Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1–3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4–6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that—compared with previous human NPC models obtained from purified NEs—the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs.DOI: http://dx.doi.org/10.7554/eLife.15015.001

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozenhydrated specimens in a variety of healthy and diseased conditions with and without treatments.

Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12–15 and 5–6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods.

Patchy particle interactions are predicted to facilitate the controlled self-assembly and arrest of particles into phase-stable and morphologically tunable "equilibrium" gels, which avoids the arrested phase separation and subsequent aging that is typically observed in traditional particle gels with isotropic interactions. Despite these promising traits of patchy particle interactions, such tunable equilibrium gels have yet to be realized in the laboratory due to experimental limitations associated with synthesizing patchy particles in high yield.Here, we introduce a supramolecular metal-coordination platform consisting of metallic nanoparticles linked by telechelic polymer chains, which validates the predictions associated with patchy particle interactions and facilitates the design of equilibrium particle hydrogels through limited valency interactions. We demonstrate that the interaction valency and self-assembly of the particles can be effectively controlled by adjusting the relative concentration of polymeric linkers to nanoparticles, which enables the gelation of patchy particle hydrogels with programmable local anisotropy, morphology, and low mechanical percolation thresholds. Moreover, by crowding the local environment around the patchy particles with competing interactions, we introduce an independent method to control the self-assembly of the nanoparticles, thereby enabling the design of highly anisotropic particle hydrogels with substantially reduced percolation thresholds. We thus establish a canonical platform that facilitates multifaceted control of the self-assembly of the patchy nanoparticles en route to the design of patchy particle gels with tunable valencies, morphologies, and percolation thresholds. These advances lay important foundations for further fundamental studies of patchy particle systems and for designing tunable gel materials that address a wide range of engineering applications.

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dimensional (3D) visualization of vitrified cells can uncover structures of subcellular 27 complexes without chemical fixation or staining. Here, we present a pipeline integrating three 28 imaging modalities to visualize the same specimen at cryogenic temperature at different scales: 29 cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron 30 microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark 31 revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast 32 cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone 33 Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be 34 applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-35 hydrated specimens in a variety of healthy and diseased conditions with and without treatments. 36 37 KEYWORDS 38 Airyscan microscopy; cryo correlative-light and electron microscopy, cryoCLEM; volume 39 cryo-focused ion bean scanning electron microscopy, cryoFIB-SEM; cryo-electron tomography, 40 cryoET; Hsp104 chaperone; protein aggregation 41 42 46 determination 1 . However, several limitations preclude its wider application, including the thickness 47 of some samples and difficulties in locating and identifying features of interest within them. To48 visualize molecular details in thicker samples by cryoET, such as regions in eukaryotic cells away 49 from the thin cell periphery, cryogenic focused ion beam scanning electron microscopy (cryoFIB-50 SEM) has been used to generate thin lamellae from vitrified cells 2-5 (i.e. thin layers through the 51 3 bulky cell) with a process called "ion beam milling", enabling many exciting biological observations 52 inside the cell 6 . However, technical challenges remain in ensuring that the milled lamellae contain 53 the features of interest. Correlative light and electron microscopy (CLEM) can overcome this 54 challenge by fluorescently labelling targets 7,8 , thereby guiding cryoFIB-SEM milling 9,10 and the 55 selection of optimal imaging areas for cryoET experiments, as well as aiding the interpretation of 56 observed features. However, even the smallest eukaryotic cells are typically several microns thick 57 and the desirable lamella thickness range is ~100-500 nm; thus, targeting nanoscale features 58 along the z direction remains extremely challenging. Here, as a proof-of-principle, we present a 59 pipeline to address this challenge by using high-resolution CryoAiryscan Confocal Microscopy 60 (CACM) to determine the z position of fluorescent targets within cells that were vitrified on electron 61 microscopy (EM) grids, followed by cryoFIB-SEM "mill and view" (MAV) imaging, which provides 62 a 3D view of whole cells with resolvable organelles as they are being milled to produce a lamella 63 containing the target of interest, and ending with visualization of molecular details of regions of 64 interest by cryoET. 65 66...

This study provides evidence for the Golgi-like activity of the multilayered interlaced network (MIN) and new ultrastructural observations of the MIN in the sporoplasm of Anncaliia algerae, a microsporidium that infects both insects and humans. The MIN is attached to the end of the polar tubule upon extrusion from the germinating spore. It surrounds the sporoplasm, immediately below its plasma membrane, and most likely maintains the integrity of the sporoplasm, as it is pulled through the everting polar tube. Furthermore, the MIN appears to deposit its dense contents on the surface of the sporoplasm within minutes of spore discharge thickening the plasma membrane. This thickening is characteristic of the developmental stages of the genus Anncaliia. The current study utilizes transmission electron microscopy (TEM), enzyme histochemistry, and high voltage TEM (HVEM) with 3D tomographic reconstruction to both visualize the structure of the MIN and demonstrate that the MIN is a Golgi-related structure. The presence of developmentally regulated Golgi in the Microsporidia has been previously documented. The current study extends our understanding of the microsporidial Golgi and is consistent with the MIN being involved in the extracellular secretion in Anncaliia algerae. This report further illustrates the unique morphology of the MIN as illustrated by HVEM using 3D tomography.