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Annual Review of Physical Chemistry Apr 2021We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and... (Review)
Review
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.
Topics: Caulobacter crescentus; Cryoelectron Microscopy; Electron Microscope Tomography; HEK293 Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Nanotechnology; Single Molecule Imaging; Subcellular Fractions
PubMed: 33441030
DOI: 10.1146/annurev-physchem-090319-051546 -
Trends in Cell Biology May 2022Highly motile and functionally diverse immune cells orchestrate effective immune responses through complex and dynamic cooperative behavior. Multiphoton intravital... (Review)
Review
Highly motile and functionally diverse immune cells orchestrate effective immune responses through complex and dynamic cooperative behavior. Multiphoton intravital microscopy (MP-IVM) presents a unique and powerful tool to study the coordinated action of immune cell interactions in situ. Here, we review the current state of intravital microscopy in deepening our understanding of the immune system and discuss its fundamental limitations. In addition, we draw insights from recent technical advances in multiplex static tissue-imaging methods and propose an approach that could enable simultaneous visualization of cellular dynamics, deep phenotyping, and transcriptional states through a new type of correlative microscopy that combines these imaging technologies with advances in complex data analysis.
Topics: Cell Communication; Humans; Immune System; Intravital Microscopy; Microscopy, Fluorescence, Multiphoton
PubMed: 34920936
DOI: 10.1016/j.tcb.2021.11.007 -
Acta Crystallographica. Section D,... May 2022The Electron Microscopy Data Bank (EMDB) is the central archive of the electron cryo-microscopy (cryo-EM) community for storing and disseminating volume maps and...
The Electron Microscopy Data Bank (EMDB) is the central archive of the electron cryo-microscopy (cryo-EM) community for storing and disseminating volume maps and tomograms. With input from the community, EMDB has developed new resources for the validation of cryo-EM structures, focusing on the quality of the volume data alone and that of the fit of any models, themselves archived in the Protein Data Bank (PDB), to the volume data. Based on recommendations from community experts, the validation resources are developed in a three-tiered system. Tier 1 covers an extensive and evolving set of validation metrics, including tried and tested metrics as well as more experimental ones, which are calculated for all EMDB entries and presented in the Validation Analysis (VA) web resource. This system is particularly useful for cryo-EM experts, both to validate individual structures and to assess the utility of new validation metrics. Tier 2 comprises a subset of the validation metrics covered by the VA resource that have been subjected to extensive testing and are considered to be useful for specialists as well as nonspecialists. These metrics are presented on the entry-specific web pages for the entire archive on the EMDB website. As more experience is gained with the metrics included in the VA resource, it is expected that consensus will emerge in the community regarding a subset that is suitable for inclusion in the tier 2 system. Tier 3, finally, consists of the validation reports and servers that are produced by the Worldwide Protein Data Bank (wwPDB) Consortium. Successful metrics from tier 2 will be proposed for inclusion in the wwPDB validation pipeline and reports. The details of the new resource are described, with an emphasis on the tier 1 system. The output of all three tiers is publicly available, either through the EMDB website (tiers 1 and 2) or through the wwPDB ftp sites (tier 3), although the content of all three will evolve over time (fastest for tier 1 and slowest for tier 3). It is our hope that these validation resources will help the cryo-EM community to obtain a better understanding of the quality and of the best ways to assess the quality of cryo-EM structures in EMDB and PDB.
Topics: Cryoelectron Microscopy; Databases, Protein; Microscopy, Electron; Protein Conformation
PubMed: 35503203
DOI: 10.1107/S205979832200328X -
Nature Communications Mar 2022Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite...
Scanning probe microscopy techniques, such as atomic force microscopy and scanning tunnelling microscopy, are harnessed to image nanoscale structures with an exquisite resolution, which has been of significant value in a variety of areas of nanotechnology. These scanning probe techniques, however, are not generally suitable for high-throughput imaging, which has, from the outset, been a primary challenge. Traditional approaches to increasing the scalability have involved developing multiple probes for imaging, but complex probe design and electronics are required to carry out the detection method. Here, we report a probe-based imaging method that utilizes scalable cantilever-free elastomeric probe design and hierarchical measurement architecture, which readily reconstructs high-resolution and high-throughput topography images. In a single scan, we demonstrate imaging with a 100-tip array to obtain 100 images over a 1-mm area with 10 pixels in less than 10 min. The potential for large-scale tip integration and the advantage of a simple probe array suggest substantial promise for our approach to high-throughput imaging far beyond what is currently possible.
Topics: Microscopy, Atomic Force; Microscopy, Scanning Probe; Microscopy, Scanning Tunneling; Nanotechnology; Proteins
PubMed: 35301324
DOI: 10.1038/s41467-022-29181-z -
Current Opinion in Structural Biology Oct 2020Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close... (Review)
Review
Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close to atomic resolution. Nevertheless, despite improvements in microscope hardware, cryo-EM software and sample preparation techniques, challenges remain for structural analysis of small-sized membrane proteins (i.e.<150 kilodalton). Here we discuss recent examples of structures of macromolecules from this category determined by cryo-EM. We analyze the underlying difficulties, the enabling technologies such as the use of antibody fragments to gain size and provide fiducials for particle alignment, and the unresolved issues like dislocation of complexes at the air-water interface. Finally, we briefly highlight the biological relevance of some of these success stories, and our predictions for the future.
Topics: Cryoelectron Microscopy; Macromolecular Substances; Membrane Proteins; Single Molecule Imaging; Software
PubMed: 32603877
DOI: 10.1016/j.sbi.2020.05.009 -
Current Opinion in Structural Biology Oct 2022Single-particle cryogenic electron-microscopy (cryo-EM) has emerged as a powerful technique for the structural characterisation of membrane proteins, especially for... (Review)
Review
Single-particle cryogenic electron-microscopy (cryo-EM) has emerged as a powerful technique for the structural characterisation of membrane proteins, especially for targets previously thought to be intractable. Taking advantage of the latest hard- and software developments, high-resolution three-dimensional (3D) reconstructions of membrane proteins by cryo-EM has become routine, with 300-kV transmission electron microscopes (TEMs) being the current standard. The use of 200-kV cryo-TEMs is gaining increasingly prominence, showing the capabilities of reaching better than 2 Å resolution for soluble proteins and better than 3 Å resolution for membrane proteins. Here, we highlight the challenges working with membrane proteins and the impact of cryo-EM, and review the technical and practical benefits, achievements and limitations of imaging at lower electron acceleration voltages.
Topics: Cryoelectron Microscopy; Membrane Proteins; Single Molecule Imaging; Software
PubMed: 36029606
DOI: 10.1016/j.sbi.2022.102440 -
Histochemistry and Cell Biology Jun 2022Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine...
Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.
Topics: Electron Microscope Tomography; Humans; Microscopy, Electron; Microscopy, Fluorescence; Optical Imaging; Workflow
PubMed: 35267057
DOI: 10.1007/s00418-022-02094-0 -
Nature Communications Mar 2023The exchange of large and complex slide microscopy imaging data in biomedical research and pathology practice is impeded by a lack of data standardization and...
The exchange of large and complex slide microscopy imaging data in biomedical research and pathology practice is impeded by a lack of data standardization and interoperability, which is detrimental to the reproducibility of scientific findings and clinical integration of technological innovations. We introduce Slim, an open-source, web-based slide microscopy viewer that implements the internationally accepted Digital Imaging and Communications in Medicine (DICOM) standard to achieve interoperability with a multitude of existing medical imaging systems. We showcase the capabilities of Slim as the slide microscopy viewer of the NCI Imaging Data Commons and demonstrate how the viewer enables interactive visualization of traditional brightfield microscopy and highly-multiplexed immunofluorescence microscopy images from The Cancer Genome Atlas and Human Tissue Atlas Network, respectively, using standard DICOMweb services. We further show how Slim enables the collection of standardized image annotations for the development or validation of machine learning models and the visual interpretation of model inference results in the form of segmentation masks, spatial heat maps, or image-derived measurements.
Topics: Humans; Microscopy; Data Science; Reproducibility of Results
PubMed: 36949078
DOI: 10.1038/s41467-023-37224-2 -
Annual Review of Biophysics May 2023Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of... (Review)
Review
Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions. By bridging structural and cell biology, cryo-FIB-ET is advancing our understanding of structure-function relationships in their native environment and becoming a tool for discovering new biology.
Topics: Electron Microscope Tomography; Cryoelectron Microscopy
PubMed: 37159298
DOI: 10.1146/annurev-biophys-111622-091327 -
Current Opinion in Structural Biology Dec 2022All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and... (Review)
Review
All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.
Topics: Cryoelectron Microscopy; Electron Microscope Tomography; Macromolecular Substances; Single Molecule Imaging
PubMed: 36323134
DOI: 10.1016/j.sbi.2022.102484