Single-molecule Imaging Research Articles

Electrochemically modulated single-molecule localization microscopy for in vitro imaging cytoskeletal protein structures

Abstract A new concept of electrochemically modulated single-molecule localization super-resolution imaging is developed. Applications of single-molecule localization super-resolution microscopy have been limited due to insufficient availability of qualified fluorophores with favorable low duty cycles. The key for the new concept is that the “On” state of a redox-active fluorophore with unfavorable high duty cycle could be driven to “Off” state by electrochemical potential modulation and thus become available for single-molecule localization imaging. The new concept was carried out using redox-active cresyl violet with unfavorable high duty cycle as a model fluorophore by synchronizing electrochemical potential scanning with a single-molecule localization microscope. The two cytoskeletal protein structures, the microtubules from porcine brain and the actins from rabbit muscle, were selected as the model target structures for the conceptual imaging in vitro. The super-resolution images of microtubules and actins were obtained from precise single-molecule localizations determined by modulating the On/Off states of single fluorophore molecules on the cytoskeletal proteins via electrochemical potential scanning. Importantly, this method could allow more fluorophores even with unfavorable photophysical properties to become available for a wider and more extensive application of single-molecule localization microscopy.

Nanoliter Plasma Sample for Disease Diagnosis by Using a Visualization Platform of Counting Extracellular Vesicle Particles.

Extracellular vesicles (EVs) are an excellent candidate for noninvasive diagnostic and prognostic assessments for liquid biopsy applications owing to reflecting the dynamic changes of pathological and physiological states in the body and exceptional stability in vivo. Herein, we developed a single-extracellular vesicle fluorescence visualization and counting platform containing three parts of the nanoliter-liquid operating platform for aspirating the sample, antibody-functionalized coverslip for capturing targeted EVs, and the single-molecule fluorescence labeling and imaging system for the visualization and counting of EVs. Compared to previously reported studies, our developed platform not only minimized operational complexity but also successfully detected single EVs using a minimal volume of 25 nL of plasma. Moreover, the results of fluorescence spot counts demonstrated that the developed platform exhibits a wide dynamic range and an excellent linear correlation between fluorescence intensity and particle numbers for the detection of CD9+ EVs from standard samples. Remarkably, our developed single EVs capture and analysis platform was successfully applied not only in the quantitative analysis of EpCAM+ EVs to CD9+ EVs for the diagnosis of breast cancer (BC) but also in the quantitative analysis of CD14+ EVs to CD9+ EVs for the diagnosis of type 2 diabetes (T2DM). Collectively, our work provides new ideas for the diagnosis of breast cancer and type 2 diabetes while developing a versatile method for distinguishing other diseases by simultaneous detection of multibiomarkers of Exos subtypes in trace samples due to the substitutability of antibodies.

3D Single-Molecule Super-Resolution Imaging of Microfabricated Multiscale Fractal Substrates for Calibration and Cell Imaging.

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open up new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity as well as axial calibration issues. In this work, we address this issue by leveraging the geometrical characteristics of fractal-like structures, which serve as axial calibration tools and modulate cell growth. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as an alternative to electron microscopy for material imaging but also as calibration tools for 3D super-resolution microscopy. We used their multiscale and known geometry to perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilized these substrates as a platform for high-resolution bioimaging. As a proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with a 3D SMLM assessment.

The actin filament pointed-end depolymerase Srv2/CAP depolymerizes barbed ends, displaces capping protein, and promotes formin processivity

Cellular actin networks exhibit distinct assembly and disassembly dynamics, primarily driven by multicomponent reactions occurring at the two ends of actin filaments. While barbed ends are recognized as the hotspot for polymerization, depolymerization is predominantly associated with pointed ends. Consequently, mechanisms promoting barbed-end depolymerization have received relatively little attention. Here, using microfluidics-assisted three-color single-molecule imaging, we reveal that cyclase-associated protein (CAP), long known for its roles in nucleotide exchange and pointed-end depolymerization, also acts as a processive depolymerase at filament barbed ends. CAP molecules track barbed ends for several minutes, inducing depolymerization rates of up to 60 subunits per second. Importantly, CAP modulates barbed-end dynamics even under cytosol-mimicking assembly promoting conditions. We further show that CAP can colocalize with both formin and capping protein (CP) at barbed ends. CAP enhances formin processivity by 10-fold, allowing CAP-formin complexes to track fast-elongating barbed ends. In contrast, CAP destabilizes CP-bound barbed ends and accelerates dissociation of CP by fourfold. Our findings, combined with CAP's previously reported activities, firmly establish CAP as a key regulator of cellular actin dynamics.

Innate Immune Memory is Stimulus Specific.

Innate immune memory (also termed trained immunity) is defined in part by its ability to cross-protect against heterologous pathogens, and can be generated by many different stimuli, suggesting a "universal" trained state. However, different stimuli could form distinct memories, leading to stimulus-specific trained responses. Here, we use primary human monocyte-derived macrophages to demonstrate phenotypic and epigenetic stimulus specificity of innate immune memory six days after initial exposure. Quantification of cytokine production with single-molecule RNA imaging demonstrates stimulus-specific patterns of response to restimulation at the single cell level. Differential licensing of inflammatory transcription factors is associated with encoding of specificities in chromatin. Trained cells show stronger responses to secondary stimuli that are more similar to the initial stimulus they experienced, suggesting a functional role for these stimulus-specific memories. Rather than activating a universal training state, our findings demonstrate that different stimuli impart specific memories that generate distinct training phenotypes in macrophages.

Assessing spatial sequencing and imaging approaches to capture the molecular and pathological heterogeneity of archived cancer tissues.

Spatial transcriptomics (ST) offers enormous potential to decipher the biological and pathological heterogeneity in precious archival cancer tissues. Traditionally, these tissues have rarely been used and only examined at a low throughput, most commonly by histopathological staining. ST adds thousands of times as many molecular features to histopathological images, but critical technical issues and limitations require more assessment of how ST performs on fixed archival tissues. In this work, we addressed this in a cancer-heterogeneity pipeline, starting with an exploration of the whole transcriptome by two sequencing-based ST protocols capable of measuring coding and non-coding RNAs. We optimised the two protocols to work with challenging formalin-fixed paraffin-embedded (FFPE) tissues, derived from skin. We then assessed alternative imaging methods, including multiplex RNAScope single-molecule imaging and multiplex protein imaging (CODEX). We evaluated the methods' performance for tissues stored from 4 to 14 years ago, covering a range of RNA qualities, allowing us to assess variation. In addition to technical performance metrics, we determined the ability of these methods to quantify tumour heterogeneity. We integrated gene expression profiles with pathological information, charting a new molecular landscape on the pathologically defined tissue regions. Together, this work provides important and comprehensive experimental technical perspectives to consider the applications of ST in deciphering the cancer heterogeneity in archived tissues. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Super-photostable organic dye for long-term live-cell single-protein imaging.

Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime than conventional organic dyes without the requirement of any anti-photobleaching additives. PF555 is an asymmetric cyanine structure in which, on one side, the indole in the conventional Cyanine-3 is substituted with 3-oxo-quinoline. PF555 provides a powerful tool for long-term live-cell single-molecule imaging, as demonstrated by the imaging of the dynamic single-molecule interactions of the epidermal growth factor receptor with clathrin-coated structures on the plasma membrane of a live cell under physiological conditions.

Dynamic interactions of retroviral Gag condensates with nascent viral RNA at transcriptional burst sites: implications for genomic RNA packaging.

Retroviruses are responsible for significant pathology in humans and animals, including the acquired immunodeficiency syndrome and a wide range of malignancies. A crucial yet poorly understood step in the replication cycle is the recognition and selection of unspliced viral RNA (USvRNA) by the retroviral Gag protein, which binds to the psi (Ψ) packaging sequence in the 5' leader, to package it as genomic RNA (gRNA) into nascent virions. It was previously thought that Gag initially bound gRNA in the cytoplasm. However, previous studies demonstrated that the Rous sarcoma virus (RSV) Gag protein traffics transiently through the nucleus, which is necessary for efficient gRNA packaging. These data formed a strong premise for the hypothesis that Gag selects nascent gRNA at transcription sites in the nucleus, the location of the highest concentration of USvRNA molecules in the cell. In support of this model, previous studies using fixed cells infected with RSV revealed that Gag co-localizes with large USvRNA nuclear foci representing viral transcriptional burst sites. To test this idea, we used single molecule labeling and imaging techniques to visualize fluorescently-tagged, actively transcribing viral genomes, and Gag proteins in living cells. Gag condensates were observed in the nucleus, transiently co-localized with USvRNA at transcriptional burst sites, forming co- localized viral ribonucleoprotein complexes (vRNPs). These results support a novel paradigm for retroviral assembly in which Gag traffics to transcriptional burst sites and interacts through a dynamic kissing interaction to capture nascent gRNA for incorporation into virions.

Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging.

Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

Advanced Aberration-corrected STEM Techniques for Atomic Imaging of Zeolites-confined Single Molecules: From Ex situ to In situ

Advanced Aberration-corrected STEM Techniques for Atomic Imaging of Zeolites-confined Single Molecules: From Ex situ to In situ

Single-molecule imaging for investigating the transcriptional control.

Transcription is an essential biological process involving numerous factors, including transcription factors (TFs) which play a central role in this process by binding to their cognate DNA motifs. Although cells must tightly regulate the kinetics of factor association and dissociation during transcription, factor dynamics during transcription remain poorly characterized, primarily because of the reliance on ensemble experiments that average out molecular heterogeneity. Recent advances in single-molecule fluorescence imaging techniques have enabled the exploration of TF dynamics at unprecedented resolution. Findings on the temporal dynamics of individual TFs have challenged classical models and provided new insights into transcriptional regulation. Single-molecule imaging has also elucidated the assembly kinetics of transcription complexes. In this review, we describe the single-molecule fluorescence imaging methods widely used to determine factor dynamics during transcription. We highlight new findings on TF binding to chromatin, TF target search, and the assembly order of transcription complexes. Additionally, we discuss the remaining challenges in achieving a comprehensive understanding of the temporal regulation of transcription.

Single-molecule electrochemical imaging of 'split waves' in the electrocatalytic (EC') mechanism.

We describe a single-molecule electrochemical imaging strategy to study the electrocatalytic (EC') mechanism. Using the fluorescent molecule ATTO647N at extremely low concentrations as the substrate, we confirmed its catalytic reduction to a nonfluorescent form in the presence of the mediator phenazine methosulfate (PMS) by imaging and counting fluorescent molecules. Conventional electrochemical current in cyclic voltammetry would not have allowed us to infer the existence of an EC' process or the PMS-mediated ATTO647N reduction. Additionally, we observed shifts in the catalytic reduction potential of ATTO647N at various mediator concentrations, which agree with the theoretical predictions by Savéant. Our work offers a new perspective on connecting single-molecule EC' behaviors with the conventional ensemble EC' mechanism, both practically and theoretically.

Dynamics of spindle assembly and position checkpoints: Integrating molecular mechanisms with computational models.

Mitotic checkpoints orchestrate cell division through intricate molecular networks that ensure genomic stability. While experimental research has uncovered key aspects of checkpoint function, the complexity of protein interactions and spatial dynamics necessitates computational modeling for a deeper, system-level understanding. This review explores mathematical frameworks-from ordinary differential equations to stochastic simulations, which reveal checkpoint dynamics across multiple scales, encompassing models ranging from simple protein interactions to whole-system simulations with thousands of parameters. These approaches have elucidated fundamental properties, including bistable switches driving spindle assembly checkpoint (SAC) activation, spatial organization principles underlying spindle position checkpoint (SPOC) signaling, and critical system-level features ensuring checkpoint robustness. This study evaluates diverse modeling approaches, from rule-based models to chemical organization theory, highlighting their successful application in predicting protein localization patterns and checkpoint response dynamics validated through live-cell imaging. Contemporary challenges persist in integrating spatial and temporal scales, refining parameter estimation, and enhancing spatial modeling fidelity. However, recent advances in single-molecule imaging, data-driven algorithms, and machine learning techniques, particularly deep learning for parameter optimization, present transformative opportunities for improving model accuracy and predictive power. By bridging molecular mechanisms with system-level behaviors through validated computational frameworks, this review offers a comprehensive perspective on the mathematical modeling of cell cycle control, with practical implications for cancer research and therapeutic development.

Signal profiles and spatial regulation of β-arrestin recruitment through Gβ5 and GRK3 at the μ-opioid receptor

The μ-opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that mediates both analgesic effects and adverse effects of opioid drugs. Despite extensive efforts to develop a signal-biased drug, drugs with sufficiently reduced side effects have not been established, in part owing to lack of comprehensive signal transducer profiles of MOR. In this study, by profiling the activity of signal transducers including G proteins and GPCR kinases (GRKs), we revealed an unprecedented mechanism of selective GRK3 activation by Gβ5, leading to β-arrestin recruitment. By utilizing multiple genome-edited cell lines and functional assays, we found that oliceridine, an FDA-approved G-protein-biased agonist, selectively activates Gαz- and GRK3-mediated signaling. Notably, among the five Gβ subtypes, only Gβ5 distinguishes GRK3 from GRK2. Using single-molecule imaging, we found that GRK3 is recruited to the plasma membrane upon MOR agonist stimulation by Gβ1 and Gβ5, yet their interaction dynamics with GRK3 and mechanisms of action are different. Furthermore, particle diffusion analysis suggests that Gβ5 is enriched in confined membrane domains, through which GRK3 is recruited to the plasma membrane in a freely diffusible state, thereby allowing GRK3 to efficiently interact with MOR. These findings provide a mechanism by which MOR agonists rely on a specific Gα–Gβ–GRK axis to induce β-arrestin recruitment.

Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.

Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.

Pulling Forces Dampen Torsional Fluctuations of Actin Filaments and Reduce Cooperative Cofilin Binding.

A variety of potential biological roles of mechanical forces have been proposed in the field of cell biology. In particular, mechanical forces alter the mechanical conditions within cells and their environment, exerting a strong effect on the reorganization of the actin cytoskeleton. Single-molecule imaging studies have provided evidence that an actin filament may act as a mechanosensor. An increase in the tension within actin filaments causes changes in their conformation and affinity to their regulatory proteins. However, our current understanding of the molecular mechanisms of the tension sensing and the affinity change of regulatory proteins is still incomplete. In this study, we employed fluorescence polarization microscopy and magnetic tweezers to directly quantify the torsional fluctuations of single actin filaments and cofilin binding to the filament under several distinct mechanical conditions. When an actin filament was severed by scratching the filament with a pipette tip, the amplitude of twisting/torsional fluctuations and the rate of cooperative cofilin binding increased. On the other hand, when a piconewton force was applied to single actin filaments by magnetic tweezers, the amplitude of twisting fluctuations and the rate of cooperative cofilin binding decreased. This may be involved in the molecular mechanism behind the mechanical force-dependent severing of actin filaments in cells.

Live-Cell Single-Molecule Imaging of Influenza A Virus-Receptor Interaction.

Influenza A viruses are a major health care burden, and their biology has been intensely studied for decades. However, many details of virus infection are still elusive, requiring the development of refined and advanced technologies. Super-resolution microscopy allows thestudy of virus replication at the scale of an infecting virus, offering an exciting perspective on previously unseen mechanistic details of infection. Here we describe the materials and procedures required to perform single-molecule imaging of virus-receptor interaction in live cells. We further provide hints and tips on how to analyze and visualize the obtained datasets.

Distinguishing local photoinduced forces from global photoacoustic interactions in homodyne infrared photoinduced force microscopy

The rise of tip-based optical force microscopy has transformed nanoscale optical and chemical imaging, enabling sub-10 nm spatial resolution by integrating optical excitation with atomic force microscopy. Photoinduced force microscopy (PiFM) stands out as a key technique, with applications in single-molecule imaging, Raman spectroscopy, and near-field electromagnetic characterization. However, the contrast mechanisms underlying homodyne PiFM, particularly in the infrared regime, remain underexplored. This study provides novel insights into the interpretation of PiF signals, focusing on homodyne IR-PiFM. Contrary to previous assumptions, we demonstrate that the linear dependence of the PiF signal on sample thickness in homodyne mode originates from global photoacoustic forces rather than localized photothermal effects. By minimizing global interactions through power reduction and tip–sample proximity, we achieve a spatial resolution of 11 nm, comparable to heterodyne PiFM. Our findings reveal that both homodyne and heterodyne modes are fundamentally sensitive to tip-enhanced near-field optical intensity, with similar dependencies on sample thickness. This work advances the understanding of the contrast mechanism of PiFM and demonstrates that both homodyne and heterodyne modes can achieve high-resolution imaging, paving the way for broader applications in nanoscale optical and chemical analysis.

Super-Resolved Fluorescence Lifetime Imaging of Single Cy3 Molecules and Quantum Dots Using Time-Correlated Single Photon Counting with a Four-Pixel Fiber Optic Array Camera.

Time-resolved single molecule localization microscopy (TR-SMLM) with a 2 × 2 pixel fiber optic array camera was combined with time-correlated single photon counting (TCSPC) to obtain super-resolved fluorescence lifetime images of individual Cy3 dye molecules and individual colloidal CdSe/CdS/ZnS core/shell/shell semiconductor quantum dots (QDs). The characteristic blinking and bleaching behavior of the Cy3 and the blinking behavior of the QD emitters were used as distinguishing optical characteristics to isolate them and determine their centroid locations with spatial resolution below the optical diffraction limit. TCSPC was used to characterize the fluorescence lifetime and intensity corresponding to each emitter location. The mean centroid locations of the QDs could be determined with a precision of ∼1-4 nm, and the mean centroid locations of the Cy3 molecules could be determined with a precision of ∼2-9 nm, depending on the number of photons collected during the observation time. In a super-resolved fluorescence lifetime image with a single Cy3 dye molecule and a single QD separated by ∼34 nm, the two emitters were distinguished based on the average photon arrival times with respect to the excitation laser pulse observed during time intervals when only one emitter was in the on state, ∼6 ns for Cy3 and ∼17 ns for the QD. The mean distance between the two emitters was determined with a precision of ∼8 nm. The feasibility of using this super-resolved fluorescence lifetime imaging technique to investigate QD-dye complexes that use Förster resonance energy transfer (FRET) and/or electron transfer to form optical biosensors is discussed.