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DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning.

Recent developments in clearing and microscopy enable 3D imaging with cellular resolution up to the whole organ level. These methods have been used extensively in neurobiology, but their uptake in other fields has been much more limited. Application of this approach to the human heart and effective use of the data acquired present challenges of scale and complexity. Four interlinked issues need to be addressed: 1) efficient clearing and labelling of heart tissue, 2) fast microscopic imaging of human-scale samples, 3) handling and processing of multi-terabyte 3D images, and 4) extraction of structural information in computationally tractable structure-based models of cardiac function. Preliminary studies show that each of these requirements can be achieved with the appropriate application and development of existing technologies.

Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.

Localization-based super-resolution microscopy techniques such as PALM, STORM, and PAINT are increasingly critical tools for biological discovery. These methods generate lists of single fluorophore positions that capture nanoscale structural details of subcellular organisation, but to develop biological insight, we must post-process and visualize this data in a meaningful way. A large number of algorithms have been developed for localization post-processing, transforming point data into representations which approximate traditional microscopy images, and performing specific quantitative analysis directly on points. Implementations of these algorithms typically stand in isolation, necessitating complex workflows involving multiple different software packages. Here we present PYMEVisualize, an open-source tool for the interactive exploration and analysis of 3D, multicolor, single-molecule localization data. PYMEVisualize brings together a broad range of the most commonly used post-processing, density mapping, and direct quantification tools in an easy-to-use and extensible package. This software is one component of the PYthon Microscopy Environment (python-microscopy.org), an integrated application suite for light microscopy acquisition, data storage, visualization, and analysis built on top of the scientific Python environment.

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 μm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

Schroeder et al. quantitatively evaluate ER architecture in live cells at a ∼50-nm resolution through stimulated emission depletion (STED) microscopy. The ER is not limited to uniform sheets and tubules; they observe dynamic, nanoscale-size holes in ER sheets termed “nanoholes,” and they characterize the effects of perturbations of reticulons, Climp63, and the microtubule cytoskeleton on ER membrane nanostructures., The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique called nested-loop ensemble PSF fitting. This approach enables extraction of the size of cellular features and the PSF width in fixed-cell and live-cell images without relying on beads or precalibration. Nested-loop ensemble PSF fitting accurately recapitulates microtubule diameter from stimulated emission depletion images and can measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. Our algorithm has been implemented as a plugin for the PYthon Microscopy Environment, a freely available and open-source software.

A new strategy is used to reveal nanometer-scale single-molecule dynamics within protein assemblies to study the eisosome: a stable, linear cluster of proteins on the yeast plasma membrane. The BAR-domain protein Pil1p binds and unbinds at eisosome ends, supporting a new model of eisosomes as dynamic oligomeric filaments., Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. Here we adapt a single-molecule strategy to perform single-molecule recovery after photobleaching (SRAP) within dense macromolecular assemblies to reveal and characterize binding and unbinding dynamics within such assemblies. We applied this method to study the eisosome, a stable assembly of BAR-domain proteins on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the main eisosome BAR-domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, localized recruitment of new Pil1p molecules with ∼30-nm precision. Comparing our data to computer simulations, we show that Pil1p exchange occurs specifically at eisosome ends and not along their core, supporting a new model of the eisosome as a dynamic filament. This result is the first direct observation of any BAR-domain protein dynamics in vivo under physiological conditions consistent with the oligomeric filaments reported from in vitro experiments.

The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97. Unexpectedly, we observed that both α and βSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst βSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, βSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with βSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, βSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in other major PSD proteins (e.g., Shank, PSD95, and SAP102), this alternative splicing may result in individual PSD proteins having divergent functional and structural roles in both physiological and pathophysiological synaptic states.

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time-and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

Actin is required for clathrin-mediated endocytosis (CME) in yeast. Experimental observations indicate that this actin assembly generates force to deform the membrane and overcome the cell’s high turgor pressure, but the precise molecular details remain unresolved. Based on previous models, we predicted that actin at endocytic sites continually polymerize and disassemble, turning over multiple times during an endocytic event. Here we applied single-molecule speckle tracking in live fission yeast to directly measure this predicted turnover within the CME assembly for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins’ motions. These results indicate that rapid and continuous turnover is a key feature of the endocytic machinery and suggest revising quantitative models of force production.

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.One Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

DNA-PAINT is an increasingly popular super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and very slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. We present a fluorogenic DNA-PAINT probe that solves these problems and demonstrate 3D imaging without the need for optical sectioning and a 26-fold increase in imaging speed over regular DNA-PAINT.

Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered. From the lipid release times we computed pore openness, the fraction of time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested, SNARE-mediated and nonspecifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels, SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t-SNAREs to the fusion site, consistent with t-SNAREs being preclustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion-pore bending energy, and indirectly by concentrating several SNAREs into individual fusion events.

dSTORM super-resolution imaging relies on switching buffers to enable dye molecules to enter and exit a metastable dark state. Current buffers have a very limited shelf life of approximately 1 day and poorly match sample refractive index, impacting negatively on measurement reproducibility and image fidelity. We present a buffer based on chemical, rather than enzymatic, oxygen scavenging which exhibits dramatically improved stability, switching speed, contrast, and index matching.

International audience; While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.

Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and the cytoplasm. They impose a diffusion barrier to macromolecules and enable the selective transport of nuclear transport receptors with bound cargo. The underlying mechanisms that establish these permeability properties remain to be fully elucidated, but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat domains of different types, such as FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining how the FG network contributes to the barrier and transport properties of the NPC, it remains unknown whether the number and/or the spatial positioning of different FG-domains along a cylindrical, ~40 nm diameter transport channel contributes to their collective properties and function. To begin to answer these questions, we have used DNA origami to build a cylinder that mimics the dimensions of the central transport channel and can house a specified number of FG-domains at specific positions with easily tunable design parameters, such as grafting density and topology. We find the overall morphology of the FG-domain assemblies to be dependent on their chemical composition, determined by the type and density of FG-repeat, and on their architectural confinement provided by the DNA cylinder, largely consistent with here presented molecular dynamics simulations based on a coarse-grained polymer model. In addition, high-speed atomic force microscopy reveals local and reversible FG-domain condensation that transiently occludes the lumen of the DNA central channel mimics, suggestive of how the NPC might establish its permeability properties.

Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.

The contractile properties of cardiac myocytes depend on the calcium (Ca2 +) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~ 57% of the perimeter of each myofibril. The average size of internal couplons is ~ 63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥ 80% of the total RyRs were detected in clusters containing ≥ 100 receptors. ~ 56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca2 +-induced Ca2 + release (100 nm) and were grouped into ‘superclusters’. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca2 + levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2 clusters/μm3) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca2 + signalling.

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect localization precision and accuracy. We present a simplified sCMOS non-uniform noise model that incorporates pixel specific read-noise, offset and sensitivity variation. Using this model we develop a new weighted least squared (WLS) fitting method designed to remove the effect of sCMOS pixel non-uniformities. Simulations with the sCMOS noise model, performed to test under which conditions sCMOS specific localization corrections are required, suggested that pixel specific offsets should always be removed. In many applications with thick biological samples photon fluxes are sufficiently high that corrections of read-noise and sensitivity correction may be neglected. When correction is required, e.g. during fast imaging in thin samples, our WLS fit procedure recovered the performance of an equivalent sensor with uniform pixel properties and the fit estimates also attained the Cramer-Rao lower bound. Experiments with sub-resolution beads and a DNA origami test sample confirmed the results of the simulations. The WLS localization procedure is fast to converge, compatible with 2D, 3D and multi-emitter localization and thus provides a computationally efficient sCMOS localization approach compatible with most SMLM modalities.

Besides its well-established role in generating COPI vesicles by recruiting coatomer at the Golgi, the small GTPase ARF1 is additionally involved in the formation of anterograde and retrograde tubular carriers at the Golgi., Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

The cardiac ryanodine receptor (RyR) plays a central role in the control of contractile function of the heart. In cardiac ventricular myocytes RyRs and associated Ca(2+) handling proteins, including membrane Ca(2+) channels, Ca(2+) pumps and other sarcolemmal and sarcoplasmic reticulum proteins interact to set the time course and amplitude of the electrically triggered cytosolic Ca(2+) transient. It has become increasingly clear that protein distribution and clustering on the nanometer scale is critical in determining the interaction of these proteins and the resulting properties of cardiac Ca(2+) handling. Such intricate near-molecular scale detail cannot be visualized with conventional fluorescence microscopy techniques (e.g. confocal microscopy) but it has recently become accessible with optical super-resolution techniques. These techniques retain the advantages of fluorescent marker technology, i.e. high specificity and excellent contrast, but have a spatial resolution approaching 10nm, i.e. objects not much further apart than 10nm can be distinguished, previously only attainable with electron microscopy. We review the use of these novel imaging techniques for the study of protein distribution in cardiac ventricular myocytes and discuss technical considerations as well as recent findings using super-resolution imaging. An emphasis is on single molecule localization based super-resolution approaches and their use to reveal the complexity of RyR cluster morphology, placement and relationship to other excitation-contraction coupling proteins. Super-resolution imaging approaches have already demonstrated their utility for the study of cardiac structure-function relationships and we anticipate that their use will rapidly increase and help improve our understanding of cardiac Ca(2+) regulation.

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. We have developed a novel, broadly applicable, fluorescent labeling and imaging protocol, called Single-molecule Recovery After Photobleaching (SRAP), which allowed us to reveal the heterogeneous dynamics of the eisosome, a multi-protein structure on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the core eisosome BAR domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, recorded the binding of individual Pil1p molecules with ~20 nm precision. Further analysis of these dynamic structures and comparison to computer simulations allowed us to show that Pil1p exchange is spatially heterogeneous, supporting a new model of the eisosome as a dynamic filament.Abbreviations usedBARBin/Amphiphysin/Rvs domainFRAPFluorescence Recovery After PhotobleachingmEGFPmonomeric Enhanced Green Fluorescent ProteinSiR647silicon-rhodamine 647TIRFTotal Internal Reflection Fluorescence

Signalling nanodomains requiring close contact between the plasma membrane and internal compartments, known as ‘junctions’, are fast communication hubs within excitable cells such as neurones and muscle. Here, we have examined two transgenic murine models probing the role of junctophilin-2, a membrane-tethering protein crucial for the formation and molecular organisation of sub-microscopic junctions in ventricular muscle cells of the heart. Quantitative single-molecule localisation microscopy showed that junctions in animals producing above-normal levels of junctophilin-2 were enlarged, allowing the re-organisation of the primary functional protein within it, the ryanodine receptor (RyR; in this paper, we use RyR to refer to the myocardial isoform RyR2). Although this change was associated with much enlarged RyR clusters that, due to their size, should be more excitable, functionally it caused a mild inhibition in the Ca2+ signalling output of the junctions (Ca2+ sparks). Analysis of the single-molecule densities of both RyR and junctophilin-2 revealed an ∼3-fold increase in the junctophilin-2 to RyR ratio. This molecular rearrangement is compatible with direct inhibition of RyR opening by junctophilin-2 to intrinsically stabilise the Ca2+ signalling properties of the junction and thus the contractile function of the cell.

Haplodeficiency of the microglia gene TREM2 increases risk for late-onset Alzheimer's disease (AD) but the mechanisms remain uncertain. To investigate this, we used high-resolution confocal and super-resolution (STORM) microscopy in AD-like mice andhuman AD tissue. We found that microglia processes, rich in TREM2, tightly surround early amyloid fibrils and plaques promoting their compaction and insulation. In Trem2- or DAP12-haplodeficient mice and in humans with R47H TREM2 mutations, microglia had a markedly reduced ability to envelop amyloid deposits. This led to an increase in less compact plaques with longer and branched amyloid fibrils resulting in greater surface exposure to adjacent neurites. This was associated with more severe neuritic tau hyperphosphorylation and axonal dystrophy around amyloid deposits. Thus, TREM2 deficiency may disrupt the formation of a neuroprotective microglia barrier that regulates amyloid compaction and insulation. Pharmacological modulation of this barrier could be a novel therapeutic strategy for AD.

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution., The intracellular applications of STED microscopy are limited by the availability of dyes. Here the authors develop a two-colour labelling strategy based on SiR and ATTO590 dyes, and apply their strategy to image various subcellular membrane compartments.

Sections from human eye tissue were analyzed with Structured Illumination Microscopy (SIM) using a specially designed microscope setup. In this microscope the structured illumination was generated with a Twyman-Green Interferometer. This SIM technique allowed us to acquire light-optical images of autofluorophore distributions in the tissue with previously unmatched optical resolution. In this work the unique setup of the microscope made possible the application of SIM with three different excitation wavelengths (488, 568 and 647 nm), thus enabling us to gather spectral information about the autofluorescence signal.

Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional (3D) localisation microscopy by engineering of the point-spread function (PSF) of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions (standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively). The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications.

A model based method for the accurate quantification of the 3D structure of fluorescently labelled cellular objects similar in size to the optical resolution limit is presented. This method is applied to both simulated confocal images of chromatin structures and to real confocal data obtained on a Fluorescence in situ Hybridization (FISH) labelled gene domain. The model assumes that the object is composed of a small number of discrete points which are convolved with the microscope point spread function to give the image. Fitting this model to image data results in a method to assess object structure which is accurate, shows a low bias, and does not require user intervention or the potentially subjective setting of a threshold.

We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often

The hypothesis that distinct chromatin domains expand and are remodelled differently when they undergo transcription, replication or cell cycle processes is well accepted. The condensation changes by which chromosomes are transformed at the metaphase–interphase transition are especially interesting and therefore extensively studied by light microscopy; however, quantitative information of the size on specific small chromatin domains during the cell cycle is scarce. In this respect, a serious problem is the determination of structural features close to the resolution limit. In this report we use a novel approach to quantify the lateral extent of the 8q24/c-myc gene domain and the centromeric region of chromosome 8 in doubly labelled normal human foreskin fibroblasts using confocal laser scanning microscopy (CLSM). The domains were analysed in both metaphase spreads and interphase nuclei. These high precision measurements revealed a somewhat smaller (few 10s of nm) lateral extension of the centromere region in metaphase compared to interphase. Surprisingly, within the same cells the lateral extension of the 8q24/c-myc region was significantly smaller in interphase than in metaphase. For comparison the centromere size was more condensed in metaphase than in interphase. This implies a different folding behaviour for specific chromatin domains with opposite condensation behaviour.

Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the ‘point-like’ components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling (‘spectral signatures’). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th–1/10th of the exciting wavelength.