Microscopy and Microanalysis

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data acquisition approaches in single particle cryo-EM do not take it into account. Here, we demonstrate how the sample thickness can be determined before data acquisition, allowing to identify optimal regions and restrict automated data collection to images with preserved high-resolution details. This quality over quantity approach, almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to lack of high-resolution information. It maximizes data collection efficiency and lowers the electron microscopy time required per dataset. This strategy is especially useful, if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck. SynopsisSample thickness is a key parameter in single particle cryo-electron microscopy. Determining sample thickness before data acquisition allows to target optimal areas and maximize data output quality of single particle cryo-electron microscopy sessions. Scripts and optimized workflows for EPU and SerialEM are presented and available as open-source.

Micro-CT has revolutionized functional morphology by enabling volumetric reconstruction of biological specimens at micrometre scales, but its accuracy is compromised by fixation artefacts. State-of-the-art in vivo imaging avoids this, but still requires subjects to be tethered, anaesthetized, or stained. Here we use ultra-fast synchrotron-based micro-CT to produce the first 3D scan of an unrestrained living organism at micrometre resolution, demonstrating the potential of this method in physiology, behaviour, and biomechanics.

Micro-CT imaging is a powerful tool for generating high resolution, isotropic three-dimensional datasets of whole, centimeter-scale model organisms that can be used for qualitative and quantitative analysis. The small size, global freshwater distribution, wide range of cell size and structures of micron scale, and common use of D. magna in toxicological and environmental studies make it an ideal model for demonstrating the potential power of micro-CT-enabled whole-organism phenotyping. This protocol details the steps involved in D. magna samples preparation for micro-CT: euthanasia, fixation, staining, and resin embedding. Micro-CT reconstructions of samples imaged using synchrotron micro-CT reveal histological (microanatomic) features of organ systems, tissues, and cells in the context of the entire organism at sub-micron resolution, and in 3 dimensionality. The enabled "3D histology" and 3D renderings can be used towards morphometric analyses across cells, tissues, and organ systems for both descriptive and hypothesis testing studies.

Advances in single-particle cryogenic electron microscopy (cryoEM) now allow for routine structure determination of well-behaved biological specimens to high-resolution. Despite advances in the electron microscope, direct electron detectors, and data processing software, the preparation of high-quality grids with thin layers of vitreous ice containing the specimen of interest in random orientations remains a critical bottleneck for many projects. Although numerous efforts have been dedicated to overcoming hurdles frequently encountered during specimen vitrification using traditional blot-and-plunge specimen preparation techniques, the development of blot-free grid preparation devices provide a unique opportunity to carefully tune ice thickness, particle density, and specimen behavior during the vitrification process for improvements in image quality. Here, we describe critical steps of high-quality grid preparation using a SPT Labtech chameleon, evaluation of grid quality/ice thickness using the chameleon software, high-throughput imaging in the electron microscope, and recommend steps for troubleshooting grid preparation when standard parameters fail to yield suitable specimen. Video LinkContents of this manuscript are available as a video tutorial. This video can be found here

Super resolution detector acquisition for cryo-EM has been used to improve the clarity of cryo-EM reconstructions. Recent reports have demonstrated achieving resolutions beyond the physical Nyquist limit using super resolution acquisition. Here, we demonstrate exceeding the physical Nyquist limitation by pre-processing the raw micrograph movies from "counting mode" data which has already reached physical Nyquist reconstruction resolution. To demonstrate functionality, micrograph movies of five datasets were pre-processed and demonstrate that it is possible to exceed the physical Nyquist limit via pixel doubling before motion correction. We call this "post-acquisition super resolution", or PASR. While this was originally developed for processing of giant virus datasets, where acquiring at high magnification is not always possible or desirable, it is also shown to work for smaller objects such as adeno-associated virus (AAV) and apoferritin, both of which are still high symmetry, and jack bean urease, with lower symmetry. PASR could reduce the magnification required to achieve desired resolutions, which may increase collection efficiency. PASR can also be of use for in vivo tomography and facilities with high storage demands. However, this method should only be used for data which is able to achieve the Nyquist limit without PASR pre-processing. It will not improve attained resolutions of data which does not already reach the Nyquist limit.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Understanding how chromatin architecture changes during cellular differentiation requires structural methods that can resolve native genomic organization at high resolution. Here, we present an AI-assisted cryo-electron tomography (cryo-ET) and segmentation workflow to quantify chromatin compaction across various stages of motor neuron differentiation from induced pluripotent stem cells (iPSC). By directly imaging extracted and vitrified chromatin, we preserve native structure and avoid artifacts from heavy metal staining and resin embedding. Using three-dimensional (3D) density analysis, we measure chromatin density and capture the progressive increase in chromatin compaction with lineage commitment. This is then correlated with population-averaged Hi-C experiments, observing consistency between the microscale higher order structure of chromatin and global contact patterns. Our approach enables direct visualization of chromatin organization under near-physiological conditions, bridging the gap between structural imaging and genome-wide contact mapping. This platform therefore establishes an AI-assisted experimental framework for linking chromatin architecture to regulatory mechanisms during differentiation.

In cryo-electron microscopy (cryo-EM), imaging of biological specimens is restricted by the limited field of view and by sample thickness. Hard X-ray imaging, with its ability to penetrate samples several tens of micrometers thick, offers a complementary approach for high-resolution visualization. A major concern is whether cryo-preserved samples can withstand the handling conditions at synchrotron facilities without excessive icing, and whether the radiation exposure during X-ray imaging compromises specimen integrity, thereby hindering subsequent attempts to achieve high-resolution 3D reconstructions via cryo-EM. To evaluate this, we deposited apoferritin samples on a cryo-EM grid, exposed them to varied X-ray doses typical for X-ray tomography experiments at a synchrotron facility, and subsequently analysed the exposed particles by cryo-EM. Despite the apparent damage sustained throughout the experiment, the samples remained amenable to cryo-EM analysis, with structural details at a resolution of [~]4 [A] at the highest absorbed X-ray dose of 100 MGy. By comparison, a similar cryo-EM dataset of the apoferritin particles that were not exposed to X-rays but were mounted on the same cryo-EM grid, resulted in a 3D reconstruction at 3.17 [A] resolution. Thus, while radiation damage may limit the high-resolution information in specimens processed by cryo-X-ray tomography, the cryo-preserved biological material exposed to these high X-ray doses can be still used for subsequent cryo-EM workflows aiming to obtain structural biology insights at intermediate to high resolution. These findings lay the groundwork for an integrated imaging workflow that combines X-ray and cryo-EM techniques to enable multiscale analysis of thick vitrified biological specimens.

Current guidelines for depositing cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) data require submission of unfiltered, unmasked, and unsharpened raw half-maps. The Fourier Shell Correlation (FSC) between the half-maps is then used as a proxy for the signal-to-noise ratio (SNR) to estimate the reconstructions resolution. This policy was introduced to enable independent validation of reported resolutions. Although developed to safeguard data integrity and minimize bias, these guidelines do not account for specific features of modern cryo-EM processing software, in particular weighting schemes that are not retained in half-map depositions and yet in general are necessary to recapitulate resolution estimates. As a results, resolution estimates and other validation statistics based on half-maps FSC may be under- or overestimated. Here, we describe the limitations of the current deposition guidelines and propose an alternative: depositing cryo-EM results in Fourier (reciprocal) space together with the mandatory deposit of molecular masks or their descriptors. This approach addresses the current limitations, preserves critical information from the reconstruction process, and better supports downstream analyses. HighlightsO_LIReciprocal-space deposition to preserve signal and uncertainty estimates. C_LIO_LIMandatory molecular masks deposition for accurate and unbiased FSC validation. C_LIO_LILimitations of half-map-based Fourier Shell Coefficients. C_LIO_LIThe difference maps can be calculated. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/690836v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1a35884org.highwire.dtl.DTLVardef@1086af4org.highwire.dtl.DTLVardef@1a5f118org.highwire.dtl.DTLVardef@177815a_HPS_FORMAT_FIGEXP M_FIG C_FIG

In electron microscopy, sparse imaging consists in the collection of a limited subset of the image pixels, which can be used to reduce electron beam damage. Scanning transmission electron microscopy (STEM) is particularly adapted to sparse imaging owing to the scanning nature of the method, scan patterns can be designed where fewer sample locations are targeted. However, since some of the pixels are not scanned, there is an inherent loss of information. Several algorithms were developed to reconstruct missing pixels with high fidelity. Whereas sparse imaging and missing pixel reconstruction in 2D experiments are mature methods, the application of sparse imaging in 3D scanning transmission electron tomography (STET) is rare and still under development. The main difficulty encountered in tomography studies is the tilt-series alignment, which must be accurate to ensure high-quality 3D reconstruction. Because sparse images contain only a certain portion of the original information, the images constituting sparse tilt-series might not share enough mutual information to guarantee an accurate alignment, even after missing pixel reconstruction. This work presents for the first time a thorough analysis of the fiducial alignment and reconstruction of sparse (cryo)STET tilt-series. Furthermore, the limits of sparse imaging are explored to estimate the minimum amount of information required to obtain good-quality 3D reconstructions. The use of a cryo-fixed biological sample is motivated by the fact that cryo-samples are typical highly beam-sensitive samples, and that the intricate nature and structure complexity of biological samples place them among the most difficult to reconstruct with high details.

Cryo-electron tomography (cryo-ET) enables high-resolution, three-dimensional imaging of cellular structures in their native, frozen state. However, image quality is limited by a trade-off between angular sampling and radiation damage, making the choice of angular increment during data collection a critical parameter, affecting tomogram quality and downstream analyses. Optimising this increment is challenging due to the high demands on microscope time, storage, and computation. In this study, we systematically evaluated tilt increments of 1{degrees}, 2{degrees}, 3{degrees}, 5{degrees}, and 10{degrees} using lamellae from Dictyostelium discoideum cells. Keeping total electron dose constant, we found that finer tilt increments (1-3{degrees}) produced better-aligned tomograms with higher signal-to-noise ratios and improved outcomes in template matching and subtomogram averaging. A 3{degrees} increment emerged as the optimal balance between data quality, alignment accuracy, dose per image, and processing efficiency. This practical recommendation supports both high-throughput and high-resolution structural studies and can guide future cryo-ET data acquisition strategies.

Cryo-electron microscopy (cryo-EM) enables the study of protein complexes, cytoskeletal elements, and organelles in three dimensions without the use of chemical fixation. Most cryo-EM studies focus on vitreously frozen individual cells separated from their native tissue contexts. This reliance on imaging of single cells is primarily due to technical challenges associated with preparing fresh tissue sections at a thinness sufficient for visualization via cryo-EM. Highly heterogenous and specialized tissues, such as brain, are especially affected by this limitation as the cellular, subcellular, and synaptic milieus can significantly vary across neuroanatomical locations. To address this limitation, we established new instrumentation and a workflow that consists of: 1) high-pressure freezing of fresh brain tissue; 2) tissue trimming followed by cryo-focused ion beam milling via the H-bar approach to generate ultrathin lamellae; and 3) cryo-EM imaging. Here, we apply this workflow to visualize the fine ultrastructural details of organelles, as well as cytoskeletal and synaptic elements that comprise the cortical neuropil within fresh, unfixed mouse brain tissue. Moreover, we present initial studies that apply principles of the above workflow to the analysis of postmortem human brain tissue. Overall, our work integrates the strengths of cryo-electron microscopy and tissue-based approaches to produce a generalizable workflow capable of visualizing subcellular structures within complex tissue environments.

BackgroundElectron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. MethodsHere, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. ResultsWe demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in [~]15 [A] resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. DiscussionThis study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.

Measurement of in-vivo chromosome conformations (structures) in single cells is a major technological goal of structural biology. If one could identify many genetic loci in a microscope image despite the limited palette of fluorescent colors used to label them, then the conformation could be solved at some resolution by connecting the dots. Computational tools for making this reconstruction are expected to produce near-perfect reconstructions when the number of fluorescent colors is high enough, irrespective of the number of loci assayed. Here we report the first experimental test of the performance of these reconstruction algorithms and check their ability to reconstruct experimentally-measured conformations. We also demonstrate the experimental metrics needed to assess reconstruction quality. Our results indicate that current sequential FISH experiments may be close to the point where the reconstructions are nearly flawless at some distance scales.

Creating a high-resolution brain atlas in diverse species offers crucial insights into general principles underlying brain function and development. A volume electron microscopy approach to generate such neural maps has been gaining importance due to advances in imaging, data storage capabilities, and data analysis protocols. Sample preparation remains challenging and is a crucial step to accelerate the imaging and data processing pipeline. Here, we introduce several replicable methods for processing the brains of the gastropod mollusc, Berghia stephanieae for volume electron microscopy. Although high-pressure freezing is the most reliable method, the depth of cryopreservation is a severe limitation for large tissue samples. We introduce a BROPA-based method using pyrogallol and methods to rapidly process samples that can save hours at the bench. This is the first report on sample preparation and imaging pipeline for volume electron microscopy in a gastropod mollusc, opening up the potential for connectomic analysis and comparisons with other phyla.

Cryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the techniques performance in structural determination, we present an SPA map at subnanometer resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle.

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom ([A]) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 [A] and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 [A], respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset ([≤]1,000 micrographs) was sufficient to generate a 3.3 [A] reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg+2. In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

The big data era in biology is underway, but the study of organismal form has been slow to capitalize on advances in imaging and computation. Modern imaging can digitize whole organisms, but low throughput has limited the effort to document morphological diversity. Within the open science initiative Antscan, we applied high-throughput synchrotron X-ray microtomography to capture phenotypes across a diverse and ecologically dominant insect group -- ants. We provide 2193 whole-body 3D ant datasets from 792 species to broadly cover the ant phylogeny with a global scope, also pairing phenomic data with genome sequencing projects. Scans acquired with standardized parameters facilitate automated analysis and free access to data can broaden the audience and incentivize methods development. Antscan presents a scalable approach to create libraries of diverse anatomies, heralding a new era of studies on the evolution, structure, and function of organismal phenotypes.

We have recently introduced a microsecond time-resolved version of cryo-electron microscopy (cryo-EM) to enable the observation of the fast conformational motions of proteins. Our technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. We have previously described two alternative implementations of the technique, using either an optical microscope or performing revitrification experiments in situ. Here, we show that it is possible to obtain near-atomic resolution reconstructions from in situ revitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within our spatial resolution. Interestingly, we observe that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation. SynopsisNear-atomic resolution reconstructions can be obtained from in situ melted and revitrified cryo samples. Revitrification results in a more homogeneous angular distribution.