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Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far, the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy. We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs µm -2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains, and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

Background: We have recently developed the correlative light and electron microscopy of hematoxylin and eosin (H&E)-stained glass slides using the ‘NanoSuit’ method. The aim of this study is to explore the utility of the new NanoSuit-correlative light and electron microscopy method combined with scanning electron microscopy-energy dispersive X-ray spectroscopy elemental analysis for the diagnosis of lanthanum phosphate deposition in the H&E-stained glass slides. Methods: Nine H&E-stained glass slides of the upper gastrointestinal tract mucosa containing the brown pigmented areas by light microscopic observation, which were suspected as lanthanum phosphate deposition, were observed and analyzed by scanning electron microscopy-energy dispersive X-ray spectroscopy using the NanoSuit-correlative light and electron microscopy method. Results: In all nine slides, the new NanoSuit-correlative light and electron microscopy method combined with scanning electron microscopy-energy dispersive X-ray spectroscopy revealed the accumulation of both lanthanum and phosphorus in the tissue area corresponding to the brown pigment deposition. In addition to the existence of lanthanum phosphate in the stomach and duodenum, known target organs, we observed deposition in the esophagus for the first time. Furthermore, we observed lanthanum phosphate deposition in the background mucosa of stomach containing primary adenocarcinoma. Conclusions: Scanning electron microscopy-energy dispersive X-ray spectroscopy analysis using the NanoSuit-correlative light and electron microscopy method is useful for the diagnosis of lanthanum phosphate deposition in the H&E-stained glass slides. Lanthanum phosphate deposition occurs not only in the stomach and duodenum but also in the esophagus.

Loss of mitochondrial membrane potential (ΔΨm) triggers dramatic structural changes in mitochondria from a tubular to globular shape, referred to as mitochondrial fragmentation; the resulting globular mitochondria are called swelled or ring/doughnut mitochondria. We evaluated the early period of structural changes during the ΔΨm loss-induced transformation after carbonyl cyanide m -chlorophenyl hydrazine (CCCP) administration using a newly developed correlative microscopic method combined with fluorescence microscopic live imaging and volume electron microscopy. We found that most mitochondria changed from a tubular shape to a globular shape without fusion or fission and typically showed ring shapes within 10 min after CCCP exposure. In contrast, most ring mitochondria did not have a true through hole; rather, they had various indents, and 47% showed stomatocyte shapes with vase-shaped cavities, which is the most stable physical structure without any structural support if the long tubular shape shortens into a sphere. Our results suggested that loss of ΔΨm triggered collapse of mitochondrial structural support mechanisms.

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting superresolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

Cryo-correlative light and electron microscopy (cryo-CLEM) is a powerful technique that combines fluorescence imaging for specific localization with electron microscopy for detailed structural analysis, enabling high-resolution exploration of synaptic structures in neurons. In this study, we employed a cryo-CLEM approach using three independent alignment markers to precisely correlate electron microscopy (EM) images with light microscopy (LM) images of neuronal synapses under cryogenic conditions. This methodology revealed a distinctive pattern of electron densities in the synaptic clefts. Additionally, we were able to capture high-resolution images of presynaptic vesicles in various states, underscoring the potential of cryo-CLEM in advancing synaptic research.

Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electrondense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material. Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using three-dimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome–organelle relationships during macroautophagy. Key words: 3D-CLEM, autophagosome, electron microscopy, endoplasmic reticulum, lysosome

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex. When viruses infect human cells, they hijack the cell’s machinery to produce the proteins they need to replicate. Retroviruses like HIV-1 do this by entering the nucleus and inserting their genetic information into the genome of the infected cell. This requires HIV-1 to convert its genetic material into DNA, which is then released from the protective shell surrounding it (known as the capsid) via a process called uncoating. The nucleus is enclosed within an envelope containing pores that molecules up to a certain size can pass through. Until recently these pores were thought to be smaller than the viral capsid, which led scientists to believe that the HIV-1 genome must shed this coat before penetrating the nucleus. However, recent studies have found evidence for HIV-1 capsid proteins and capsid structures inside the nucleus of some infected cells. This suggests that the capsid may not be removed before nuclear entry or that it may even play a role in helping the virus get inside the nucleus. To investigate this further, Müller et al. attached fluorescent labels to the newly made DNA of HIV-1 and some viral and cellular proteins. Powerful microscopy tools were then used to monitor the uncoating process in various cells that had been infected with the virus. Müller et al. found large amounts of capsid protein inside the nuclei of all the infected cells studied. During the earlier stages of infection, the capsid proteins were mostly associated with viral DNA and the capsid structure appeared largely intact. At later time points, the capsid structure had been broken down and the viral DNA molecules were gradually separating themselves from these remnants. These findings suggest that the HIV-1 capsid helps the virus get inside the nucleus and may protect its genetic material during conversion into DNA until right before integration into the cell’s genome. Further experiments studying this process could lead to new therapeutic approaches that target the capsid as a way to prevent or treat HIV-1.

Recent developments in serial-section electron microscopy allow the efficient generation of very large image data sets but analyzing such data poses challenges for software tools. Here we introduce Volume Annotation and Segmentation Tool (VAST), a freely available utility program for generating and editing annotations and segmentations of large volumetric image (voxel) data sets. It provides a simple yet powerful user interface for real-time exploration and analysis of large data sets even in the Petabyte range.