Super-resolution Light Research Articles

Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.

To Pinpoint the Location and the Orientation of Proteins Associated with Dense-Core Vesicles (DCVs) using CLEM

Exocytosis of catecholamine (adrenaline and noradrenaline) from adrenal chromaffin cells is important for stress responses and insulin release from pancreatic beta cells is critical in regulating blood glucose levels. These exocytic processes are highly regulated by a dozens of key proteins in eukaryotic cells. While the origin, transport, and delivery of cargo-loaded vesicles (dense-core vesicles) and the proteins involved is well studied, how these proteins are organized with respect to dense-core vesicles (DCVs) at the nanoscale remains to be explored. Here, we directly image several proteins that are key regulators of the transport, docking, and priming of DCVs at the plasma membrane. Specifically, correlative super-resolution (dSTORM) light and platinum replica transmission electron microscopy (CLEM) imaging was performed on PC12 cells (a rat adrenal chromaffin-derived neuroendocrine cell line) that were transfected with proteins of interest. We aligned super-resolution dSTORM images of Rab proteins (Rab27a and Rab3a) and Rab effector proteins (granuphilin and Rabphilin) with corresponding TEM images of the plasma membrane and observed the precise correlation between proteins and vesicles at the plasma membrane. The nano-scale molecular architecture of DCVs generated from this correlative imaging will help us understand how key proteins guide the docking, priming, and fusion of DCVs in endocrine cells.

Imaging the Nanoscale Structure of Endocytosis with Correlative Super-Resolution Light and Electron Microscopy

Platinum replicas of unroofed cells can be imaged with an electron microscope to produce high resolution images of the structure of the cytoplasmic side of plasma membrane. It has been challenging, however, to identify proteins within these samples. Super-resolution localization microscopy can image fluorescently-labeled molecules with better than 20 nm precision. We have developed a correlative super-resolution light and platinum replica EM method (CLEM) that couples these two complementary methods to produce images where identified proteins are mapped within the dense structural context of the cell. This correlative method is uniquely suited to investigate the nanometer-scale molecular organization of the plasma membrane and associated organelles such as endocytic and exocytic vesicles. Using this CLEM method, we studied 19 key proteins involved in clathrin-mediated endocytosis. Our data provide a comprehensive molecular architecture of endocytic structures at the nanoscale. We discover that endocytic proteins distribute into three distinct spatial zones; inside, outside, and at the edge of the clathrin coat. The presence and amount of many factors within these zones change during organelle maturation. We propose that the formation and curvature of single clathrin-coated pits is regulated by the recruitment, re-organization, and loss of proteins within these three nanoscale zones during endocytosis.

SuperCLEM: an accessible correlative light and electron microscopy approach for investigation of neurons and glia in vitro.

ABSTRACTThe rapid evolution of super-resolution light microscopy has narrowed the gap between light and electron microscopy, allowing the imaging of molecules and cellular structures at high resolution within their normal cellular and tissue context. Multimodal imaging approaches such as correlative light electron microscopy (CLEM) combine these techniques to create a tool with unique imaging capacity. However, these approaches are typically reserved for specialists, and their application to the analysis of neural tissue is challenging. Here we present SuperCLEM, a relatively simple approach that combines super-resolution fluorescence light microscopy (FLM), 3D electron microscopy (3D-EM) and rendering into 3D models. We demonstrate our workflow using neuron-glia cultures from which we first acquire high-resolution fluorescent light images of myelinated axons. After resin embedding and re-identification of the region of interest, serially aligned EM sections are acquired and imaged using a serial block face scanning electron microscope (SBF-SEM). The FLM and 3D-EM datasets are then combined to render 3D models of the myelinated axons. Thus, the SuperCLEM imaging pipeline is a useful new tool for researchers pursuing similar questions in neuronal and other complex tissue culture systems.

High resolution imaging of the cortex isolated from sea urchin eggs and embryos.

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10–20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy.

Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ∼13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).

Coupling the Structural and Functional Assembly of Synaptic Release Sites.

Information processing in our brains depends on the exact timing of calcium (Ca2+)-activated exocytosis of synaptic vesicles (SVs) from unique release sites embedded within the presynaptic active zones (AZs). While AZ scaffolding proteins obviously provide an efficient environment for release site function, the molecular design creating such release sites had remained unknown for a long time. Recent advances in visualizing the ultrastructure and topology of presynaptic protein architectures have started to elucidate how scaffold proteins establish “nanodomains” that connect voltage-gated Ca2+ channels (VGCCs) physically and functionally with release-ready SVs. Scaffold proteins here seem to operate as “molecular rulers or spacers,” regulating SV-VGCC physical distances within tens of nanometers and, thus, influence the probability and plasticity of SV release. A number of recent studies at Drosophila and mammalian synapses show that the stable positioning of discrete clusters of obligate release factor (M)Unc13 defines the position of SV release sites, and the differential expression of (M)Unc13 isoforms at synapses can regulate SV-VGCC coupling. We here review the organization of matured AZ scaffolds concerning their intrinsic organization and role for release site formation. Moreover, we also discuss insights into the developmental sequence of AZ assembly, which often entails a tightening between VGCCs and SV release sites. The findings discussed here are retrieved from vertebrate and invertebrate preparations and include a spectrum of methods ranging from cell biology, super-resolution light and electron microscopy to biophysical and electrophysiological analysis. Our understanding of how the structural and functional organization of presynaptic AZs are coupled has matured, as these processes are crucial for the understanding of synapse maturation and plasticity, and, thus, accurate information transfer and storage at chemical synapses.

Single-molecule localization microscopy as a promising tool for γH2AX/53BP1 foci exploration

Quantification and structural studies of DNA double strand breaks (DSBs) are an essential part of radiobiology because DSBs represent the most serious damage introduced to the DNA molecule by ionizing radiation. Although standard immunofluorescence confocal microscopy has demonstrated its usefulness in a large number of research studies, it lacks the resolution required to separate individual, closely associated DSBs, which appear after cell exposure to high linear energy transfer (high-LET) radiation and can be visualized as clusters or streaks of radiation-induced repair foci (IRIFs). This prevents our deeper understanding of DSB induction and repair. Recent breakthroughs in super-resolution light microscopy, such as the development of single-molecule localization microscopy (SMLM), offer an optical resolution of approximately an order of magnitude better than that of standard confocal microscopy and open new horizons in radiobiological research. Unlike electron microscopy, SMLM (also referred to as “nanoscopy”) preserves the natural structure of biological samples and is not limited to very thin sample slices. Importantly, SMLM not only offers a resolution on the order of approximately 10 nm, but it also provides entirely new information on the biochemistry and spatio-temporal organization of DSBs and DSB repair at the molecular level. Nevertheless, it is still challenging to correctly interpret these often surprising nanoscopy results. In the present article, we describe our first attempts to use SMLM to explore γH2AX and 53BP1 repair foci induced with 15N high-LET particles.

Super-Resolution Correlative Light and Electron Microscopy (SR-CLEM) Reveals Novel Ultrastructural Insights Into Dendritic Cell Podosomes.

Podosomes are multimolecular cytoskeletal structures that coordinate the migration of tissue-resident dendritic cells (DCs). They consist of a protrusive actin-rich core and an adhesive integrin-rich ring that contains adaptor proteins such as vinculin and zyxin. Individual podosomes are typically interconnected by a dense network of actin filaments giving rise to large podosome clusters. The actin density in podosome clusters complicates the analysis of podosomes by light microscopy alone. Here, we present an optimized procedure for performing super-resolution correlative light and electron microscopy (SR-CLEM) to study the organization of multiple proteins with respect to actin in podosome clusters at the ventral plasma membrane of DCs. We demonstrate that our procedure is suited to correlate at least three colors in super-resolution Airyscan microscopy with scanning electron microscopy (SEM). Using this procedure, we first reveal an intriguing complexity in the organization of ventral and radiating actin filaments in clusters formed by DCs which was not properly detected before by light microscopy alone. Next, we demonstrate a differential organization of vinculin and zyxin with respect to the actin filaments at podosomes. While vinculin mostly resides at sites where the actin filaments connect to the cell membrane, zyxin is primarily associated with filaments close to and on top of the core. Finally, we reveal a novel actin-based structure with SEM that connects closely associated podosome cores and which may be important for podosome topography sensing. Interestingly, these interpodosomal connections, in contrast to the radiating and ventral actin filaments appear to be insensitive to inhibition of actin polymerization suggesting that these pools of actin are not dynamically coupled. Together, our work demonstrates the power of correlating different imaging modalities for studying multimolecular cellular structures and could potentially be further exploited to study processes at the ventral plasma membrane of immune cells such as clathrin-mediated endocytosis or immune synapse formation.

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters.

DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

Direct imaging of uncoated biological samples enables correlation of super-resolution and electron microscopy data

A simple method for imaging biological tissue samples by electron microscopy and its correlation with super-resolution light microscopy is presented. This room temperature protocol, based on protecting thin biological specimens with methylcellulose and imaging with low voltage scanning electron microscopy, circumvents complex classical electron microscopy sample preparation steps requiring dehydration, resin embedding and use of contrast agents. This technique facilitates visualization of subcellular structures e.g. synaptic clefts and synaptic vesicles in mouse brain tissue and the organization of mitochondrial cristae in the zebrafish retina. Application of immunogold protocols to these samples can determine the precise localization of synaptic proteins and, in combination with super-resolution light microscopy methods clearly pinpoints the subcellular distribution of several proteins in the tissue. The simplicity of the method, including section collection on a silicon wafer, reduces artefacts and correlates protein location with sample morphology.

A microscopy-based approach for studying meiosis in live and fixed human oocytes.

Human eggs frequently carry an incorrect number of chromosomes, which is a leading cause of pregnancy loss and congenital disorders. The origins of high aneuploidy rates in human eggs have remained largely unclear. This is due to two main reasons: first, the availability of human eggs is limited so that studies of fixed human eggs typically involve very small numbers and limited quantifications. Second, methods for studying meiosis in live human eggs have been missing. The ever rising prevalence of Assisted Reproductive Technologies has facilitated a recent breakthrough in the field. The mechanistic basis of meiosis in humans can now be examined directly in live eggs. Here, we present a robust method for culturing human eggs in vitro and describe how meiotic processes in human eggs can be studied in real time using fluorescent reporters. We further describe methods for the in-depth analysis of immunolabeled eggs by super-resolution light microscopy.

Super-resolution longitudinally polarized light needle achieved by tightly focusing radially polarized beams

Based on the vector diffraction theory, a super-resolution longitudinally polarized optical needle with ultra-long depth of focus (DOF) is generated by tightly focusing a radially polarized beam that is modulated by a self-designed ternary hybrid (phase/amplitude) filter (THF). Both the phase and the amplitude patterns of THF are judiciously optimized by the versatile particle swarm optimization (PSO) searching algorithm. For the focusing configuration with a combination of a high numerical aperture (NA) and the optimized sine-shaped THFs, an optical needle with the full width at half maximum (FWHM) of 0.414λ and the DOF of 7.58λ is accessed, which corresponds to an aspect ratio of 18.3. The demonstrated longitudinally polarized super-resolution light needle with high aspect ratio opens up broad applications in high-density optical data storage, nano-photolithography, super-resolution imaging and high-efficiency particle trapping.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (−195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Functional insights into pathogen biology from 3D electron microscopy.

In recent years, novel imaging approaches revolutionised our understanding of the cellular and molecular biology of microorganisms. These include advances in fluorescent probes, dynamic live cell imaging, superresolution light and electron microscopy. Currently, a major transition in the experimental approach shifts electron microscopy studies from a complementary technique to a method of choice for structural and functional analysis. Here we review functional insights into the molecular architecture of viruses, bacteria and parasites as well as interactions with their respective host cells gained from studies using cryogenic electron tomography and related methodologies.

Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.

Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo.

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP).