Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Easy, Efficient and Electroporation-Based SiMS Microscopy (eSiMS) <s> Lamellipodia are thin, veil-like extensions at the edge of cells that contain a dynamic array of actin filaments. We describe an approach for analyzing spatial regulation of actin polymerization and depolymerization in vivo in which we tracked single molecules of actin fused to the green fluorescent protein. Polymerization and the lifetime of actin filaments in lamellipodia were measured with high spatial precision. Basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip. Most of the actin filaments in the lamellipodium were generated by polymerization away from the tip. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Easy, Efficient and Electroporation-Based SiMS Microscopy (eSiMS) <s> Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. <s> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Easy, Efficient and Electroporation-Based SiMS Microscopy (eSiMS) <s> 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. <s> BIB003
In 2014, we introduced a new SiMS method, which is easy-to-use for researchers without prior background in SiMS and that achieves high spatiotemporal resolution BIB002 . We call the new method eSiMS. In comparison with eSiMS, the original SiMS method demands experience to find cells containing EGFP-tagged probes at the optimal level BIB001 . Although the low expression levels of genetically-labeled proteins are achieved under the control of the defective CMV promoter, only a minor population of cells expresses EGFP-tagged proteins at a sufficiently low level. This technical difficulty for SiMS has prevented wider use of this technique in the research community. In eSiMS, we employed an actin probe that is chemically labeled with a red fluorescent DyLight 550 dye (Thermo Fisher Scientific, Waltham, MA, USA) on lysine side chains. Electroporation-mediated delivery of DyLight 550-actin (DL550-actin) enables the labeling of cells with 100% efficiency at the optimal low density (Figure 2A ). Note that excessively high density  In 2014, we introduced a new SiMS method, which is easy-to-use for researchers without prior background in SiMS and that achieves high spatiotemporal resolution BIB002 . We call the new method eSiMS. In comparison with eSiMS, the original SiMS method demands experience to find cells containing EGFP-tagged probes at the optimal level BIB001 . Although the low expression levels of genetically-labeled proteins are achieved under the control of the defective CMV promoter, only a minor population of cells expresses EGFP-tagged proteins at a sufficiently low level. This technical difficulty for SiMS has prevented wider use of this technique in the research community. In eSiMS, we employed an actin probe that is chemically labeled with a red fluorescent DyLight 550 dye (Thermo Fisher Scientific, Waltham, MA, USA) on lysine side chains. Electroporation-mediated delivery of DyLight 550-actin (DL550-actin) enables the labeling of cells with 100% efficiency at the optimal low density (Figure 2A ). Note that excessively high density causes overlapping of SiMS, which prevents accurate tracking of individual molecules. Too low a density makes SiMS imaging difficult due to the photobleaching of fluorophore during the finding of cells process and focus adjustment. In addition, too low a density limits the number of observations of speckles in each experiment. An example of optimal density for SiMS analysis are shown in Figure 2A ,B, whereas the "optimal density" could be variable depending on the purpose of SiMS measurement. Furthermore, DL550-actin labels cellular actin networks with improved photostability and brightness compared to EGFP-actin. These favorable properties of DL550-actin the extend time window of the eSiMS analysis. In combination with the Speckle TrackerJ software, the eSiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of 8-8.5 nm at 100 ms exposure time ( Figure 2B ). The basic protocol of eSiMS microscopy is introduced in our recent publications BIB003 BIB002 . Comparison between the original SiMS method and the eSiMS method is summarized in Table 1 . Note that DyLight 550 is the replacement of discontinued DyLight 549 that we had used in our previous study BIB002 . Table 1 . Comparison between the original SiMS method BIB001 and the eSiMS method BIB002 .