Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Previous observations indicated that the lamellipodium ("leading edge") of fibroblasts contains a dense meshwork, as well as numerous bundles (microspikes) of actin filaments. Most, if not all, of the filaments have a uniform polarity, with the "barbed" end associated with the membrane. I investigated whether and how actin subunits exchange in this region by microinjecting living gerbil fibroma cells (IMR-33) with actin that had been labeled with iodoacetamidotetramethylrhodamine. After incorporation of the labeled actin into the lamellipodium, I used a laser microbeam to photobleach a 3-4-micron region at and surrounding a microspike, without disrupting the integrity of the structure. I then recorded the pattern of fluorescence recovery and analyzed it using a combination of TV image intensification and digital image processing techniques. Fluorescence recovery was first detected near the edge of the cell and then moved toward the cell's center at a constant rate of 0.79 +/- 0.31 micron/min. When only part of the lamellipodium near the edge of the cell was photobleached, the bleached spot also moved toward the cell's center and through an area unbleached by the laser beam. These results indicated that steady state incorporation of actin subunits occurred predominantly at the membrane-associated end of actin filaments, and that actin subunits in the lamellipodium underwent a constant movement toward the center of the cell. I suggest that treadmilling, possibly in combination with other molecular interactions, may provide an effective mechanism for the movement of actin subunits and the protrusion of cytoplasm in the lamellipodium of fibroblasts. <s> BIB001 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells. <s> BIB002 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits. <s> BIB003 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge. <s> BIB004 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton. <s> BIB005 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells. <s> BIB006 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> The reconstitution of microbial rocketing motility in vitro with purified proteins has recently established definitively that no myosin motor is required for protrusion. Instead, actin polymerization, in conjunction with a small number of proteins, is sufficient. A dendritic pattern of nucleation controlled by the Arp2/3 complex provides an efficient pushing force for lamellipodial motility. <s> BIB007 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <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> BIB008 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling. <s> BIB009 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. <s> BIB010 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Fluorescent speckle microscopy (FSM) is a technology used to analyze the dynamics of macromolecular assemblies in vivo and in vitro. Speckle formation by random association of fluorophores with a macromolecular structure was originally discovered for microtubules. Since then FSM has been expanded to study other cytoskeleton and cytoskeleton-binding proteins. Specialized software has been developed to convert the stochastic speckle image signal into spatiotemporal maps of polymer transport and turnover in living cells. These maps serve as a unique quantitative readout of the dynamic steady state of the cytoskeleton and its responses to molecular and genetic interventions, allowing a systematic study of the mechanisms of cytoskeleton regulation and its effect on cell function. Here, we explain the principles of FSM imaging and signal analysis, outline the biological questions and corresponding methodological advances that have led to the current state of FSM, and give a glimpse of new FSM modalities under development. <s> BIB011 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Cells generate diverse microtubule populations by polymerization of a common α/β-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations. <s> BIB012 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> The actin cytoskeleton drives cell locomotion and tissue remodeling. The invention of live-cell fluorescence single-molecule imaging opened a window for direct viewing of the actin remodeling processes in the cell. Since then, a number of unanticipated molecular functions have been revealed. One is the mechanism of F-actin network breakdown. In lamellipodia, one third of newly polymerized F-actin disassembles within 10 seconds. This fast F-actin turnover is facilitated by the filament severing/disrupting activity involving cofilin and AIP1. Astoundingly fast dissociation kinetics of the barbed end interactors including capping protein suggests that F-actin turnover might proceed through repetitive disruption/reassembly of the filament near the barbed end. The picture of actin polymerization is also being revealed. At the leading edge of the cell, Arp2/3 complex is highly activated in a narrow edge region. In contrast, mDia1 and its related Formin homology proteins display a long-distance directional molecular movement using their processive actin capping ability. Recently, these two independently-developed projects converged into a discovery of the spatiotemporal coupling between mDia1-mediated filament nucleation and actin disassembly. Presumably, the local concentration fluctuation of G-actin regulates the actin nucleation efficiency of specific actin nucleators including mDia1. Pharmacological perturbation and quantitative molecular behavior analysis synergize to reveal hidden molecular linkages in the actin turnover cycle and cell signaling. <s> BIB013 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Cells must rapidly remodel the actin filament network to achieve various cellular functions. Actin filament turnover is a dynamic process that plays crucial roles in cell adhesion, locomotion, cytokinesis, endocytosis, phagocytosis, tissue remodeling, etc., and is regulated by cell signaling cascades. Success in elucidating dynamic biological processes such as actin-based motility relies on the means enabling real time monitoring of the process. The invention of live-cell fluorescence single-molecule imaging has opened a window for direct viewing of various actin remodeling processes. In general, assembly and dissociation of actin and its regulators turned out to occur at the faster rates than previously estimated by biochemical and structural analyses. Cells undergo such fast continuous exchange of the components perhaps not only to drive actin remodeling but also to facilitate rapid response in many other cell mechanics and signaling cascades. This chapter describes how epifluorescence single-molecule imaging which visualizes deeper area than the TIRF microscopy is achieved in XTC cells, the currently best platform for this approach. <s> BIB014 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis. <s> BIB015 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Live-cell single-molecule imaging is a powerful tool to elucidate the in vivo biochemistry of cytoskeletal proteins. However, it is often somewhat difficult to interpret how a bulk population of the observed molecule might behave as a whole. We review our recent studies in which the combination of image analysis with modeling and bulk kinetics measurements such as FRAP (fluorescence recovery after photobleaching) clarified basic problems in the regulation of actin remodeling pathways. <s> BIB016 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <s> Single fluorescent-molecule video imaging and tracking in living cells are revolutionizing our understanding of molecular interactions in the plasma membrane and intracellular membrane systems. They have revealed that molecular interactions occur surprisingly dynamically on much shorter time scales (≪1s) than those expected from the results by conventional techniques, such as pull-down assays (minutes to hours). Single-molecule imaging has unequivocally showed that G-protein-coupled receptors (GPCRs) undergo dynamic equilibrium between monomers and dimers, by enabling the determination of the 2D monomer-dimer equilibrium constant, the dimer dissociation rate constant (typically ∼10s(-1)), and the formation rate constant. Within one second, GPCRs typically undergo several cycles of monomer and homo-dimer formation with different partners. <s> BIB017 </s> Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution <s> Introduction <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> BIB018
Remodeling of the cortical cytoskeleton plays a central role in cell locomotion, cytokinesis, endocytosis, phagocytosis and tissue organization. To elucidate the regulation of cell morphogenesis, real-time monitoring of molecular behavior in the actin cytoskeleton offers tremendous potential. The development of live-cell fluorescence single-molecule imaging has opened a window for the direct viewing of assembly and remodeling processes of the actin filament network. Many of these processes have turned out to occur much faster than predicted by in vitro biochemical data BIB013 . Live-cell single-molecule imaging has provided critical information on not only remodeling of the actin cytoskeleton BIB013  BIB010 , but also the motion of plasma membrane proteins BIB009 BIB017 and dynamics of molecular motor proteins  BIB012 . Prior to the development of this approach, two methods were mainly employed to monitor the dynamics of actin filament turnover in live cells. In one method, researchers observed the site of incorporation of microinjected labeled actin probes BIB002 BIB003 . These studies revealed that actin incorporates fastest into the tip of pseudopods, such as filopodia and lamellipodia. The other methods are photoactivation of fluorescence (PAF) of caged fluorescent actin and fluorescence recovery after photobleaching (FRAP) of fluorescently-labeled actin. In an early FRAP study BIB001 , the retrograde actin flow, which is the centripetal movement of the actin network ubiquitously observed at the periphery of cultured cells, was visualized. Another study employing photoactivation of caged fluorescent actin BIB004 further revealed that the majority of F-actin disassembles before reaching the lamellipodium base in migrating fish keratocytes. Although these seminal studies revealed the dynamic actin filament turnover and the retrograde actin flow in lamellipodia, there was a long-standing debate as to whether treadmilling governs BIB005 BIB007  actin filament turnover in lamellipodia or actin undergoes frequent disassembly and reassembly within the body of lamellipodia BIB004 . A further development of FRAP and PAF is fluorescence localization after photobleaching (FLAP), in which actin are labeled with two distinct fluorophores: one to be photobleached and the other to act as a reference label  . The use of a reference fluorophore allows the tracking of the distribution of the labeled actin by simple image differencing and thus enables measurement of fast redistribution dynamics  BIB015 . As discussed in detail in previous studies BIB013 BIB016 , FRAP, PAF and FLAP experiments may exhibit potential problems, such as low spatial resolution, insufficiency in monitoring subpopulation, local reincorporation of disassembled probes and photodamage during the photobleaching procedure. These problems may have given rise to the distinct results and interpretations in the FRAP and PAF studies. On the other hand, single-molecule imaging directly visualizes assembly, dwell time and movement of individual fluorescently labeled molecules. One can also follow the change in the molecular behavior over time by only intermittently acquiring the images. In 1998, fluorescent speckle microscopy (FSM), which visualizes movement of cytoskeletal polymers in living cells with higher resolution than the above microscopy techniques, was introduced BIB006 . The principle of FSM is that incorporation of fluorescently labeled subunits at low concentrations provides fiduciary marks, which are referred to as speckles, on polymers. By further lowering the density of the labeled actin probes, we were able to demonstrate the actin "speckles" consisting of a single enhanced green fluorescent protein (EGFP) tagged actin molecule in live cells BIB008 . We call our observation target "single-molecule speckle (SiMS)" to distinguish from the "speckles" characterized by Waterman-Storer and her colleagues BIB006 BIB011 . In the original FSM method, "speckle" was claimed to contain 2 to 10 fluorescent molecules in resolvable image units whereas we track the behavior of individual fluorescent molecules. Through statistic analysis of a large number of molecular events, researchers can extract exact kinetic parameters in molecular assembly and disassembly reactions in vivo. To correctly perform SiMS analysis in live cells, one must carefully design the observation conditions to minimize the photodamage of the sample due to the strong illumination required for single-molecule visualization and error tracking of SiMS behavior. A decade of experience has steadily improved the feasibility of SiMS microscopy BIB014 BIB018 . Especially the use of electroporation of fluorescent proteins for cell labeling and the introduction of new photostable chemical dyes extends the applications of SiMS microscopy to cellular structure that could not previously be imaged using this technique. In this article, we briefly review principles and applications of SiMS microscopy. We also summarize our recent progress in SiMS methods to provide background information for our accompanying paper introducing new near infrared actin probes.