamashiro, Sawako
AU - Mizuno, Hiroaki
AU - Watanabe, Naoki
N1 - Funding Information:
This work was supported by the Human Frontiers Science Program Grant No. RGP0061∕2009-C (N.W. and D.V.); NEXT program grant No. LS013 from the Cabinet Office, Government of Japan (N.W.); a grant from Takeda Science Foundation (N.W.) and a Grant-in-Aid for Scientific Research on Innovative Areas (S.Y.).
Publisher Copyright:
© 2015 Elsevier Inc.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - Actin
KW - Computer-assisted tracking of speckles
KW - Electroporation
KW - Nanometer-scale displacement analysis
KW - Single-molecule speckle microscopy
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U2 - 10.1016/bs.mcb.2014.10.013
DO - 10.1016/bs.mcb.2014.10.013
M3 - Article
AN - SCOPUS:84921834289
VL - 125
SP - 43
EP - 59
JO - Methods in Cell Biology
JF - Methods in Cell Biology
SN - 0091-679X
ER -