Near-infrared actin probes for deep-cell Single-Molecule Speckle (SiMS) microscopy

抄録

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate cellular processes at the molecular level in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy has been 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. We have recently introduced 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 (Yamashiro and Watanabe, Sensors, E1545, 2017). In live cells, single molecules of CF680R-labeled actin (CF680R-actin) can be visualized with low autofluorescence background, which is approximately 13% of eSiMS imaging with DyLight 550-labeled actin. CF680R-actin incorporated into actin structures and showed excellent brightness and photostability suitable for single-molecule imaging. CF680R-actin enabled monitoring of actin SiMS in actomyosin bundles associated with adherens junctions (AJs) located at 3.5 to 4 μm above the basal surfaces of epithelial monolayers. Furthermore, CF680R-actin with green and red fluorescent probes extends application of eSiMS microscopy to three-color imaging. More recently, we found that genetically-labeled actin with NIR fluorescent protein, miRFP703 (Shcherbakova et al., Nat Commun., 7:12405, 2016), is also applicable to SiMS imaging. These favorable properties of NIR actin probes extend the application of SiMS microscopy to actin turnover and flow analyses in deep cellular structures.