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Sensors 2017, 17(7), 1545; https://doi.org/10.3390/s17071545

An Infrared Actin Probe for Deep-Cell Electroporation-Based Single-Molecule Speckle (eSiMS) Microscopy

1
Laboratory of Single-Molecule Cell Biology, Kyoto University Graduate School of Biostudies, Kyoto 606-8501, Japan
2
Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
*
Author to whom correspondence should be addressed.
Received: 1 May 2017 / Revised: 18 June 2017 / Accepted: 22 June 2017 / Published: 1 July 2017
(This article belongs to the Special Issue Single-Molecule Sensing)
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Abstract

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation. CF680R-labeled actin (CF680R-actin) incorporated into actin structures and showed excellent brightness and photostability suitable for single-molecule imaging. Importantly, the intensity of autofluorescence with respect to SiMS brightness was reduced to approximately 13% compared to DyLight 550-labeled actin (DL550-actin). CF680R-actin enabled the monitoring of actin SiMS in actomyosin bundles associated with adherens junctions (AJs) located at 3.5–4 µm above the basal surfaces of epithelial monolayers. These favorable properties of CF680R-actin extend the application of eSiMS to actin turnover and flow analyses in deep cellular structures. View Full-Text
Keywords: single-molecule imaging; actin dynamics; near infrared probe single-molecule imaging; actin dynamics; near infrared probe
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Yamashiro, S.; Watanabe, N. An Infrared Actin Probe for Deep-Cell Electroporation-Based Single-Molecule Speckle (eSiMS) Microscopy. Sensors 2017, 17, 1545.

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