Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Semi-Automatic Imaging Analysis Tool, Speckle TrackerJ <s> Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm(2)/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Semi-Automatic Imaging Analysis Tool, Speckle TrackerJ <s> Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin. <s> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Semi-Automatic Imaging Analysis Tool, Speckle TrackerJ <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> BIB003 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Semi-Automatic Imaging Analysis Tool, Speckle TrackerJ <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> BIB004
To assist in the analysis of SiMS data, the imaging analysis tool, Speckle TrackerJ BIB001 has been developed by our collaborator Dimitrios Vavylonis and his colleagues at Lehigh University. The tool is a freely available open source ImageJ plugin [44] . Speckle TrackerJ enables the recording of the position, appearance and disappearance of numerous speckles through time. The software is designed to allow manual operation of a number of error-correction commands in combination with computer-based particle tracking. With the user's supervision/assistance, Speckle TrackerJ greatly helps the collection of reliable results from images of mixed qualities. In addition, measurement of speckles' centroids by using the two-dimensional Gaussian fit model of Speckle TackerJ facilitates high-resolution tracking of nanometer-scale displacement analysis BIB003 (Figure 2B ). The method to use Speckle TrackerJ is described on the above website, and data analyses using Speckle TrackerJ were introduced in our recent publications BIB004 BIB003 BIB001 BIB002 . Other automatic particle detection and tracking software currently available from various resources can track individual molecules fairly precisely in ideal situations, but the frequency of tracking errors often becomes intolerable, influenced by the variations in clarity and density of single-molecule images. 