Smart microscopy is transforming life sciences by automating experimental imaging workflows and enabling real-time adaptation based on feedback from images and other data streams. This shift increases throughput, improves reproducibility, and expands the functional capabilities of microscopes. However, the current landscape is highly fragmented. Academic researchers often develop custom solutions for specific scientific needs, while industry offerings are typically proprietary and tied to specific hardware. This diversity, while fostering innovation, also creates major challenges in interoperability, reproducibility, and standardization, which slows progress and adaption. This article presents a collaborative effort between academic and industry leaders to survey the current state of smart microscopy, highlight representative implementations, and identify common technical and organizational barriers. We propose a framework for greater interoperability based on shared standards, modular software design, and community-driven development. Our goal is to support collaboration across the field and lay the groundwork for a more connected, reusable, and accessible smart microscopy ecosystem. We conclude with a call to action for researchers, hardware developers, and institutions to join in building an open, interoperable foundation that will unlock the full potential of smart microscopy in life science research.

Super-resolution microscopy (SRM) has revolutionized life sciences by overcoming the diffraction limit, enabling the visualization of biological structures at the nanoscale. Expansion Microscopy (ExM) has emerged as a powerful and accessible technique that enhances resolution by physically enlarging the specimen. Importantly, the principles of ExM provide a unique foundation for combinations with SRM methods, pushing the boundaries of achievable resolution. This review explores the fundamental principles of ExM and examines its successful integration with various SRM techniques, including fluorescence fluctuation-based SRM, structured illumination microscopy, stimulated emission depletion microscopy, and single-molecule localization microscopy. We discuss the applications, strengths, limitations, and resolutions achieved by these combined approaches, providing a comprehensive guide for researchers to select the most suitable method for their specific scientific needs. Key considerations when combining ExM with SRM include the impact on fluorophores, the requirement for specialized buffers, and the challenges posed by the sensitivity of expanded hydrogels to temperature and hydration. Strategies to address these challenges, such as optimized labeling techniques and gel re-embedding, are discussed in detail. This review aims to assist researchers in navigating the rapidly evolving landscape of ExM and SRM, facilitating the development of tailored imaging pipelines to advance our understanding of biological systems at the nanoscale.

Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining both, we achieve images with a resolution of 25 nm in combination with diffraction-limited microscopes. Our step-by-step instructions include the expansion of cells and tissue samples, the optimization of multispectral microscopy parameters and the implementation of quality control metrics to minimize artifacts. We further cover the use of quantitative tools such as NanoJ-SQUIRREL, which enable the assessment of resolution improvements and image fidelity. We discuss key considerations for each stage, including sample preparation, image acquisition, computational processing and downstream analysis. Potential pitfalls and troubleshooting strategies are also addressed. This protocol can be used for imaging a variety of sample types with multiple fluorescent labels. With nanoscale spatial resolution and molecular specificity, expansion-enhanced super-resolution radial fluctuations microscopy provides a flexible, accessible approach for investigating cellular ultrastructure, protein localization and interaction networks, suitable for applications in cell biology, histopathology and biomedical research. The procedure requires 3–4 d to complete, involving ~7–9 h of total bench, imaging and processing time and only requires basic expertise in tissue handling, molecular and cell biology, and microscopy. The combination of expansion microscopy with enhanced super-resolution radial fluctuations analysis enhances the image resolution of a conventional microscope to 25 nm in clinical and experimental samples.

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the method’s potential and practical limitations. ### Competing Interest Statement The authors have declared no competing interest. European Union, https://ror.org/019w4f821, 101180631, 101099654 European Research Council, 101001332 European Molecular Biology Organization, EMBO-2020-IG-4734 Agence Nationale de la Recherche, ANR-21-ESRE-0002 Fundação para a Ciência e Tecnologia, CEECIND/01480/2021, UIDB/04612/2020, UIDP/04612/2020 Biotechnology and Biological Sciences Research Council, BB/X511092/1, UKRI715 Royal Society, RGF/EA/181021

Optical microscopy is an indispensable tool in life sciences research, but conventional techniques require compromises between imaging parameters like speed, resolution, field-of-view, and phototoxicity. To overcome these limitations, data-driven microscopes incorporate feedback loops between data acquisition and analysis. This review overviews how machine learning enables automated image analysis to optimise microscopy in real-time. We first introduce key data-driven microscopy concepts and machine learning methods relevant to microscopy image analysis. Subsequently, we highlight pioneering works and recent advances in integrating machine learning into microscopy acquisition workflows, including optimising illumination, switching modalities and acquisition rates, and triggering targeted experiments. We then discuss the remaining challenges and future outlook. Overall, intelligent microscopes that can sense, analyse, and adapt promise to transform optical imaging by opening new experimental possibilities.

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.

The node of Ranvier is the key element in saltatory conduction along myelinated axons, but its specific protein organization remains elusive in the human species. To shed light on nanoscale anatomy of the human node of Ranvier in health and disease, we assessed human nerve biopsies of patients with polyneuropathy by super-resolution fluorescence microscopy. We applied direct stochastic optical reconstruction microscopy (dSTORM) and supported our data by high-content confocal imaging combined with deep learning-based analysis. As a result, we revealed a ∼ 190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules in human peripheral nerves. In patients with polyneuropathy, periodic distances increased at the paranodal region of the node of Ranvier, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. High content analysis showed that such paranodal disorganization occurred especially in acute and severe axonal neuropathy with ongoing Wallerian degeneration and related cytoskeletal damage. We provide nanoscale and protein-specific evidence for the prominent, but vulnerable role of the node of Ranvier for axonal integrity. Furthermore, we show that super-resolution imaging can identify, quantify and map elongated periodic protein distances and protein interaction in histopathological tissue samples. We thus introduce a promising tool for further translational applications of super resolution microscopy.

Understanding the structure of supramolecular complexes provides insight into their functional capabilities and how they can be modulated in the context of disease. Super-resolution microscopy (SRM) excels in performing this task by resolving ultrastructural details at the nanoscale with molecular specificity. However, technical limitations, such as underlabelling, preclude its ability to provide complete structures. Single-particle analysis (SPA) overcomes this limitation by combining information from multiple images of identical structures and producing an averaged model, effectively enhancing the resolution and coverage of image reconstructions. This review highlights important studies using SRM-SPA, demonstrating how it broadens our knowledge by elucidating features of key biological structures with unprecedented detail.

To shed light on nanoscale pathologies in patients with polyneuropathy, we assessed human nerve biopsies by super-resolution fluorescence microscopy. We focused on both physiological protein arrangement and pathological ultrastructural changes at the node of Ranvier, a crucial region of the peripheral myelinated axon. Direct stochastic optical reconstruction microscopy (dSTORM) revealed a ∼190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules. Periodic distances increased at the paranodal region of the node of Ranvier in patients with polyneuropathy, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis of human nerve biopsies revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. Super-resolution dual-color colocalization data was supported by high-content confocal imaging combined with deep learning-based analysis, indicating that paranodal elongation occurs especially in acute and severe axonal neuropathy, as a possible correlate of Wallerian degeneration and related cytoskeletal damage. Our findings show that super-resolution imaging can identify, quantify and map elongated periodic protein distances in peripheral nerve biopsies for pathophysiological studies and direct implications for diagnostic assessment.Graphical abstractPathological alterations of ultrastructural protein arrangement at the paranodal region of the node of Ranvier in polyneuropathy.Super-resolution microscopy allows for assessing ultrastructural protein arrangement and pathological alterations in patients with polyneuropathy. In human healthy nodes, axoglial and axoskeletal proteins follow a 190 nm periodic arrangement (left). In pathologically altered nodes (right), periodic protein distances of axonal ß2-spectrin elongate, in combination with elongation and partial loss of the axoglial complex of Caspr-1 and neurofascin-155. The axoglial complex itself colocalizes closely even in pathologically altered nodes. A complete loss of the axoglial complex could be the ultrastructural correlate of the detachment of paranodal myelin loops. Scale bar 2 μm.

We propose symmetrical cationic trimethine cyanine dyes with β-substituents in the polymethine chain based on modified benzothiazole and benzoxazole heterocycles as probes for the detection and visualization of live and fixed cells by fluorescence microscopy. The spectral-luminescent properties of trimethine cyanines have been characterized for free dyes and in the presence of nucleic acids (NA) and globular proteins. The studied cyanines are low to moderate fluorescent when free, but in the presence of NA, they show an increase in emission intensity up to 111 times; the most pronounced emission increase was observed for the dyes T-2 in the presence of dsDNA and T-1 with RNA. Spectral methods showed the binding of all dyes to nucleic acids, and different interaction mechanisms have been proposed. The ability to visualize cell components of the studied dyes has been evaluated using different human cell lines (MCF-7, A2780, HeLa, and Hs27). We have shown that all dyes are cell-permeant staining nucleus components, probably RNA-rich nucleoli with background fluorescence in the cytoplasm, except for the dye T-5. The dye T-5 selectively stains some structures in the cytoplasm of MCF-7 and A2780 cells associated with mitochondria or lysosomes. This effect has also been confirmed for the normal type of cell line-human foreskin fibroblasts (Hs27). The costaining of dye T-5 with MitoTracker CMXRos Red demonstrates specificity to mitochondria at a concentration of 0.1 μM. Colocalization analysis has shown signals overlapping of dye T-5 and MitoTracker CMXRos Red (Pearson's Coefficient value = 0.92 ± 0.04). The photostability study shows benzoxazole dyes to be up to ∼7 times more photostable than benzothiazole ones. Moreover, studied benzoxazoles are less cytotoxic at working concentrations than benzothiazoles (67% of cell viability for T-4, T-5 compared to 12% for T-1, and ∼30% for T-2, T-3 after 24 h). Therefore, the benzoxazole T-4 dye is proposed for nucleic acid detection in vitro and intracellular fluorescence imaging of live and fixed cells. In contrast, the benzoxazole dye T-5 is proposed as a good alternative to commercial dyes for mitochondria staining in the green-yellow region of the spectrum.

Interrogating platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke, or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed αIIbβ3 receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of αIIbβ3 clusters in resting platelets, as well as in activated platelets, indicating that they contribute to the rapid platelet response during platelet clotting.

The unicellular eukaryote Saccharomyces cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi - including yeast - and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts is a crucial step to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae , with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae .

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.

The maker movement has reached the optics labs, empowering researchers to actively create and modify microscope designs and imaging accessories. 3D printing has especially had a disruptive impact on the field, as it entails an accessible new approach in fabrication technologies, namely additive manufacturing, making prototyping in the lab available at low cost. Examples of this trend are taking advantage of the easy availability of 3D printing technology. For example, inexpensive microscopes for education have been designed, such as the FlyPi (1). Also, the highly complex robotic microscope OpenFlexure (2) represents a clear desire for the democratisation of this technology. 3D printing facilitates new and powerful approaches to science and promotes collaboration between researchers, as 3D designs are easily shared. This holds the unique possibility to extend the open-access concept from knowledge to technology, allowing researchers from everywhere to use and extend model structures. Here we present a review of additive manufacturing applications in microscopy, guiding the user through this new and exciting technology and providing a starting point to anyone willing to employ this versatile and powerful new tool.

The maker movement has reached the optics labs, empowering researchers to create and modify microscope designs and imaging accessories. 3D printing has a disruptive impact on the field, improving accessibility to fabrication technologies in additive manufacturing. This approach is particularly useful for rapid, low-cost prototyping, allowing unprecedented levels of productivity and accessibility. From inexpensive microscopes for education such as the FlyPi to the highly complex robotic microscope OpenFlexure, 3D printing is paving the way for the democratization of technology, promoting collaborative environments between researchers, as 3D designs are easily shared. This holds the unique possibility of extending the open-access concept from knowledge to technology, allowing researchers everywhere to use and extend model structures. Here, it is presented a review of additive manufacturing applications in optical microscopy for life sciences, guiding the user through this new and exciting technology and providing a starting point to anyone willing to employ this versatile and powerful new tool.

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting. Blot clots can save lives when stopping blood loss after an injury, but also kill when blocking a major vessel. The highly abundant and densely distributed GPIIb/IIIa receptors are one reason why the underlying key distributions and interactions, in particular the relevance of integrin clustering, are not fully understood. Such dense receptor scenarios are difficult to assess even by super-resolution fluorescence microscopy. Here, we quantify various receptor interactions, and demonstrate that expansion microscopy can pinpoint such challenging interactions where conventional methods fail in such dense 3D scenarios with highly abundant receptors. We successfully combine dual-color expansion and confocal microscopy with colocalization analysis and assess platelet receptor organization without the need of a super-resolution microscope. We reveal that GPIIb/IIIa receptors are organized in pre-formed clusters in resting platelets - a pattern that orchestrates platelet clotting. We show that 4x expansion is most straight-forward for platelet imaging, while 10x expansion provides highest precision which turned out to be absolutely necessary for the most difficult of the scenarios described here. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/431449v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1963a29org.highwire.dtl.DTLVardef@1c48dd9org.highwire.dtl.DTLVardef@13cd7ceorg.highwire.dtl.DTLVardef@17c4a55_HPS_FORMAT_FIGEXP M_FIG C_FIG Nonstandard Abbreviations and AcronymsGPIX: glycoprotein IX ExM: expansion microscopy Key PointsO_LIMapping of the very dense, highly abundant platelet receptor landscape requires 10x Expansion Microscopy C_LIO_LIExM reveals that GPIIb/IIIa receptors are organized in pre-formed clusters in resting platelets. C_LI

This is a depository for two single molecule localisation microscopy datasets of nuclear pore complex (NPC) structures for single particle averaging. The data was published in: Heil, H.S., Schreiber, B., Götz, R. et al. Sharpening emitter localisation in front of a tuned mirror. Light Sci Appl 7, 99 (2018). https://doi.org/10.1038/s41377-018-0104-z Both datasets have two different levels of localisation precision as one is a conventional STORM experiment and the second a mirror-enhanced STORM experiment. A detailed description of the sample preparation and imaging conditions can be found in the related publication. In short the NPC structures are placed on the surface of a glas coverslip or nano-mirror coated coverslip by manual isolation and spreading of nuclear envelopes from xenopus laevis oocytes, fixed and stained by indirect immunolabeling. The primary antibody targets GP210, the secondary F(ab')2 fragment is conjugated with Alexa Fluor 647. In this depository I'm providing the raw images data, localisation data and super-resolved reconstruction for the two experiments, as well as the localisation data and super-resolved reconstruction of single NPC rings. I'm also providing a MatLab script that allows to select single NPC positions in the super-resolved image and export the localization data of the single NPC ROI: P01_ImageAlignment_PickElements.m Information about the dataset is also available here: NPC Image Alignment Dataset_Info.pdf. Image parameters: 102 nm pixel size, EM Gain 100, Photoelectrons per A/D count: 15.01 Column structure of the localisation text files: Id,Frame, x [nm], y [nm], sigma [nm], intensity [photon], offset [photon], bkgstd [photon], chi2, Uncertainty [nm], detections Files: NPCData_glass_EPI.tif -> NPC on glass coverslip, low power EPI illumination, widefield image, 20 ms exposure NPCData_glass_STORM.tif -> NPC on glass coverslip, high power EPI illumination, 5 ms exposure, 20000 frames NPCData_glass_STORM_loc.csv -> ThunderSTORM Localisation data of NPCData_glass_STORM.tif, parameters specified NPCData_glass_STORM_loc-protocol.txt NPCData_glass_STORM_20xNormalizedGaussian.tif -> 20x Nomalized Gaussian reconstruction of localization data from NPCData_glass_STORM.tif (ThunderSTORM), pixelsize 5.1 nm NPCData_glass_STORM_singleRings.zip -> Localisation data and 20x 20x Nomalized Gaussian reconstruction of single NPC ROIs picked out of the NPCData_glass_STORM dataset, ROI size is 240*240 nm2 NPCData_nanomirror_EPI.tif -> NPC on nanomirror coated coverslip, low power EPI illumination, widefield image, 20 ms exposure NPCData_nanomirror_STORM.tif -> NPC on nanomirror coated coverslip, high power EPI illumination, 5 ms exposure, 20000 frames NPCData_nanomirror_STORM_loc.csv -> ThunderSTORM Localisation data of NPCData_nanomirror_STORM.tif, parameters specified NPCData_nanomirror_STORM_loc-protocol.txt NPCData_nanomirror_STORM_20xNormalizedGaussian.tif -> 20x Nomalized Gaussian reconstruction of localisation data from NPCData_nanomirror_STORM.tif (ThunderSTORM), pixelsize 5.1 nm NPCData_nanomirror_STORM_singleRings.zip -> Localisation data and 20x 20x Nomalized Gaussian reconstruction of single NPC ROIs picked out of the NPCData_nanomirror_STORM dataset, ROI size is 240*240 nm2

Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.