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Single-particle tracking techniques enable investigation of the complex functions and interactions of individual particles in biological environments. Many such techniques exist, each demonstrating trade-offs between spatiotemporal resolution, spatial and temporal range, technical complexity, and information content. To mitigate these trade-offs, we enhanced a confocal laser scanning microscope with an asynchronous read-out single-photon avalanche diode array detector. This detector provides an image of the particle’s emission, precisely reflecting its position within the excitation volume. This localization is utilized in a real-time feedback system to drive the microscope scanning mechanism and ensure the particle remains centered inside the excitation volume. As each pixel is an independent single-photon detector, single-particle tracking is combined with fluorescence lifetime measurement. Our system achieves 40 nm lateral and 60 nm axial localization precision with 100 photons and sub-millisecond temporal sampling for real-time tracking. Offline tracking can refine this precision to the microsecond scale. We validated the system’s spatiotemporal resolution by tracking fluorescent beads with diffusion coefficients up to 10  μ m 2 /s. Additionally, we investigated the movement of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the marker expressed on a membrane protein. We expect that this implementation will open other correlative imaging and tracking studies. Here, the authors upgrade a confocal laser scanning microscope with a single-photon array detector, achieving 40 nm lateral and 60 nm axial localisation precision with 100 photons and a sub-millisecond temporal sampling for real-time single-particle tracking with fluorescence lifetime measurement.

Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles. The wide variety of cellular processes involving biomolecular condensation makes their quantification a challenging task. Here, the authors present an integrated platform based on single-photon microscopy to study complex biomolecular processes.

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories. The authors developed an open-source, low-cost, multi-channel time-tagging module for fluorescence lifetime image scanning microscopy and correlation spectroscopy that can tag in parallel multiple single-photon events with 30 ps precision.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m 6 A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m 6 A downregulation. Notably, cells expressing mutant FUS were characterized by higher m 6 A levels suggesting a possible link between m 6 A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS. In Amyotrophic Lateral Sclerosis (ALS), formation of cytoplasmic inclusions by mutant protein aggregation is observed. Here the authors show that these inclusions dissolve faster when m 6 A RNA modification is inhibited in ALS cellular models.

Protein dynamics in the synaptic bouton are still not well understood, despite many quantitative studies of synaptic structure and function. The complexity of the synaptic environment makes investigations of presynaptic protein mobility challenging. Here, we present an in vitro approach to create a minimalist model of the synaptic environment by patterning synaptic vesicles (SVs) on glass coverslips. We employed fluorescence correlation spectroscopy (FCS) to measure the mobility of monomeric enhanced green fluorescent protein (mEGFP)-tagged proteins in the presence of the vesicle patterns. We observed that the mobility of all eleven measured proteins is strongly reduced in the presence of the SVs, suggesting that they all bind to the SVs. The mobility observed in these conditions is within the range of corresponding measurements in synapses of living cells. Overall, our simple, but robust, approach should enable numerous future studies of organelle-protein interactions in general.

Image Scanning Microscopy (ISM) enables good signal-to-noise ratio (SNR), super-resolution and high information content imaging by leveraging array detection in a laser-scanning architecture. However, the SNR is still limited by the size of the detector, which is conventionally small to avoid collecting out-of-focus light. Nonetheless, the ISM dataset inherently contains the axial information of the fluorescence emitters. We leverage this knowledge to achieve computational optical sectioning without sacrificing the conventional benefits of ISM. We invert the physical model to fuse the raw dataset into a single image with improved sampling, SNR. lateral resolution, and optical sectioning. We provide a complete theoretical framework and validate our approach with experimental images of biological samples acquired with a custom setup equipped with a single photon avalanche diode (SPAD) array detector. Furthermore, we generalize our method to other imaging techniques, such as multi-photon excitation fluorescence microscopy and fluoresce lifetime imaging. To enable this latter, we take advantage of the single-photon timing ability of SPAD arrays, accessing additional sample information. Our method outperforms conventional reconstruction techniques and opens new perspectives for exploring the unique spatio-temporal information provided by SPAD array detectors.

Image Scanning Microscopy (ISM) enables good signal-to-noise ratio (SNR), super-resolution and high information content imaging by leveraging array detection in a laser-scanning architecture. However, the SNR is still limited by the size of the detector, which is conventionally small to avoid collecting out-of-focus light. Nonetheless, the ISM dataset inherently contains the axial information of the fluorescence emitters. We leverage this knowledge to achieve computational optical sectioning without sacrificing the conventional benefits of ISM. We invert the physical model to fuse the raw dataset into a single image with improved sampling, SNR. lateral resolution, and optical sectioning. We provide a complete theoretical framework and validate our approach with experimental images of biological samples acquired with a custom setup equipped with a single photon avalanche diode (SPAD) array detector. Furthermore, we generalize our method to other imaging techniques, such as multi-photon excitation fluorescence microscopy and fluoresce lifetime imaging. To enable this latter, we take advantage of the single-photon timing ability of SPAD arrays, accessing additional sample information. Our method outperforms conventional reconstruction techniques and opens new perspectives for exploring the unique spatio-temporal information provided by SPAD array detectors.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m A downregulation. Notably, cells expressing mutant FUS were characterized by higher m A levels suggesting a possible link between m A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

The cellular accumulation of alpha-synuclein (aS) aggregates is a hallmark of several neurodegenerative diseases. Recent studies suggest that the aberrant transition of monomeric aS into solid-like aggregates may occur through an intermediate liquid-like state, where the protein partitions between dense and dilute phases. Although aS is not typically recognized as an RNA-binding protein, it can bind RNA under aggregation conditions, but its impact on aS liquid-like phases remains unexplored. Employing a combination of fluorescence spectroscopy techniques, we investigated aS dynamics in both phases in the presence of RNA. Our analysis revealed the formation of nanoclusters involved in initiating phase separation and uncovered heterogeneity within the dense phase, discovering that aS molecules exist in two distinct mobility states. Additionally, we demonstrated that RNA induces morphological changes and promotes the liquid-to-solid transition of aS dense phase. These findings underscore the active role of RNA in modulating aS phase transitions.

Neuronal communication relies on precisely maintained synaptic vesicle (SV) clusters, which assemble via liquid-liquid phase separation (LLPS). This process requires synapsins, the major synaptic phosphoproteins, which are known to bind actin. The reorganization of SVs, synapsins and actin is a hallmark of synaptic activity, but their interplay is still unclear. Here, we combined the reconstitution approaches, expansion microscopy, super-resolution imaging and cryo-electron tomography to dissect the roles of synapsin-SV condensates in the organization of the presynaptic actin cytoskeleton. Our data indicate that LLPS of synapsin initiates actin polymerization, allowing for SV:synapsin:actin assemblies to facilitate the mesoscale organization of SV clusters along axons mimicking the native presynaptic organization in both lamprey and mammalian synapses. Understanding the relationship between the actin network and synapsin-SVs condensates is an essential building block on a roadmap to unravel how coordinated neurotransmission along the axon enables circuit function and behavior.