Super-resolution Light Research Articles

Application of super-resolution microscopy to the study of B and T lymphocyte mitochondria morphology and metabolism

Abstract High-resolution microscopy techniques have advanced our understanding of lymphocyte biology. An emerging focus in the field of immunometabolism on activation-induced metabolic reprogramming and its effects on immune cell function will require in-depth analyses of mitochondria. However, even with newer imaging technologies, obtaining super-resolution images of lymphocyte mitochondria remains challenging due to their small cytoplasmic sizes. Here, we compared standard confocal laser microscopy with three commonly used super-resolution methods to analyze activation-induced mitochondrial changes in B and T cells. Naïve B cells were stimulated in vitro via Toll-like receptor 9 and/or the B cell receptor, and naïve T cells were differentiated in vitro into activated or T regulatory phenotypes. The cells were imaged using standard laser confocal microscopy, AiryScan, stimulated emission depletion (STED) microscopy, and computerized tomography electron microscopy (TEM). Mitochondrial morphology was best resolved by staining both the mitochondrial membrane and matrix simultaneously. In preliminary analyses, we observed activation-induced changes in the distribution of lymphocyte mitochondria. Of the imaging methods used, only TEM allowed for 3D reconstructions of the mitochondria’s internal structure, which revealed swelling of the inner matrix and significant loss of cristae in B cells, but not in T cells. In conclusion, the super-resolution light microscopy techniques we developed appear to be powerful tools for immunometabolism studies of lymphocyte mitochondria, having demonstrated clear visualization even at the molecular level, while TEM answered key questions about intra-mitochondrial morphology.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Determining the bacterial cell biology of Planctomycetes

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.

Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells.

Dozens of proteins capture, polymerize and reshape the clathrin lattice during clathrin-mediated endocytosis (CME). How or if this ensemble of proteins is organized in relation to the clathrin coat is unknown. Here, we map key molecules involved in CME at the nanoscale using correlative super-resolution light and transmission electron microscopy. We localize 19 different endocytic proteins (amphiphysin1, AP2, β2-arrestin, CALM, clathrin, DAB2, dynamin2, EPS15, epsin1, epsin2, FCHO2, HIP1R, intersectin, NECAP, SNX9, stonin2, syndapin2, transferrin receptor, VAMP2) on thousands of individual clathrin structures, generating a comprehensive molecular architecture of endocytosis with nanoscale precision. We discover that endocytic proteins distribute into distinct spatial zones in relation to the edge of the clathrin lattice. The presence or concentrations of proteins within these zones vary at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization and loss of proteins within these partitioned nanoscale zones.

Examination of Antigen-Induced Endocytic Structure Formation in B Lymphocytes

B lymphocytes are an essential component of the adaptive immune response against pathogens. The ability of a B cell lineage to adapt to better recognize a given antigen depends critically upon the ability of each B cell to bind and internalize antigen through its B cell receptor (BCR). The mechanism of antigen internalization by B cells appears to require cooperation between BCR signaling, the actomyosin network and clathrin-mediated endocytic processes. However, it remains unclear how these actors are coordinated to achieve efficient and controlled uptake of structurally heterogeneous antigens. To better understand this process, we set out to directly observe antigen-induced endocytic structures in the human DG-75 B cell line using a combination of conventional and super-resolution microscopy techniques including confocal laser scanning microscopy (CLSM), total-internal reflected fluorescence microscopy (TIRF-M), and correlative super-resolution light and electron microscopy (CLEM). Cells expressing GFP-tagged BCR and red fluorescent protein-tagged endocytic proteins were stimulated with anti-human IgM Fab’2 to model BCR clustering and internalization. We observed recruitment of early adaptors of clathrin mediated endocytosis – FCHO1, Eps15, Epsin, CALM, and clathrin itself – to Fab’2-induced BCR clusters by TIRF-M and CLSM. CLEM revealed that BCR clusters are heterogeneous in size and frequently partition with clathrin on larger membrane invaginations. This work reveals novel structural features of antigen-induced endocytic structures and expands our understanding of the mechanism by which B cells may accommodate antigen of varying size during internalization.

The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

CLAFEM: Correlative light atomic force electron microscopy.

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

3D subcellular localization with superresolution array tomography on ultrathin sections of various species.

Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterwardby electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.

Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells.

Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.

Real‐Time Controls of Designer Surface Plasmon Polaritons Using Programmable Plasmonic Metamaterial

Manipulating the dispersion behaviors of electromagnetic (EM) waves at subwavelength scale provides many exciting physical phenomena and functionalities such as the phase matching, gain enhancement, super resolution, and slow light. Among the dispersion manipulations, surface plasmon polaritons (SPPs) are typical EM modes to control the wave flows owing to their sensitivity to the designed decorations on the metal–dielectric interfaces. However, either on metal–dielectric interfaces or decoration surfaces, it is challenging to construct reconfigurable and programmable SPPs to control SPP waves dynamically in real time. Here, the authors propose the concepts of coding and programmable designer SPPs, which are realized by an ultrathin corrugated metallic strip loaded with active devices and a digital system. Based on the big freedom provided by the coding representation, the authors experimentally demonstrate that a simple programmable SPP system can reach at least three different digital‐analog functionalities: (1) SPP logical gate based on 1‐bit coding; (2) SPP digital phase shifter based on 2‐bit coding; and (3) controllable SPP slow waves based on 4‐bit coding. Such functionalities can be switched in real time using a single prototype and the digital control system.

Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy.

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.

Shh and ZRS enhancer colocalisation is specific to the zone of polarising activity.

Limb-specific Shh expression is regulated by the (∼1 Mb distant) ZRS enhancer. In the mouse, limb bud-restricted spatiotemporal Shh expression occurs from ∼E10 to E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS colocalisation only in the Shh-expressing regions of the limb bud, in a conformation consistent with enhancer-promoter loop formation. However, in all tissues and at all developmental stages analysed, Shh-ZRS spatial distances were still consistently shorter than those to a neural enhancer located between Shh and ZRS in the genome. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS colocalisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh and ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate the expression of Shh.

VirusMapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy.

The nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. Their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. The recent development of super-resolution (SR) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. In addition, these techniques provide the added benefit of molecular specific labelling and the capacity to investigate viral structural dynamics using live-cell microscopy. However, there is a lack of robust analytical tools that allow for precise mapping of viral structure within the setting of infection. Here we present an open-source analytical framework that combines super-resolution imaging and naïve single-particle analysis to generate unbiased molecular models. This tool, VirusMapper, is a high-throughput, user-friendly, ImageJ-based software package allowing for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. We demonstrate the usability of VirusMapper by applying it to SIM and STED images of vaccinia virus in isolation and when engaged with host cells. VirusMapper allows for the generation of accurate, high-content, molecular specific virion models and detection of nanoscale changes in viral architecture.

Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Abstract B26: Centriole amplification during mammalian multiciliated cell development reveals a novel centrosome asymmetry

Abstract The centrosome comprises a mother and a daughter centriole, both regarded as equivalent in their ability to form new centrioles. Their symmetric duplication during the cell cycle is crucial for proper genetic partitioning between the daughter cells. In multiciliated cells, hundreds of centrioles are thought to appear de novo, i.e. independently from the centrosome, to nucleate the same number of motile cilia and propel physiological fluids. The origin of these centrioles remains unknown. Here we provide the cellular mechanism driving large-scale centriole production in multiciliated cells and identify the daughter centriole of the centrosome as the nucleation point for 95% of the new centrioles. Using live imaging combined with correlative super-resolution light and electron microscopy microscopy, we show that centriole amplification proceeds through a recurrent mechanism whereby the daughter centriole concomitantly generates the new centrioles and their cytoplasmic shuttles known as deuterosomes. By revealing this novel centrosome cycle, our work challenges the postulate that large-scale centriole production is a de novo mechanism in mammalian cells. In addition, it uncovers a fundamental asymmetry between mother and daughter centrioles in the control of centriole biogenesis and number. These results may have critical implications in the understanding of the etiology of cilia-related diseases, brain defects, and tumor formation. Citation Format: Adel Al Jord, Nathalie Spassky, Alice Meunier. Centriole amplification during mammalian multiciliated cell development reveals a novel centrosome asymmetry. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B26.

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Stochastic Single-Molecule Dynamics of Synaptic Receptor Domains

Neurotransmitter receptor molecules, concentrated in postsynaptic membrane domains along with scaffold and other kinds of molecules, are crucial for signal transmission across chemical synapses. Using superresolution light microscopy it has been found that individual receptor molecules have very quick turnover rates of the order of seconds, while synaptic receptor domains are globally stable over months or even longer periods of time. Through an interplay between experiments on a minimal model system and theoretical modeling it has been demonstrated that the diffusion and reaction properties of receptors and scaffolds at the membrane are necessary and sufficient for the formation of stable receptor-scaffold domains of the characteristic size observed in nerve cells. However, existing theoretical models of synaptic receptor domains are limited to the mean-field level, and thus cannot account for the stochastic trajectories of individual synaptic molecules observed in experiments on nerve cells. Here we formulate and study a stochastic lattice gas model of synaptic receptor domains which allows us to overcome this challenge. Using kinetic Monte Carlo simulations we show that our stochastic lattice gas model reproduces the experimentally observed patterns of receptor-scaffold domains of a stable characteristic size, while also allowing for a coupling of reaction and diffusion noise to the nonlinear reaction and diffusion dynamics of receptors and scaffolds in the highly crowded membrane environments provided by synaptic domains. Our stochastic lattice gas model allows us to make detailed comparisons with experimental data on the stochastic properties of synaptic domains, and provides a bridge connecting the rapid and highly stochastic dynamics that rule the molecular realm of cell membranes to the global stability of synaptic receptor domains essential for the biological function of synapses.

Deep nuclear invaginations are linked to cytoskeletal filaments - integrated bioimaging of epithelial cells in 3D culture.

The importance of context in regulation of gene expression is now an accepted principle; yet the mechanism by which the microenvironment communicates with the nucleus and chromatin in healthy tissues is poorly understood. A functional role for nuclear and cytoskeletal architecture is suggested by the phenotypic differences observed between epithelial and mesenchymal cells. Capitalizing on recent advances in cryogenic techniques, volume electron microscopy and super-resolution light microscopy, we studied human mammary epithelial cells in three-dimensional (3D) cultures forming growth-arrested acini. Intriguingly, we found deep nuclear invaginations and tunnels traversing the nucleus, encasing cytoskeletal actin and/or intermediate filaments, which connect to the outer nuclear envelope. The cytoskeleton is also connected both to other cells through desmosome adhesion complexes and to the extracellular matrix through hemidesmosomes. This finding supports a physical and/or mechanical link from the desmosomes and hemidesmosomes to the nucleus, which had previously been hypothesized but now is visualized for the first time. These unique structures, including the nuclear invaginations and the cytoskeletal connectivity to the cell nucleus, are consistent with a dynamic reciprocity between the nucleus and the outside of epithelial cells and tissues.