Stochastic Optical Reconstruction Microscopy Imaging Research Articles

Optimized Stochastic Optical Reconstruction Microscopy for Imaging Chromatin Structure in Pathological Tissue.

Direct visualization of higher-order chromatin structure at the molecular scale is of great importance for understanding the impact of chromatin organization on gene expression in many biological processes. Understanding the changes in chromatin structure during pathological processes requires the use of in vivo models and clinical samples, and formalin-fixed, paraffin-embedded (FFPE) tissue is the most widespread form of preservation. Here we describe the details of PathSTORM, an optimized stochastic optical reconstruction microscopy (STORM) protocol for high-quality super-resolution imaging of densely packed higher-order chromatin organization in pathological tissue. We discuss detailed methods for fluorescence staining of DNA and histone proteins, as well as the key technical factors for obtaining high-quality STORM images in pathological tissue samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Fluorescence staining of chromatin in pathological tissue Basic Protocol 2: STORM data processing Support Protocol 1: Drift correction Support Protocol 2: Image reconstruction Support Protocol 3: Hematoxylin & eosin (H&E) staining.

2010 - IDENTIFYING NECESSARY CHROMATIN STATES AND TARGETS FOR HEMOGENIC SPECIFICATION AND REPROGRAMMING

Hematopoietic stem cells (HSCs) originate from a subset of endothelium in the embryo known as hemogenic endothelium. Hemogenic endothelium must undergo an endothelial to hematopoietic transition (EHT) to form HSCs. EHT requires the activity of the transcription factor (TF) RUNX1. Ectopic pan-endothelial expression of RUNX1 in mouse embryos is able to specify embryonic endothelium as hemogenic. However, as compared to embryonic endothelium, RUNX1 is not as effective at specifying fetal endothelium as hemogenic. To understand the difference in the competency of embryonic versus fetal endothelium to be specified as hemogenic, we examined differences in chromatin at these two developmental stages. ATAC-seq revealed that RUNX1 appears to be specifying embryonic endothelium through a canonical EHT program. To orchestrate this EHT transcriptional program, RUNX1 participates with other hematopoietic TFs, such as FLI1, ETS1, SPI1, and GFI1. Following RUNX1 induction in embryonic endothelium, immune genes and motility related genes are upregulated. In fetal endothelium, however, RUNX1 induction results in a more complex picture. To determine if these differences were due to underlying chromatin differences between fetal and embryonic endothelium, we examined control embryonic and fetal endothelium using stochastic optical reconstruction microscopy (STORM). STORM imaging suggests that there are global differences in chromatin structure between fetal and embryonic endothelium. Fetal endothelium appears to have more densely packed chromatin that is located more peripherally than embryonic endothelium. Taken together, these data show that the reduced specification efficiency by RUNX1 observed between embryonic and fetal endothelium is due, at least in part, to chromatin structure.

Replication Origins Exposed on the Surface of a Replication Domain by Transcription Elongation are Preferentially Fired for DNA Replication

In mammals, DNA replication is initiated at discrete loci known as replication origins. Among totally ∼250,000 potential replication origins, only a small subset is activated and used for complete replication of the whole genome during any S phase, but the selection mechanism remains elusive. Using Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging, we mapped the spatial distribution of replication origins relative to individual replication domains (RDs). Metabolic pause labeling revealed that DNA replication mainly initiated at the spatial boundary of replication domains. Further, we choose two adjacent RDs and used fluorescence in situ hybridization (FISH) to fluorescently label the RDs and replication origins of them. Interestingly, we found that predominant origins of the early S phase replication domain (ESRD) which were actually inside of the RD became exposed on the surface of the RD from middle G1 phase to the beginning of S phase (G1/S phase), not the case with dormant origins of ESRD or predominant origins of the late S phase replication domain (LSRD). To understand what drives ESRD predominant origins out, we inhibited transcription elongation or knocked down CTCF or cohesin and got spatial redistribution of predominant origins disappeared. To know why origins who were located outside could be fired, we labeled proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 2 (MCM2) and CCCTC-binding factor (CTCF) in G1 phase and found PCNA clusters distributed outside of replication domains while MCM2 or CTCF distributed randomly. Taken together, our STORM images reveal that DNA replication initiates at origins exposed on the surface of replication domains where replication proteins distribute. Replication Origins selection may be a result of their spatial redistribution mediated by cooperation of transcription elongation and CTCF-cohesin formed loops.

Optimizing Astigmatism for 3D Stochastic Optical Reconstruction Microscopy

Single molecule tracking and localization has become a powerful strategy for noninvasive imaging of nanoscale structures in biology. The aim of this study is to develop a scanning astigmatism module for 3D Stochastic-Optical-Reconstruction-Microscopy (STORM). For 3D STORM imaging, a weak cylindrical lens is placed in the imaging path to induce astigmatism. The specific location of the lens in the optical path can affect the extent of correction and in our approach, the ability to scan the lens position allows for finer control and thus optimization for the reconstruction. Our design uses an Arduino-based circuit along with a stepper motor controlled by a Python interface to position the cylindrical lens. The ellipticity and orientation of fluorophores’ localization are obtained using the ImageJ data analysis plugin ThunderSTORM. We demonstrate the feasibility of this approach through 3D reconstruction of the mitochondrial morphology of HeLa cells. Improvement in the visualization of cellular structures and their relationships with nanoscale resolution in all three dimensions will contribute to a better understanding of molecular processes occurring in cells.

GPR124 facilitates pericyte polarization and migration by regulating the formation of filopodia during ischemic injury.

Prolonged occlusion of multiple microvessels causes microvascular injury. G protein-coupled receptor 124 (GPR124) has been reported to be required for maintaining central nervous system (CNS) angiogenesis and blood-brain barrier integrity. However, the molecular mechanisms by which GPR124 regulates pericytes during ischemia have remained elusive.Methods: A microsphere embolism-induced ischemia model was used to evaluate the expression of GPR124 following microsphere embolism. Immunocytochemistry and stochastic optical reconstruction microscopy imaging were used to assess the expression and distribution of GPR124 in human brain vascular pericytes (HBVPs) and after the treatment with 3-morpholino-sydnonimine (SIN-1) or oxygen-glucose deprivation (OGD). The effect of GPR124 knockdown or overexpression on HBVP migration was analyzed in vitro using wound healing assays and a microfluidic device. GPR124 loss-of-function studies were performed in HBVPs and HEK293 cells using CRISPR-Cas9-mediated gene deletion. Time-lapse imaging was used to assess dynamic changes in the formation of filopodia in an individual cell. Finally, to explore the functional domains required for GPR124 activity, deletion mutants were constructed for each of the N-terminal domains.Results: GPR124 expression was increased in pericytes following microsphere embolism. Morphological analysis showed localization of GPR124 to focal adhesions where GPR124 bound directly to the actin binding protein vinculin and upregulated Cdc42. SIN-1 or OGD treatment redistributed GPR124 to the leading edges of HBVPs where GPR124 signaling was required for pericyte filopodia formation and directional migration. Partial deletion of GPR124 domains decreased SIN-1-induced filopodia formation and cell migration.Conclusion: Taken together, our results provide the first evidence for a role of GPR124 in pericyte migration under ischemic conditions and suggest that GPR124 was essential for Cdc42 activation and filopodia formation.

Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy (STORM)

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs) and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of optical and electrical microscopy, the ultra-structure of ESG has not been revealed until recent development of super high resolution fluorescence optical microscope, STORM. We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC (bEnd3, mouse brain microvascular endothelial cells) monolayers. After confluent, the bEnd3 cells were immunolabeled with anti-HS, followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated HA binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM with a 100 x/1.49 oil immersed lens. Multiple Reporters of ATTO488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA. The field of 256 × 256 (40 × 40 μm2) of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame. The figure demonstrates the STORM images for the HA (top view, red, top row on the right) and HS (green, top row in the middle) and overlay (top row on the left) of the ESG focused at the bottom surface of the bEnd3 cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. The figure shows that HA is a long molecule weaving into a network, which is horizontal to the EC cell surface. In contrast, HS is a shorter molecule, which is perpendicular to the cell surface. The height of the HS is ~600 nm. HA and HS seem to overlap with each other at the EC surface. The revealed ultra-structure of ESG by STORM suggests that HS plays a major role in mechanosensing and HA plays a major role in forming the molecular sieve. Support or Funding Information NIH 1SC1CA153325-01 and NSF MRI CBET 1337746 STORM images showing distribution of ATTO 488 labeled anti-HS (green, middle) and Alexa Fluor 647 labeled anti-HA (red, right) and overlay (left) in the ESG of bEnd3 cell monolayer, focused on the bottom surface of the cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Repurposing a photosynthetic antenna protein as a super-resolution microscopy label

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.

Adaptive optics stochastic optical reconstruction microscopy (AO-STORM) by particle swarm optimization.

Stochastic optical reconstruction microscopy (STORM) can achieve resolutions of better than 20nm imaging single fluorescently labeled cells. However, when optical aberrations induced by larger biological samples degrade the point spread function (PSF), the localization accuracy and number of localizations are both reduced, destroying the resolution of STORM. Adaptive optics (AO) can be used to correct the wavefront, restoring the high resolution of STORM. A challenge for AO-STORM microscopy is the development of robust optimization algorithms which can efficiently correct the wavefront from stochastic raw STORM images. Here we present the implementation of a particle swarm optimization (PSO) approach with a Fourier metric for real-time correction of wavefront aberrations during STORM acquisition. We apply our approach to imaging boutons 100 μm deep inside the central nervous system (CNS) of Drosophila melanogaster larvae achieving a resolution of 146 nm.

Cell-Shaping Micropatterns for Quantitative Super-Resolution Microscopy Imaging of Membrane Mechanosensing Proteins.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Stochastic Optical Reconstruction Microscopy (STORM).

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc.

Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.

Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

Reconstruction of super-resolution STORM images using compressed sensing based on low-resolution raw images and interpolation.

Single-molecule-localization-based super-resolution microscopic technologies, such as stochastic optical reconstruction microscopy (STORM), require lengthy runtimes. Compressed sensing (CS) can partially overcome this inherent disadvantage, but its effect on super-resolution reconstruction has not been thoroughly examined. In CS, measurement matrices play more important roles than reconstruction algorithms. Larger measurement matrices have better restricted isometry properties (RIPs). This paper proposes, analyzes, and compares uses of higher resolution cameras and interpolation to achieve better outcomes. Statistical results demonstrate that super-resolution reconstructions is significantly improved by interpolating low-resolution STORM raw images and using point-spread-function-based measurement matrices with better RIPs. The analysis of publically accessible experimental data confirms this conclusion.

随机光学重构显微成像技术及其应用

随机光学重构显微成像技术及其应用

Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

Unidirectional Living Growth of Self-Assembled Protein Nanofibrils Revealed by Super-resolution Microscopy.

Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchange kinetics, and polymerization mechanism for fibrils composed of a de novo designed recombinant triblock protein polymer. This macromolecule features a silk-inspired self-assembling central block composed of GAGAGAGH repeats, which are known to fold into a β roll with turns at each histidine and, once folded, to stack, forming a long, ribbon-like structure. We find several properties that allow the growth of patterned protein nanofibrils: the self-assembly takes place on only one side of the growing fibrils by the essentially irreversible addition of protein polymer subunits, and these fibril ends remain reactive indefinitely in the absence of monomer ("living ends"). Exploiting these characteristics, we can grow stable diblock protein nanofibrils by the sequential addition of differently labeled proteins. We establish control over the block length ratio by simply varying monomer feed conditions. Our results demonstrate the use of engineered protein polymers in creating precisely patterned protein nanofibrils and open perspectives for the hierarchical self-assembly of functional biomaterials.

Super Resolution Imaging of Nanoparticles Cellular Uptake and Trafficking.

Understanding the interaction between synthetic nanostructures and living cells is of crucial importance for the development of nanotechnology-based intracellular delivery systems. Fluorescence microscopy is one of the most widespread tools owing to its ability to image multiple colors in native conditions. However, due to the limited resolution, it is unsuitable to address individual diffraction-limited objects. Here we introduce a combination of super-resolution microscopy and single-molecule data analysis to unveil the behavior of nanoparticles during their entry into mammalian cells. Two-color Stochastic Optical Reconstruction Microscopy (STORM) addresses the size and positioning of nanoparticles inside cells and probes their interaction with the cellular machineries at nanoscale resolution. Moreover, we develop image analysis tools to extract quantitative information about internalized particles from STORM images. To demonstrate the potential of our methodology, we extract previously inaccessible information by the direct visualization of the nanoparticle uptake mechanism and the intracellular tracking of nanoparticulate model antigens by dendritic cells. Finally, a direct comparison between STORM, confocal microscopy, and electron microscopy is presented, showing that STORM can provide novel and complementary information on nanoparticle cellular uptake.

Issue Cover (November 2015)

Front cover: Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image. Shown here is a two-color STORM image of mitochondria with both the inner and outer mitochondrial membranes labeled (inner membrane: ATP Synthase, red; outer membrane: TOM20, green). These membranes are in close proximity to each other but can be clearly resolved using STORM. Read the full article ‘Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods’ by J. Tam and D. Merino (J. Neurochem. 2015, vol. 135 (4), pp. 643–658) on doi: 10.1111/jnc.13257

Studying neuronal microtubule organization and microtubule-associated proteins using single molecule localization microscopy.

The formation and maintenance of highly polarized neurons critically depends on the proper organization of the microtubule (MT) cytoskeleton. In axons, MTs are uniformly oriented with their plus-end pointing outward whereas in mature dendrites MTs have mixed orientations. MT organization and dynamics can be regulated by MT-associated proteins (MAPs). Plus-end tracking proteins are specialized MAPs that decorate plus-ends of growing MTs and regulate neuronal polarity, neurite extension, and dendritic spine morphology. Conventional fluorescence microscopy enables observation of specific cellular components through molecule-specific labeling but provides limited resolution (∼250 nm). Therefore, electron microscopy has until now provided most of our knowledge about the precise MT organization in neurons. In the past decade, super-resolution fluorescence microscopy techniques have emerged that circumvent the diffraction limit of light and enable high-resolution reconstruction of the MT network combined with selective protein labeling. However, preserving MT ultrastructure, MAP binding, high labeling density, and antibody specificity after fixation protocols is still quite challenging. In this chapter, we provide an optimized protocol for two-color direct stochastic optical reconstruction microscopy imaging of neuronal MTs together with their growing plus-ends to probe MT architecture and polarity.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.