Stochastic Reconstruction Microscopy Research Articles

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13-100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Disruption of the PDZ domain–binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis, and reward motivation

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain–binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM).

Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary data are available at Bioinformatics online.

Investigation of the Spatio-Temporal Dynamics of GLUT4 in Cardiomyocytes

Individuals with Type 2 diabetes mellitus (T2DM) are at significantly increased risk of cardiovascular disease and the development of diabetic cardiomyopathy. One of the major hallmarks of T2DM is a significant decrease in myocardial glucose uptake. Glucose transport in fat, skeletal and cardiac muscle is mediated by the glucose transporter GLUT4 that is mobilized to the cell surface from intracellular stores in response to insulin. Recently, total internal reflection microscopy was used to reveal that insulin not only regulates GLUT4 translocation to the plasma membrane (PM) but also its distribution within the PM. The emergence of super resolution microscopy techniques has allowed cell biologists to examine characteristics of molecules such as GLUT4 organization in the PM at the single-molecule level beyond the diffraction limit of conventional light microscopy. Stochastic reconstruction microscopy (STORM) investigated GLUT4 dispersal in basal and insulin-stimulated adipocytes and suggested that dispersal of GLUT4 within the plasma membrane was a key action of insulin. In my project, I have applied STORM with state of the art nanobody technology to investigate spatio-temporal dynamics of GLUT4 dispersal and significantly reduce fluorescent label linkage error using cardiomyocytes as a model system. Super resolution images showed that GLUT4 clusters are present in the basal state and retained at the plasma membrane fusion site; by contrast insulin stimulated a different kind of exocytosis that dispersed GLUT4 to the PM consistent with the findings of previous studies. GLUT4 trafficking is more complex in muscle tissues and therefore our ongoing work is focused on elucidating GLUT4 dispersal in cardiomyocytes. In particular key research questions include does exercise regulate GLUT4 dispersal, how specific is the dispersal process and it is impaired in conditions of insulin resistance.

Polymodal Mechanism for TWIK-Related K+ Channel Inhibition by Local Anesthetic.

Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.

Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopy

Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms including blindness. The detailed structures of these organelles and the functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We have combined cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of mouse retinas. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening space. Within this framework we report the spatial distributions of intraflagellar transport proteins and proteins of the BBSome complex in WT and Bardet-Biedl syndrome model mice.

Self-Assembly of Mesoscopic Peptide Surfactant Fibrils Investigated by STORM Super-Resolution Fluorescence Microscopy.

Super-resolution fluorescence microscopy, specifically stochastic reconstruction microscopy (STORM), and atomic force microscopy (AFM) were used to image the self-assembly processes of the peptide surfactant I3K. The peptide surfactants self-assembled into giant helical fibrils with diameters between 5 and 10 nm with significant helical twisting. The resolution of the STORM images was 30 nm, calculated using the Fourier ring correlation method. STORM compares favorably with AFM for the calculation of contour lengths (∼6 μm) and persistence lengths (10.1 ± 1.2 μm) due to its increased field of view (50 μm), and its ability to image bulk morphologies away from surfaces under ambient solution conditions. Two-color STORM experiments were performed to investigate the dynamic process of self-assembly after mixing of two separately labeled samples, and the results revealed the formation of long nanofibers via end-to-end connections of short ones. No evidence was found for significant monomer exchange between the samples, and the self-assembled structures were very stable and long-lived.

Storm-Lite: An Inexpensive Tool to Demonstrate Stochastic Super Resolution Microscopy Techniques in the Classroom

Studying sub-cellular structures is central to biophysics research. However, diffraction limits the resolution in light microscopy to a few hundred nanometers and the finest structures of interest can no longer be resolved. To address this obstacle, a variety of “super-resolution” microscopy techniques have been developed. These techniques make use of the photophysics of fluorescent molecules to increase the resolution to several tens of nanometers.We describe here a device that will allow classrooms worldwide to easily simulate super-resolution microscopy techniques such as photoactivation localization microscopy (PALM) and stochastic reconstruction microscopy (STORM). These techniques are extremely expensive and cumbersome to demonstrate in a classroom setting, limiting students’ exposure to them. By contrast, our device is inexpensive, lightweight, and portable, using only an LED array to simulate the fluorophores, frosted glass to simulate the resolution limit, and a camera and user interface developed for the Raspberry Pi to process the images. All aspects of the device and software design will be made publically available in the hopes that others will build and use this setup in their teaching of super resolution techniques at colleges and universities worldwide.

Development of Correlative Cryo-soft X-ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells.

We have developed a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identified and localized aggregations of carbon-rich materials. STORM identified specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging.

Sequential superresolution imaging of multiple targets using a single fluorophore.

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.

Correlative iPALM and Platinum Replica Electron Tomography Pinpoints Endocytic Proteins on the Mammalian Cell Cortex in 3D

Super-resolution localization microscopy techniques like photoactivated localization microscopy (PALM) and stochastic reconstruction microscopy (STORM) can be used to locate proteins in cells to a resolution of 10 nm which, along with other recent super-resolution techniques, has revolutionized the field of fluorescence microscopy. However, even super-resolution fluorescence microscopy lacks the ability to see detailed structure surrounding the proteins in the way that electron microscopy (EM) has been able to do for decades. Here, we combine interferometric PALM (iPALM) with EM tomography of mammalian cell cortex platinum replicas to localize clathrin-associated proteins on clathrin coated structures in three dimensions at a resolution ∼20 nm. We specifically pinpoint the position of clathrin and epsin on clathrin coated pits and find that epsin is positioned on the outer perimeter of clathrin coated plaques and along the height of the sidewalls of clathrin-coated pits. We continue to enhance this method with changes in sample preparation and we are using the method to study the nanoscale organization of membrane-associated proteins in multiple different mammalian cell lines.

Role of Ankyrin-G in the Preservation of Intercellular Junctions. Visualization in the Nanoscale Using Direct Stochastic Optical Reconstruction Microscopy

Background Despite advances in technology, resolution of fluorescent microscopy images (including confocal, deconvolution, or TIRF) remains limited to 210–250 nm. This prevents quantitative analysis of images to discern critical parameters such as protein cluster size, abundance, and separation from molecular partners. This limitation is apparent in the study of intercellular junction proteins. Gap junctions and desmosomes are defined molecular clusters, but their dimensions are in the nanoscale and, as such, escape quantitative analysis by immunofluorescence. To overcome this limitation, we implemented a novel method of imaging and analysis called direct stochastic reconstruction microscopy (dSTORM) and analyzed the effect that loss of Ankyrin-G (AnkG) expression exerts on connexin43 (Cx43) and plakophilin-2 (PKP2) plaques at the site of cell apposition. Methods Neonatal rat ventricular myocytes were labeled with antibodies to Cx43 and PKP2. Cells were treated with siRNA for AnkG, or non-targeted constructs. A total of 2.000 images were obtained to detect on–off cycles of emission, and reconstructed images were analyzed using custom-made software. Results Images yielded resolutions of 10–20 nm. We measured size, shape, fluorescence intensity profile, and abundance of clusters of Cx43 and PKP2, as well as the distance separating them. Loss of AnkG increased Cx43 cluster numbers from 16 ± 1 to 29 ± 8 clusters per region of interest. This seemed concurrent with a decrease in size of individual clusters. Moreover, in control, 16.6% ± 1.4 (34/204) clusters were within 100 nm from each other; the number increased to 32.8% ± 5.5 (145/433) in cells lacking AnkG. Similar results were observed for PKP2 plaques. Finally, 45% (168/334) of all Cx43 plaques were within 20 nm of a PKP2 plaque, and the number was unaffected by AnkG expression (274/580). Conclusions Our data show that a large fraction of Cx43–PKP2 molecules are within distances compatible with direct intermolecular interaction. Moreover, the separation between Cx43 and PKP2 is unaffected by AnkG expression, whereas loss of AnkG leads to fragmented Cx43 and PKP2 plaques. We demonstrate the applicability of dSTORM to study cardiac proteins within a resolution previously reserved for electron microscopy.

Super-Resolution Imaging Reveals a Multi-Array Arrangement of Catsper Channel on the Sperm Tail

Increase in intracellular Ca2+ initiates hyperactive motility of sperm, the high amplitude and often asymmetric movement of the tail. Cationic channel in sperm (CatSper) genes encode a complex of Ca2+ channel in sperm required for hyperactivation and male fertility. However, it is unclear how the CatSper-mediated Ca2+ entry increases flagellar bending and induces hyperactivated motility. Using high-resolution fluorescence microscopy based on high-accuracy localization of photoswitchable fluorophores, or stochastic reconstruction microscopy (STORM), combined with electron microscopy, we examined three-dimensional distribution of CatSper channels and flagellar proteins on the sperm tail. The CatSper channels form a unique three-dimensional multi-array arrangement, which may explain the characteristic patterns of hyperactivated motility.

Live-Cell Super-Resolution Fluorescence Imaging at High Spatiotemporal Resolutions

Super-resolution fluorescence microscopy accomplished nanometer resolutions in optical imaging along with multi-color and three-dimensional capability in fixed specimens. For live cells, time and spatial resolutions has been limited by the intrinsic tradeoff between the two resolutions. Using bright, fast switching cyanine dyes, we achieved high spatiotemporal resolutions with stochastic reconstruction microscopy (STORM). We obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of clathrin-coated pits and their transferrin cargo labeled with photoswitchable dyes either directly or via SNAP tags in living cells. The high photon output and fast switching rate of the cyanine dye enabled us to demonstrate 2D imaging at spatial resolutions of ∼25 nm and temporal resolutions as fast as 0.5 sec, which represent a 2-3 fold improvement in spatial resolution and 50-100 fold improvement in temporal resolution compared to previously reported resolutions obtained using single-molecule-localization based super-resolution approaches. Furthermore, we demonstrate live-cell 3D volumetric super-resolution imaging for the first time, with a spatial resolution of ∼30 nm in the lateral directions and ∼50 nm in the axial direction at time resolutions down to ∼1 sec. Two-color 3D super-resolution imaging in live cells was also achieved using photoswitchable probes with distinct emission spectra. In addition, we obtained similarly high spatial and temporal resolutions imaging other important cellular structures. These imaging capabilities open a new window for resolving ultrastructures in living cells.

Theoretical Limits To Errors, Acquisition Rates, And Resolution In Microscopy Of Switchable Fluorophores: Replacing The Diffraction Limit With The Algorithm Limit

We calculate error rates, and their effects on imaging speed and resolution, in techniques that overcome the diffraction limit by using switchable fluorescent molecules. Recent experimental work has beaten the diffraction limit in fluorescence microcopy by activating and localizing subsets of the fluorescent molecules in the specimen, and repeating this process until all of the molecules have been imaged. Examples include PhotoActivation Localization Microscopy (PALM), STOchastic Reconstruction Microscopy (STORM), and microscopy of blinking quantum dots. In all these techniques there is a tradeoff between speed (activating more molecules per imaging cycle) and error rates (activating nearby molecules and producing overlapping images that hide information on molecular positions), and so intelligent image-processing approaches are needed to identify and reject bright spots containing multiple molecules. We show that there is a maximum acquisition rate determined by this trade-off, and that how closely one can approach this acquisition rate depends on the capabilities of the algorithm used to distinguish single-molecule spots from multi-molecule spots. In particular, we calculate the error rates of commonly-used algorithms that use the shape of the bright spot rather than the overall intensity. This technique is used in STORM because fluorescent dyes have fluorescence efficiencies that can be strongly affected by the local environment. We show that the capabilities of these algorithms, in combination with the target contrast between fluorophores and background, determine whether the resolution is limited by the capabilities of the algorithm or the number of photons collected, leading to photon-limited and algorithm-limited resolution regimes. Finally, we consider algorithms that can infer molecular positions from images of overlapping blurs, and derive the dependence of the minimimum acquisition time on algorithm performance for this class of algorithms.