Super-resolution Stimulated Emission Depletion Research Articles

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

β-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

#369 Anti-nephrin antibodies identify patients with steroid-resistant nephrotic syndrome responsive to immunosuppressants

Abstract Background and Aims The presence of circulating anti-nephrin antibodies has been recently reported in some patients with nephrotic syndrome (NS) and minimal change lesions, implying an autoimmune podocytopathy subset. We hypothesized that the presence of anti-nephrin antibodies would identify a subset of patients with steroid resistant NS (SRNS) and explain their response to second-line immunosuppressant therapy before and after kidney transplantation. Method We evaluated 31 patients with available kidney biopsy samples of our previously described prospective cohort of SRNS patients, genetically tested with whole exome sequencing at Meyer University Hospital IRCCS. By using high-resolution confocal microscopy, we assessed the co-localization between IgG and nephrin staining on kidney biopsy. To unequivocally establish anti-nephrin specificity of the deposited IgG, we performed super-resolution STimulated Emission Depletion (STED) microscopy. Results We observed anti-nephrin antibodies in 35.5% of patients with SRNS irrespective of podocytopathy lesion pattern. Antibody-positive (Antibody+) patients had a more severe NS at onset and better response to immunosuppression therapy compared to antibody-negative (Antibody-) patients. Only 1/11 Antibody+ patient developed chronic kidney disease compared to 9/20 Antibody- patients over a 10-year period. The single multidrug-resistant Antibody+ patient reached kidney failure had a pathogenic variant in the autosomal dominant-transmitted LMX1B. The patient experienced recurrence of NS after kidney transplantation with a positive response to plasmapheresis and rituximab. The allograft biopsy confirmed the presence of anti-nephrin antibodies. Conclusion The observed variability in patients with SRNS, influenced by a combination of genetic factors and nephrin autoimmunity, likely contributes to the diverse long-term outcomes and variations in responses to second-line immunosuppressant therapy.

A modular framework for multi-scale tissue imaging and neuronal segmentation.

The development of robust tools for segmenting cellular and sub-cellular neuronal structures lags behind the massive production of high-resolution 3D images of neurons in brain tissue. The challenges are principally related to high neuronal density and low signal-to-noise characteristics in thick samples, as well as the heterogeneity of data acquired with different imaging methods. To address this issue, we design a framework which includes sample preparation for high resolution imaging and image analysis. Specifically, we set up a method for labeling thick samples and develop SENPAI, a scalable algorithm for segmenting neurons at cellular and sub-cellular scales in conventional and super-resolution STimulated Emission Depletion (STED) microscopy images of brain tissues. Further, we propose a validation paradigm for testing segmentation performance when a manual ground-truth may not exhaustively describe neuronal arborization. We show that SENPAI provides accurate multi-scale segmentation, from entire neurons down to spines, outperforming state-of-the-art tools. The framework will empower image processing of complex neuronal circuitries.

Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues.

Influence of different surface cleaning methods on STED-FCS and scanning STED-FCS calibration measurements.

Molecular mobility is an important measure in biological functionality, as molecules have to diffuse to meet and interact and perform actions. Measurement of mobility requires specific tools such as fluorescence correlation spectroscopy (FCS). Especially, combination with superresolution stimulated emission depletion microscopy (STED-FCS), whether in a point- or beam-scanning mode, has proven valuable for determination of anomalous diffusion. STED-FCS however relies on an accurate calibration of the effective observation spot formed for different laser powers of the additional STED laser. This poster article highlights the need for calibration measurements and outlines that rather simple procedures involving acetone cover-glass surface cleaning only, instead of piranha cover-glass surface cleaning, and point instead of more complex scanning STED-FCS are sufficient for calibration.

Single domain antibodies as promising tools for the selective detection and neutralization of neurotoxic Aβ42 oligomers in the cerebrospinal fluid of Alzheimer’s disease patients

AbstractBackgroundExtracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein are the main hallmarks of Alzheimer’s disease (AD). Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents. The heterogeneous structures and the transient nature of these oligomers make their isolation and characterization very challenging. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early diagnosis and therapy for AD.MethodsPerforming an in vitro and in vivo screening of different sdAbs, we selected those targeting Aβ42 oligomers or fibrils with high specificity using dot‐blot, ELISA assay and the super resolution stimulated emission depletion (STED) microscopy. The potential of sdAbs was also investigated in preventing Aβ42 oligomer‐induced cytotoxicity in cultured neuronal cells. Then, the sdAbs were used to selectively detect Aβ42 species in the cerebrospinal fluid (CSF) of AD patients and control subjects and to neutralize their associated harmful effects following cell exposure to CSF samples.ResultssdAbs can selectively detect Aβ42 oligomers or fibrils both in vitro and in cultured cells, preventing the neurotoxicity induced by small oligomers. Moreover, sdAbs can significantly identify Aβ42 oligomers in the CSF of AD patients counteracting their induced toxicity in our cell models.ConclusionsAll these data provide a solid foundation for the development of sdAbs‐based immunodiagnostic tools that can selectively detect toxic aggregates in human body fluids for an early differential diagnosis of protein deposition diseases, such as AD. Furthermore, our results contribute to the improvement of the current therapeutic approaches against AD.The study was supported by Airalzh (AGYR 2020 to R.C.).

Analyzing the cellular plasma membrane by fast and efficient correlative STED and platinum replica EM

The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5–15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.

Structural changes in perineuronal nets and their perforating GABAergic synapses precede motor coordination recovery post stroke

BackgroundStroke remains one of the leading causes of long-term disability worldwide, and the development of effective restorative therapies is hindered by an incomplete understanding of intrinsic brain recovery mechanisms. Growing evidence indicates that the brain extracellular matrix (ECM) has major implications for neuroplasticity. Here we explored how perineuronal nets (PNNs), the facet-like ECM layers surrounding fast-spiking interneurons, contribute to neurological recovery after focal cerebral ischemia in mice with and without induced stroke tolerance.MethodsWe investigated the structural remodeling of PNNs after stroke using 3D superresolution stimulated emission depletion (STED) and structured illumination (SR-SIM) microscopy. Superresolution imaging allowed for the precise reconstruction of PNN morphology using graphs, which are mathematical constructs designed for topological analysis. Focal cerebral ischemia was induced by transient occlusion of the middle cerebral artery (tMCAO). PNN-associated synapses and contacts with microglia/macrophages were quantified using high-resolution confocal microscopy.ResultsPNNs undergo transient structural changes after stroke allowing for the dynamic reorganization of GABAergic input to motor cortical L5 interneurons. The coherent remodeling of PNNs and their perforating inhibitory synapses precedes the recovery of motor coordination after stroke and depends on the severity of the ischemic injury. Morphological alterations in PNNs correlate with the increased surface of contact between activated microglia/macrophages and PNN-coated neurons.ConclusionsOur data indicate a novel mechanism of post stroke neuroplasticity involving the tripartite interaction between PNNs, synapses, and microglia/macrophages. We propose that prolonging PNN loosening during the post-acute period can extend the opening neuroplasticity window into the chronic stroke phase.Graphical

Rapid Internalization of Nanoparticles by Human Cells at the Single Particle Level.

Nanoparticle uptake by cells has been studied for applications both in nanomedicine and in nanosafety. While the majority of studies have focused on the biological mechanisms underlying particle internalization, less attention has been given to questions of a more quantitative nature, such as how many nanoparticles enter cells and how rapidly they do so. To address this, we exposed human embryonic kidney cells to 40-200 nm carboxylated polystyrene nanoparticles and the particles were observed by live-cell confocal and super-resolution stimulated emission depletion fluorescence microscopy. How long a particle remained at the cell membrane after adsorbing onto it was monitored, distinguishing whether the particle ultimately desorbed again or was internalized by the cell. We found that the majority of particles desorb, but interestingly, most of the particles that are internalized do so within seconds, independently of particle size. As this is faster than typical endocytic mechanisms, we interpret this observation as the particles entering via an endocytic event that is already taking place (as opposed to directly triggering their own uptake) or possibly via an as yet uncharacterized endocytic route. Aside from the rapidly internalizing particles, a minority of particles remain at the membrane for tens of seconds to minutes before desorbing or being internalized. We also followed particles after cell internalization, observing particles that appeared to exit the cell, sometimes as rapidly as within tens of seconds. Overall, our results provide quantitative information about nanoparticle cell internalization times and early trafficking.

The interwoven fibril-like structure of amyloid-beta plaques in mouse brain tissue visualized using super-resolution STED microscopy

BackgroundStandard neuropathologic analysis of Alzheimer’s brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth.ResultsIn our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5–10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM.ConclusionsThe utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aβ) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer’s disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer’s research.

Single domain antibodies as new detectors of neurotoxic Aβ42 oligomers

AbstractBackgroundThe presence of extracellular amyloid β (Aβ) plaques in the brain parenchyma and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein is the main hallmark of Alzheimer’s disease (AD). Aβ aggregation consists of a complex chain of nucleation events producing soluble oligomer intermediates, which are the major neurotoxic agents. The transient nature of these oligomers makes very challenge their isolation and characterization. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early accurate diagnosis and therapy for AD.MethodAn in vitro and in vivo screening of different sdAbs was performed, selecting those targeting Aβ42 oligomers with high specificity using immunoassays and the super resolution stimulated emission depletion (STED) microscopy. The ability of sdAbs to prevent Aβ42‐induced cytotoxicity in neuronal cells was investigated. Then, the sdAbs were used to detect and quantify the levels of Aβ42 assemblies in the cerebrospinal fluid (CSF) of AD patients and controls.ResultSdAbs can detect neurotoxic Aβ assemblies and prevent the associated neuronal dysfunction in cell models. Furthermore, sdAbs significantly discriminated Aβ aggregates in the CSF.ConclusionAll these data provide solid grounds for the understanding of the molecular basis of oligomers toxicity, thus establishing a compelling structure‐toxicity relationship. Moreover, sdAbs‐based approaches hold a great potential as they allow detection in complex mixtures such as a biological sample for an early differential diagnosis of AD, also providing therapeutic benefits.The study was supported by Airalzh (AGYR 2020 to R.C.).

68 Characterization of Bovine Extracellular Vesicles Isolated from Colostrum

Abstract Bovine colostrum is composed of a variety of biologically active compounds including microRNAs (miRNA), which are found within extracellular vesicles (EVs). Newborn calves are agammaglobulinemic. Hence, the ability of the bovine neonate to fight off pathogens is therefore almost entirely dependent upon colostrum ingestion. Extracellular vesicle-derived miRNAs may have a crucial role in the regulation of the immune response. Thus, we aimed to characterize the EVs morphology from bovine colostrum using stimulated emission depletion (STED) super resolution microscopy. Bovine colostrum samples (approx. 3-4 h after parturition) were collected from primiparous Holstein dairy cows (n = 10) at a commercial dairy farm. Colostrum samples (15 mL) were subjected to a series of centrifugation steps to achieve defatted supernatant and to label EV pellets with EV specific markers. The EV pellets were resuspended in 100µL of 1 x PBS for EV staining methods or resuspended in 1 mL of trizol reagent for further miRNA isolation. The EVs suspensions were incubated with a primary polyclonal antibody anti-CD63 and with a secondary antibody (Alexa Fluor 594) for approximately 90 minutes each. Labeled EV-antibody complex were analyzed using confocal and STED microscopy. Based on the confocal and super resolution STED imaging capability obtained from the EVs-derived from bovine colostrum (Figure 1), labeled EVs with anti-CD63 and the enhanced resolution of STED over conventional confocal imaging, allowed to image the EVs at their actual size (30-200 nm). Further in-depth view on EV morphology, marker proteins expressed on the membrane, and size will contribute to our understanding of EVs-derived from colostrum and their functions. This will be crucial in studies to evaluate EVs uptake by intestinal cells for the correct identification of compartments involved in subcellular trafficking.

Super-resolution STED imaging in the inner and outer whole-mount mouse retina.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

Decrypting the spatial relationship between peripheral chromatin and nuclear lamina in Hutchinson-Gilford Progeria Syndrome using super-resolution microscopy techniques.

The genetic condition known as Hutchinson-Gilford Progeria Syndrome (HGPS) causes the early onset of aging symptoms and premature death. HGPS is caused by a single nucleotide mutation in the LMNA gene, resulting in a truncated form of the protein Lamin A known as ΔLA50 or Progerin. This protein modification causes aberrant lamina network organization inducing nuclear and DNA structure abnormalities, leading to the loss of peripheral heterochromatin. In this study, we examine variations in chromatin compaction in a cellular model of HPGS using Expansion Microscopy (ExM). This super-resolution microscopy technique is based on embedding biological samples into a water-absorbant polymer network that, following mechanical homogenization, enables a four-fold linear expansion in water, allowing nanoscale biophysical studies using conventional microscopes. The image analysis of the HGPS cell model shows a significant variation in chromatin compaction that seems to disrupt its physical association with the nuclear lamina. Furthermore, this loss of compaction leads to a detectable spatial separation between the lamina network, mainly composed of the mutated form Progerin, and the peripheral heterochromatin. Therefore, to quantify this distance variation caused by Progerin-induced chromatin disorganization, we analyzed a batch of control cells expressing wild-typer lamin A and HGPS cells, finding a one-order of magnitude difference between the two sets. In order to validate these results, we perform super-resolution Stimulated Emission Depletion (STED) imaging. The next step will be performing Correlative Light Electron Microscopy (CLEM) on non-expanded cells to push the resolution of our analysis even forward and to investigate chromatin compaction on a nanoscale level.

Hyaluronan nanoscale clustering and Hyaluronan synthase 2 expression are linked to the invasion of child fibroblasts and infantile fibrosarcoma in vitro and in vivo

Infantile fibrosarcoma is a rare childhood tumour that originates in the fibrous connective tissue of the long bones for which there is an urgent need to identify novel therapeutic targets. This study aims to clarify the role of the extracellular matrix component hyaluronan in the invasion of child fibroblasts and Infantile fibrosarcoma into the surrounding environment. Using nanoscale super-resolution STED (Stimulated emission depletion) microscopy followed by computational image analysis, we observed, for the first time, that invasive child fibroblasts showed increased nanoscale clustering of hyaluronan at the cell periphery, as compared to control cells. Hyaluronan was not observed within focal adhesions. Bioinformatic analyses further revealed that the increased nanoscale hyaluronan clustering was accompanied by increased gene expression of Hyaluronan synthase 2, reduced expression of Hyaluronidase 2 and CD44, and no change of Hyaluronan synthase 1 and Hyaluronidases 1, 3, 4 or 5. We further observed that the expression of the Hyaluronan synthase 1, 2 and 3, and the Hyaluronidase 3 and 5 genes was linked to reduced life expectancy of fibrosarcoma patients. The invasive front of infantile fibrosarcoma tumours further showed increased levels of hyaluronan, as compared to the tumour centre. Taken together, our findings are consistent with the possibility that while Hyaluronan synthase 2 increases the levels, the Hyaluronidases 3 and 5 reduce the weight of hyaluronan, resulting in the nanoscale clustering of hyaluronan at the leading edge of cells, cell invasion and the spread of Infantile fibrosarcoma.

Visualization of Exo- and Endocytosis Membrane Dynamics with Super-Resolution STED Microscopy.

Recent advances in stimulated emission depletion (STED) microscopy offer an unparalleled avenue to study membrane dynamics of exo- and endocytosis, such as fusion pore opening, pore expansion, constriction, and closure, as well as the membrane transformation from flat-shaped to round-shaped vesicles in real time. Here we depict a method of using the state-of-the-art STED microscopy to image these membrane dynamics in bovine chromaffin cells. This method can potentially be applied to study other membrane structure dynamics in other cell model system.

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

Low-power compact continuous-wave stimulated emission depletion microscopy.

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.

Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation

BackgroundIt is becoming increasingly clear that biological metals such as iron, copper or zinc are involved in synaptic functions, and in particular in the mechanisms of synaptogenesis and subsequent plasticity. Understanding the role of metals on synaptic functions is a difficult challenge due to the very low concentration of these elements in neurons and to the submicrometer size of synaptic compartments. New methodTo address this challenge we have developed a correlative nano-imaging approach combining metal and protein detection. First, stimulated emission depletion (STED) microscopy, a super resolution optical microscopy technique, is applied to locate fluorescently labeled proteins. Then, synchrotron radiation induced X-ray fluorescence (SXRF) is performed on the same regions of interest, e.g. synaptic compartments. ResultsWe present here the principle scheme that allows this correlative nano-imaging and its experimental validation. We applied this correlative nano-imaging to the study of the physiological distribution of metals in synaptic compartments of primary rat hippocampal neurons. We thus compared the nanometric distribution of metals with that of synaptic proteins, such as PSD95 or cytoskeleton proteins. Comparison with existing method(s)Compared to correlative imaging approaches currently used to characterize synaptic structures, such as electron microscopy correlated with optical fluorescence, our approach allows for ultra-sensitive detection of trace metals using highly focused synchrotron radiation beams. ConclusionWe provide proof-of-principle for correlative imaging of metals and proteins at the synaptic scale and discuss the present limitations and future developments in this area.