Stochastic Optical Reconstruction Microscopy Imaging Research Articles

Electrochemically controlled blinking of fluorophores for quantitative STORM imaging

Stochastic optical reconstruction microscopy (STORM) allows wide-field imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long-lived OFF state and an emissive ON state. Although STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artefacts from the repetitive random blinking of dyes. Here we show that fluorophores can be electrochemically switched for STORM imaging (EC-STORM), with excellent control over the switching kinetics, duty cycle and recovery yield. Using EC-STORM, we demonstrate molecular counting by using electrochemical potential to control the photophysics of dyes. The random blinking of dyes is suppressed by a negative potential but the switching-ON event can be activated by a short positive-potential pulse, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate EC-STORM of tubulin in fixed cells with a spatial resolution as low as ~28 nm and counting of single Alexa 647 fluorophores on various DNA nanoruler structures. This control over fluorophore switching will enable EC-STORM to be broadly applicable in super-resolution imaging and molecular counting.

Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown. Here, we developed a microanalytical proteomics pipeline using capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) in the discovery setting to quantify developmental changes in the chief circadian pacemaker, the suprachiasmatic nucleus (SCN), before and after the onset of photoreceptor-dependent visual function. Nesting CE-ESI with trapped ion mobility spectrometry time-of-flight (TOF) mass spectrometry (TimsTOF PRO) doubled the number of identified and quantified proteins compared to the TOF-only control on the same analytical platform. From 10 ng of peptide input, corresponding to <∼0.5% of the total local tissue proteome, technical triplicate analyses identified 1894 proteins and quantified 1066 proteins, including many with important canonical functions in axon guidance, synapse function, glial cell maturation, and extracellular matrix refinement. Label-free quantification revealed differential regulation for 166 proteins over development, with enrichment of axon guidance-associated proteins prior to eye-opening and synapse-associated protein enrichment after eye-opening. Super-resolution imaging of select proteins using STochastic Optical Reconstruction Microscopy (STORM) corroborated the MS results and showed that increased presynaptic protein abundance pre/post eye-opening in the SCN reflects a developmental increase in synapse number, but not presynaptic size or extrasynaptic protein expression. This work marks the first development and systematic application of TimsTOF PRO for CE-ESI-based microproteomics and the first integration of microanalytical CE-ESI TimsTOF PRO with volumetric super-resolution STORM imaging to expand the repertoire of technologies supporting analytical neuroscience.

Super-resolution imaging reveals the relationship between CaMKIIβ and drebrin within dendritic spines

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIβ subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIβ binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIβ within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIβ co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIβ and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIβ was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIβ in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIβ is in a standby state before its activation.

Nanoscale reorganisation of synaptic proteins in Alzheimer's disease.

Synaptic strength depends strongly on the subsynaptic organisation of presynaptic transmitter release and postsynaptic receptor densities, and their alterations are expected to underlie pathologies. Although synaptic dysfunctions are common pathogenic traits of Alzheimer's disease (AD), it remains unknown whether synaptic protein nano-organisation is altered in AD. Here, we systematically characterised the alterations in the subsynaptic organisation in cellular and mouse models of AD. We used immunostaining and super-resolution stochastic optical reconstruction microscopy imaging to quantitatively examine the synaptic protein nano-organisation in both Aβ1-42-treated neuronal cultures and cortical sections from a mouse model of AD, APP23 mice. We found that Aβ1-42-treatment of cultured hippocampal neurons decreased the synaptic retention of postsynaptic scaffolds and receptors and disrupted their nanoscale alignment to presynaptic transmitter release sites. In cortical sections, we found that while GluA1 receptors in wild-type mice were organised in subsynaptic nanoclusters with high local densities, receptors in APP23 mice distributed more homogeneously within synapses. This reorganisation, together with the reduced overall receptor density, led to reduced glutamatergic synaptic transmission. Meanwhile, the transsynaptic alignment between presynaptic release-guiding RIM1/2 and postsynaptic scaffolding protein PSD-95 was reduced in APP23 mice. Importantly, these reorganisations were progressive with age and were more pronounced in synapses in close vicinity of Aβ plaques with dense cores. Our study revealed a spatiotemporal-specific reorganisation of synaptic nanostructures in AD and identifies dense-core amyloid plaques as the major local inductor in APP23 mice.

Abstract 4253: Pan-cancer analysis of XPO1 R749Q mutations across 217,570 patients reveals association with high tumor mutational burden and therapy resistance

Abstract Background: XPO1 is a nuclear export receptor responsible for exporting &amp;gt;200 proteins out of the nucleus, including many cancer-related proteins. We previously reported recurrent hotspot mutations occurring in XPO1 in cancer and identified XPO1 R749Q variant only in solid tumors. To determine the prevalence of XPO1 mutations across cancer types, we performed a large-scale genomic analysis of 217,570 patients with cancer to identify and characterize XPO1 variants from real-world patient tumors. Methods: Solid tumor samples representing 14 cancer types were submitted to Caris Life Sciences (Phoenix, AZ) for Next-generation sequencing of DNA (592 genes or WES). TMB-High (defined as &amp;gt;10 mt/MB) and dMMR/MSI-H was tested by IHC/NGS. We engineered CRISPR knock-in HCT116 and LS-174T colon cancer isogenic cell lines bearing XPO1 R749Q mutations and Stochastic Optical Reconstruction Microscopy (STORM) analysis was performed. Structural modeling was performed with published XPO1 structures by mapping XPO1 mutations from the MSKCC-IMPACT dataset. Results: A total of 96 patients with XPO1 R749Q mutations were identified (50% occurred in endometrial cancer and 23% occurred in colorectal cancer). TMB-H was observed in 92% of XPO1 R749Q mt tumors (9% were dMMR/MSI-H), while 86% of XPO1 R749Q mt tumors were POLE-mutated within the endonuclease domain. Median variant allele frequency for XPO1 R749Q mt and POLE mt were 25% and 29%, respectively. STORM imaging revealed that XPO1 R749Q mutant cells had significant localization of XPO1 in the cytoplasm compared to XPO1 WT cells, especially at the outside edge of the nuclear pores. Structural modeling predicted that XPO1 R749Q mt affected the regulatory H9-loop of XPO1 favoring increased shuttling and retention in the cytoplasm. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteomes confirmed that XPO1 R749Q mt cells had increased export of proteins compared to XPO1 WT cells. A library screen of &amp;gt;200 FDA-approved drugs revealed a strong therapeutic resistance of XPO1 R749Q cells, especially to chemotherapies used in the treatment of colon cancer. XPO1 inhibition with selinexor synergized with chemotherapy in XPO1 R749Q mt cells in vitro and overcame resistance to irinotecan in vivo in xenograft mice models. Conclusion: This study sheds novel insights into the role of nuclear export in cancers. Specifically, XPO1 R749Q mutations are enriched in TMB-H endometrial and colon cancers and increase nuclear export of key proteins that confer resistance to therapies using DNA-damaging agents. Our current work aims to identify novel therapies that can overcome resistance to DNA-damaging therapies seen in XPO1 mutant cells, such as immune checkpoint inhibitor (ICI) therapy given the co-occurrence with POLE mutations and recent reports of high level of response to ICI therapy in TMB-H and POLE mt colorectal cancers. Citation Format: Tulasigeri M. Totiger, Yasmine Baca, Wannasiri Chiraphapphaiboon, Sana Chaudhry, Skye Montoya, Monika Chojnacka, Gabriel Gaidosh, Jumana Afaghani, Maurizio Affer, Christopher D. Armstrong, Ryan Notti, Jenna Zabroski, Jacob Jahn, Vindhya Nawaratne, Ramiro Verdun, Hai Dang Nguyen, Chadi Nabhan, Thomas J. Herzog, Phil Walker, Andrew Elliott, Emil Lou, Wafik S. El-Deiry, Edith Mitchell, Jose Antonio Rodriguez, Justin Taylor. Pan-cancer analysis of XPO1 R749Q mutations across 217,570 patients reveals association with high tumor mutational burden and therapy resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4253.

Super-Resolution Fluorescence Imaging for Semiconductor Nanoscale Metrology and Inspection.

The increase in the number and complexity of process levels in semiconductor production has driven the need for the development of new measurement methods that can evaluate semiconductor devices at the critical dimensions of fine patterns and simultaneously inspect nanoscale contaminants or defects. However, conventional optical inspection methods often fail to resolve device patterns or defects at the level of tens of nanometers required for device development owing to their diffraction-limited resolutions. In this study, we used the stochastic optical reconstruction microscopy (STORM) technique to image semiconductor nanostructures with feature sizes as small as 30 nm and detect individual 20 nm-diameter contaminants. STORM imaging of semiconductor nanopatterns is based on the development of a selective labeling method of fluorophores for a negative silicon oxide surface using the charge interaction of positive polyethylenimine molecules. This study demonstrates the potential of STORM for nanoscale metrology and in-line defect inspection of semiconductor integrated circuits.

Correlated STORM-homoFRET imaging reveals highly heterogeneous membrane receptor structures

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

Ultrastructural visualization of chromatin in cancer pathogenesis using a simple small-molecule fluorescent probe.

Imaging chromatin organization at the molecular-scale resolution remains an important endeavor in basic and translational research. Stochastic optical reconstruction microscopy (STORM) is a powerful superresolution imaging technique to visualize nanoscale molecular organization down to the resolution of ~20 to 30 nm. Despite the substantial progress in imaging chromatin organization in cells and model systems, its routine application on assessing pathological tissue remains limited. It is, in part, hampered by the lack of simple labels that consistently generates high-quality STORM images on the highly processed clinical tissue. We developed a fast, simple, and robust small-molecule fluorescent probe—cyanine 5–conjugated Hoechst—for routine superresolution imaging of nanoscale nuclear architecture on clinical tissue. We demonstrated the biological and clinical significance of imaging superresolved chromatin structure in cancer development and its potential clinical utility for cancer risk stratification.

A Palette of Fluorescent Aβ42 Peptides Labelled at a Range of Surface-Exposed Sites.

Fluorescence-based single molecule techniques provide important tools towards understanding the molecular mechanism of complex neurodegenerative diseases. This requires efficient covalent attachment of fluorophores. Here we create a series of cysteine mutants (S8C, Y10C, S26C, V40C, and A42C) of A42, involved in Alzheimer’s disease, based on exposed positions in the fibril structure and label them with the Alexa-fluorophores using maleimide chemistry. Direct stochastic optical reconstruction microscopy imaging shows that all the labelled mutants form fibrils that can be detected by virtue of Alexa fluorescence. Aggregation assays and cryo-electron micrographs establish that the careful choice of labelling position minimizes the perturbation of the aggregation process and fibril structure. Peptides labelled at the N-terminal region, S8C and Y10C, form fibrils independently and with wild-type. Peptides labelled at the fibril core surface, S26C, V40C and A42C, form fibrils only in mixture with wild-type peptide. This can be understood on the basis of a recent fibril model, in which S26, V40 and A42 are surface exposed in two out of four monomers per fibril plane. We provide a palette of fluorescently labelled A42 peptides that can be used to gain understanding of the complex mechanisms of A42 self-assembly and help to develop a more targeted approach to cure the disease.

Structure-Based Analysis of Protein Cluster Size for Super-Resolution Microscopy in the Nervous System

To overcome the diffraction limit and resolve target structures in greater detail, far-field super-resolution techniques such as stochastic optical reconstruction microscopy (STORM) have been developed, and different STORM algorithms have been developed to deal with the various problems that arise. In particular, the effect of the local structure is an important issue. For objects with closely correlated distributions, simple Gaussian-based localization algorithms often used in STORM imaging misinterpret overlapping point spread functions (PSFs) as one, which limits the ability of super-resolution imaging to resolve nanoscale local structures and leads to inaccurate length measurements. The STORM super-resolution images of biological specimens from the cluster-forming proteins in the nervous system were reconstructed for localization-based analysis. Generally, the localization of each fluorophore was determined by two-dimensional Gaussian function fitting. Further, the physical shape of the cluster structure information was incorporated into the size parameter of the localization structure analysis in order to generate structure-based fitting algorithms. In the present study, we proposed a novel, structure-based, super-resolution image analysis method: structure-based analysis (SBA), which combines a structural function and a super-resolution localization algorithm. Using SBA, we estimated the size of fluorescent beads, inclusion proteins, and subtle synaptic structures in both wide-field and STORM images. The results show that SBA has a comparable and often superior performance to the commonly used full width at half maximum (FWHM) parameter. We demonstrated that SBA is able to estimate molecular cluster sizes in far-field super-resolution STORM images, and that SBA was comparable and often superior to FWHM. We also certified that SBA provides size estimations that corroborate previously published electron microscopy data.

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton.

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

[Figure: see text].

STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena

The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet–Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking.

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

Syndecan-4 Modulates Cell Polarity and Migration by Influencing Centrosome Positioning and Intracellular Calcium Distribution.

Efficient cell migration requires cellular polarization, which is characterized by the formation of leading and trailing edges, appropriate positioning of the nucleus and reorientation of the Golgi apparatus and centrosomes toward the leading edge. Migration also requires the development of an asymmetrical front-to-rear calcium (Ca2+) gradient to regulate focal adhesion assembly and actomyosin contractility. Here we demonstrate that silencing of syndecan-4, a transmembrane heparan sulfate proteoglycan, interferes with the correct polarization of migrating mammalian myoblasts (i.e., activated satellite stem cells). In particular, syndecan-4 knockdown completely abolished the intracellular Ca2+ gradient, abrogated centrosome reorientation and thus decreased cell motility, demonstrating the role of syndecan-4 in cell polarity. Additionally, syndecan-4 exhibited a polarized distribution during migration. Syndecan-4 knockdown cells exhibited decreases in the total movement distance during directional migration, maximum and vectorial distances from the starting point, as well as average and maximum cell speeds. Super-resolution direct stochastic optical reconstruction microscopy images of syndecan-4 knockdown cells revealed nanoscale changes in the actin cytoskeletal architecture, such as decreases in the numbers of branches and individual branch lengths in the lamellipodia of the migrating cells. Given the crucial importance of myoblast migration during embryonic development and postnatal muscle regeneration, we conclude that our results could facilitate an understanding of these processes and the general role of syndecan-4 during cell migration.

Photochromic Fluorescent Probe Strategy for the Super-resolution Imaging of Biologically Important Biomarkers.

Here, we report a β-galactosidase (β-Gal)-responsive photochromic fluorescent probe, NpG, that was designed to prebind to human serum albumin (HSA) to form the probe/protein hybrid, NpG@HSA. The formation of NpG@HSA led to an increase in fluorescence emission (520 nm) corresponding to the binding of the fluorescent naphthalimide unit with HSA. In addition, this enabled visualization of the spiropyran fluorescence emission in aqueous media. Our probe/protein hybrid approach afforded a unique imaging platform with enhanced cell permeability and solubility that was capable of visualizing the cellular uptake of NpG@HSA before its activation by β-Gal. The β-Gal-mediated cleavage of the galactose unit within the NpG@HSA hybrid resulted in the formation of NpM@HSA and an increase in red fluorescence emission (620 nm). The resultant merocyanine unit was then able to undergo photoisomerization (merocyanine ↔ spiropyran) to facilitate STORM (i.e., stochastic optical reconstruction microscopy) imaging with minimal phototoxicity and excellent photostability/reversibility. Using STORM, NpG@HSA was able to determine the subcellular distribution of β-Gal activity between cell lines with nanoscale precision. We believe that this system represents a versatile imaging platform for the design of photochromic fluorescent probes suitable for illuminating the precise location of disease-specific biomarkers in various cellular processes.

STochastic Optical Reconstruction Microscopy (STORM) reveals the nanoscale organization of pathological aggregates in human brain.

AimsHistological analysis of brain tissue samples provides valuable information about the pathological processes leading to common neurodegenerative disorders. In this context, the development of novel high‐resolution imaging approaches is a current challenge in neuroscience.MethodsTo this end, we used a recent super‐resolution imaging technique called STochastic Optical Reconstruction Microscopy (STORM) to analyse human brain sections. We combined STORM cell imaging protocols with neuropathological techniques to image cryopreserved brain samples from control subjects and patients with neurodegenerative diseases.ResultsThis approach allowed us to perform 2D‐, 3D‐ and two‐colour‐STORM in neocortex, white matter and brainstem samples. STORM proved to be particularly effective at visualizing the organization of dense protein inclusions and we imaged with a <50 nm resolution pathological aggregates within the central nervous system of patients with Alzheimer’s disease, Parkinson’s disease, Lewy body dementia and fronto‐temporal lobar degeneration. Aggregated Aβ branches appeared reticulated and cross‐linked in the extracellular matrix, with widths from 60 to 240 nm. Intraneuronal Tau and TDP‐43 inclusions were denser, with a honeycomb pattern in the soma and a filamentous organization in the axons. Finally, STORM imaging of α‐synuclein pathology revealed the internal organization of Lewy bodies that could not be observed by conventional fluorescence microscopy.ConclusionsSTORM imaging of human brain samples opens further gates to a more comprehensive understanding of common neurological disorders. The convenience of this technique should open a straightforward extension of its application for super‐resolution imaging of the human brain, with promising avenues to current challenges in neuroscience.

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.