Microscopy and Microanalysis

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.

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.

Cellular cryo electron tomography explores tissue and cells in their unstained flash-frozen native state, revealing in situ the structure of macromolecules together with their local environment and interactions with partners, also known as molecular sociology. To obtain thin samples, cryo-FIB milling is nowadays the most popular method, with impressive successes. The alternative, cryo-ultramicrotomy, is often overlooked on account of poorly reproducible attachment of cryo-sections to their support, resulting in extremely low throughput. We optimized the workflow, focusing on section adhesion and their support grids. We thus increased vitreous sections cryo electron tomography throughput to equal that of thin film, with typically several tens to hundreds of cryo-tomograms per sample. This open the way to new advances in cellular cryo electron tomography, as the method is devoid of beam damage, can provide large surfaces and serial sections of any type of sample from cells to tissues. In addition, section thickness can be tuned down to 30-50 nm, which may be an advantage for imaging small molecular complexes, such as DNA and nucleosomes.

Mitochondrial division is a fundamental biological process that is crucial to cellular functionality and vitality. The prevailing hypothesis of Drp1 regulation with the involvement of ER and cytoskeleton does not account for all the observations. Following up our previous study in HeLa cells which led to the new hypothesis of mitochondrial division by budding, we employed in-situ Cryo-Electron Tomography (Cryo-ET) to visualize mitochondrial budding in intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, supporting the new hypothesis. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, no rings, or ring-like structures, or ER wrapping is presented in the budding regions suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles, distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. We propose these interacting vesicles may have mitochondrial origins.

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.

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.

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur, which is part of our cisTEM software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant et al., 2015). However, Unblur cannot accommodate motion due to more localized sample bending and distortions. Here, we present Unbend, extending Unblur by incorporating local motion correction using a three-dimensional cubic spline model. The 3D spline model is constructed using cubic B-splines along the exposure time axis, and bicubic B-splines within movie frames. Unbend is integrated into our cisTEM software with a new local motion visualization panel within the cisTEM graphical user interface. We processed movie frames from various in-situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method, which operates independently of the motion correction process, to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3-8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to [~]300%. The total and Von Mises equivalent strain shows a deformation scale of less than 1% in most of the samples, confirming that our model induces minimal additional distortion. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information - known as multimodal imaging or correlative microscopy (CM) - has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artefacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To further explore the potential of cryo-fixation, we developed a high-pressure freezing (HPF) system that allows vitrification under different environmental parameters and applied it in different CLEM workflows. In this chapter, we introduce our novel HPF live instrument with a focus on its coupling to a light microscope. We elaborate on the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. We will address the adaptation of HPF to novel correlation workflows related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support microscopy-driven structural studies. Here, we present an open-source desktop application tool, CorRelator, to bridge between cryo-FLM and cryo-EM/ET data collection instruments. CorRelator was designed to be flexible for both on-the-fly and post-acquisition correlation schemes. The CorRelator workflow is easily adapted to any fluorescence and transmission electron microscope (TEM) system configuration. CorRelator was benchmarked under cryogenic and ambient temperature conditions using several FLM and TEM instruments, demonstrating that CorRelator is a rapid and efficient application for image and position registration in CLEM studies. CorRelator is a cross-platform software featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

Ptychographic single-particle analysis (SPA) is a promising technique for high-resolution biological imaging but is still limited by sub-nanometer resolution. In this study, we identified and investigated a critical issue termed sampling mismatch in ptychography that is caused by inaccuracies in the scanning step size and the pixel size of convergent beam electron diffraction (CBED) images. This mismatch induces pixel-size deviations in the reconstructed micrographs and modulates information transfer through a mismatch-induced modulation function (MIMF), which is characterized by phase reversals at specific spatial frequencies of the micrographs. These phase reversals, which vary with the defocus, cause destructive interference when merging micrographs, fundamentally limiting the resolution of SPA. We proposed a correction strategy and demonstrated, on the T. Acidophilum 20S proteasome and apoferritin datasets, that correcting sampling parameters eliminates signal distortions and improves resolution for [~]1.5 [A]. These findings underscore the necessity for the precise control and calibration of the scanning system to achieve high-resolution ptychographic SPA.

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.

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.

Cryo-EM has revolutionized structural biology, especially for flexible and heterogeneous samples, although access to high end microscopes that enable these studies remains a bottleneck. While 300 keV microscopes have been the go-to for high-resolution structural determination, they are expensive and restricted to institutional and national facilities needing specialized expertise, with access falling far short of the demand. Here, we present the user-managed operation of a cheaper 100 keV electron microscope within a structural biology laboratory enabling close integration with protein production, biochemical and biophysical studies. We provide details and considerations for the installation of the microscope, its day-to-day maintenance, and operations. Using virus surface glycoproteins as case studies, we illustrate the workflow from grid screening, data collection, and data processing, and provide examples of data quality. This user-administered setup provides a training platform for researchers at all levels, with beginners in cryo-EM achieving proficiency to independently operate the microscope within a month of regular use and training. We have demonstrated routine high-quality low-resolution reconstructions using a Ceta CMOS camera and high-resolution reconstructions enabling building of atomic models using a Falcon C direct detector. While there are several examples of facilities that manage cryo-EM and individual laboratories leveraging cryo-EM, we provide here the first demonstration of a modern group independently doing both successfully, something that has been talked about frequently but rarely seen.

Due to recent technological advances, in situ structural cell biology is becoming a high throughput microscopy technique as all the steps of the workflow, from sample preparation to data analysis, are executed faster, more reliable and more reproducible. Sample thinning by cryoFIB-SEM is an essential tool in preparing electron transparent lamellae of biological specimens suitable for further characterization by cryoET. Modern cryoFIB-SEM instruments can be operated remotely and are capable of automated and unsupervised lamellae preparation. To take full advantage of these developments they need a constant supply of LN2 to maintain cryogenic conditions inside the microscope chamber. Here, we introduce a custom automated LN2 refill system that is compatible with gas cooled cryostages, supports long-term cryoFIB-SEM operations and liberates the user from highly repetitive and manual work. We believe this solution can be utilized with other cryoSEM or cryoFIB-SEM devices requiring N2 gas-flow cooling.

O_LIEfficient comparisons of biological color patterns are critical for understanding the mechanisms by which organisms evolve in ecosystems, including sexual selection, predator-prey interactions, and thermoregulation. However, elongate or spiral-shaped organisms do not conform to the standard orientation and photographic techniques required for automated analysis. Currently, large-scale color analysis of elongate animals requires time-consuming manual landmarking, which reduces their representation in coloration research despite their ecological importance. C_LIO_LIWe present Batch-Mask: an automated and customizable workflow to facilitate the analysis of large photographic data sets of non-standard biological subjects. First, we present a user guide to run an open-source region-based convolutional neural network with fine-tuned weights for identifying and isolating a biological subject from a background (masking). Then, we demonstrate how to combine masking with existing manual visual analysis tools into a single streamlined, automated workflow for comparing color patterns across images. C_LIO_LIBatch-Mask was 60x faster than manual landmarking, produced masks that correctly identified 96% of all snake pixels, and produced pattern energy results that were not significantly different from the manually landmarked data set. C_LIO_LIThe fine-tuned weights for the masking neural network, user guide, and automated workflow substantially decrease the amount of time and attention required to quantitatively analyze non-standard biological subjects. By using these tools, biologists will be able to compare color, pattern, and shape differences in large data sets that include significant morphological variation in elongate body forms. This advance will be especially valuable for comparative analyses of natural history collections, and through automation can greatly expand the scale of space, time, or taxonomic breadth across which color variation can be quantitatively examined. C_LI

In situ cryoET (cryoelectron tomography) and cryo-FIB/SEM (cryo-focused ion beam/scanning electron microscopy) volume-EM (electron microscopy) imaging provide spatiotemporal snapshots of biological systems in their near-native aqueous environment. Freezing and subsequent thinning of thick biological specimens prior to cryo-imaging is a time-consuming and challenging task that requires state-of-art methodology. As a result, cryo-imaging reports obtained from non-trivial specimens including mammalian brain tissues are scarce and their physiological relevance remains to be determined. Here, we benchmarked plunge freezing with a variety of cryoprotectants that allow for mouse brain tissue vitrification of up to about 100 microns thick and across several brain regions while keeping the tissue functional. By utilizing the knock-in (KI) mouse model with fluorescent astrocytes we have performed targeted cryo-FIB/SEM volume-EM imaging as well as targeted high-resolution cryoET imaging. Prior to cryoET, we have successfully generated lamellae in a semi-automated fashion on both LMIS (liquid metal ion source)- and plasma-based cryo-FIB/SEM instrumentation thus expanding applicability of our pipeline. We visualized the NVU (neurovascular unit) and astrocytes processes and validated the physiological relevance of our outputs based on the morphology of the corresponding cellular and subcellular features. The pipeline utilizes common vitrification setups and can be potentially extended toward alternative tissue specimens. Ultimately, we expect our approach to become an important step towards democratization of physiologically relevant in situ cryo-imaging studies.

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.

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.

ABSTACTCryo-electron tomography (cryo-ET) provides a promising technique to study high resolution structures of macromolecules in situ, opening a new era of structural biology. One major bottleneck of this technique is to prepare suitable cryo-lamellas of cell/tissue samples. The emergence of cryo-focused ion beam (cryo-FIB) milling technique provides a good solution of this bottleneck. However, there are still large limitations of using cryo-FIB to prepare cryo-lamella of tissue specimen because the thickness of tissue increases the difficulty of specimen freezing and cryo-FIB milling. Here we report a new workflow, VHUT-cryo-FIB (Vibratome - High pressure freezing - Ultramicrotome Trimming - cryo-FIB), aiming for efficient preparation of frozen hydrated tissue lamella for subsequent cryo-ET data collection. This workflow includes tissue slicing using vibratome, high pressure freezing, ultramicrotome cryo-trimming, cryo-FIB milling and the subsequent cryo-electron microscopy (cryo-EM). The modification of equipment in this workflow is highly eliminated. We developed two strategies with a special cryo-holder tip or carrier for loading cryo-lamella into side entry cryo-holder or Autoloader catridge. We tested this workflow using the tissue sample of rat skeleton muscle and spinach leaf and collected high quality cryo-ET tilt series, which enabled us to obtain an in situ structure of spinach ribosome by sub-tomogram averaging.

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem on dose-sensitive samples. Here, we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application, showing it can tile nearly perfectly and deliver cryo-EM imaging with a resolution comparable to conventional setups.