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Articoli di riviste sul tema "Cryo-CLEM"

Autore: Grafiati

Pubblicato: 14 settembre 2024

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1

Metskas, Lauren Ann, e John A. G. Briggs. "Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy". Microscopy and Microanalysis 25, n. 4 (14 maggio 2019): 942–49. http://dx.doi.org/10.1017/s1431927619000606.

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AbstractCorrelated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

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2

Moser, Felipe, Vojtěch Pražák, Valerie Mordhorst, Débora M. Andrade, Lindsay A. Baker, Christoph Hagen, Kay Grünewald e Rainer Kaufmann. "Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy". Proceedings of the National Academy of Sciences 116, n. 11 (26 febbraio 2019): 4804–9. http://dx.doi.org/10.1073/pnas.1810690116.

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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 super-resolution 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.

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3

Li, Shuoguo, Gang Ji, Xiaojun Huang, Lei Sun, Jianguo Zhang, Wei Xu e Fei Sun. "A New Solution of Non-integrated Correlative Light and Electron Microscopy Based on High-vacuum Optical Platform". Microscopy and Microanalysis 22, S3 (luglio 2016): 248–49. http://dx.doi.org/10.1017/s1431927616002099.

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Abstract Correlative light and electron microscopy (CLEM) offers a means of guiding the search for the unique or rare events by fluorescence microscopy (FM) and allows electron microscopy (EM) to zoom in on them for subsequent EM examination in three-dimensions (3D) and with nanometer-scale resolution. FM visualizes the localization of specific antigens by using fluorescent tags or proteins in a large field-of-view to study their cellular function, whereas EM provides the high level of resolution for complex structures. And cryo CLEM combines the advantages of maintaining structural preservation in a near-native state throughout the entire imaging process and by avoiding potentially harmful pre-treatments, such as chemical fixation, dehydration and staining with heavy metals. Besides for frozen-hydrated biological samples, CLEM combines the advantages of a close-to-life preservation of biological materials by keeping them embedded in vitreous ice throughout the entire imaging process and the frozen-hydrated condition is very suitable to maintain fluorescent signals. In recent years, many new instruments and software which intended to optimize the workflow and to obtain better experimental results of CLEM have been presented or even commoditized. While, the specimen damage during transfer from FM to EM and the resolution of CLEM were still need to be improved. Here we set up a High-vacuum Optical Platform to develop CLEM imaging technology (HOPE), which was designed to realize high-vacuum optical ( fluorescent) imaging for cryo-sample on EM cryo-holder (e.g. Gatan 626). A non-integrated high-vacuum cryo-optical stage, which adapted to the EM cryo holder, was fixed on epi-fluorescence microscope (or super-resolution microscope) to obtain fluorescent images. And then the EM cryo holder would be transferred to EM for collection of EM data. This protocol was aimed to minimize the specimen damage during transfer from FM to EM and it was versatile to expend to different types of light microscopy or electron microscopy. Our HOPE had already passed correlative imaging test, and the results showed that it was convenient and effective.

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4

Hampton, Cheri M. "Practical Strategies for cryo-CLEM Experiments". Microscopy and Microanalysis 23, S1 (luglio 2017): 1400–1401. http://dx.doi.org/10.1017/s1431927617007668.

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5

Thomas, Connon I., Nicolai T. Urban, Ye Sun, Lesley A. Colgan, Xun Tu, Ryohei Yasuda e Naomi Kamasawa. "Cryo-Confocal Imaging for CLEM Mapping in Brain Tissues". Microscopy Today 29, n. 5 (settembre 2021): 34–39. http://dx.doi.org/10.1017/s1551929521001073.

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Abstract:In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.

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6

Kamp, Arnold, Martijn van Nugteren, Hildo Vader, Michael Schwertner, Duncan Stacey, Roman Koning e Bram Koster. "Automated Cryo-plunging Robot to Prepare Samples for Single Particle Analysis (SPA), Cryo-EM, Cryo-ET, Cryo-fluorescence and Cryo-CLEM". Microscopy and Microanalysis 26, S2 (30 luglio 2020): 2732–33. http://dx.doi.org/10.1017/s1431927620022606.

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7

Paraan, Reza, Victoria Hewitt, Yusuke Hirabayashi, Franck Polleux, Clint Potter e Bridget Carragher. "Characterization of ER-mitochondria contact sites using cryo-CLEM". Microscopy and Microanalysis 27, S1 (30 luglio 2021): 1712–13. http://dx.doi.org/10.1017/s1431927621006255.

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8

Schwertner, Michael, e Duncan Stacey. "Cryo-Correlative Light and Electron Microscopy (Cryo-CLEM): Specimen Workflow Paths and Recent Instrument Developments". Microscopy and Microanalysis 21, S3 (agosto 2015): 1565–66. http://dx.doi.org/10.1017/s1431927615008600.

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9

Sexton, Danielle L., Steffen Burgold, Andreas Schertel e Elitza I. Tocheva. "Super-resolution confocal cryo-CLEM with cryo-FIB milling for in situ imaging of Deinococcus radiodurans". Current Research in Structural Biology 4 (2022): 1–9. http://dx.doi.org/10.1016/j.crstbi.2021.12.001.

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10

Shahmoradian, Sarah, Jim Monistrol, Hung Tri Tran, Valerie Perez, Jenny Jiou, Jaime Vaquer-Alicea e Marc Diamond. "Abstract 2445 Elucidating Tau Fibril Formation using Correlative Cryo-CLEM in situ". Journal of Biological Chemistry 300, n. 3 (marzo 2024): 107098. http://dx.doi.org/10.1016/j.jbc.2024.107098.

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11

de Beer, Marit, Rona Roverts, Xavier Heiligenstein, Edwin Lamers, Nico Sommerdijk e Anat Akiva. "Visualizing Biological Tissues: A Multiscale Workflow from Live Imaging to 3D Cryo-CLEM". Microscopy and Microanalysis 27, S2 (novembre 2021): 11–12. http://dx.doi.org/10.1017/s1431927621013635.

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12

Casanova, G., F. Nolin, L. Wortham, D. Ploton, V. Banchet e J. Michel. "Shrinkage of freeze-dried cryosections of cells: Investigations by EFTEM and cryo-CLEM". Micron 88 (settembre 2016): 77–83. http://dx.doi.org/10.1016/j.micron.2016.06.005.

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13

Capitanio, Cristina, Anna Bieber e Florian Wilfling. "How Membrane Contact Sites Shape the Phagophore". Contact 6 (gennaio 2023): 251525642311624. http://dx.doi.org/10.1177/25152564231162495.

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During macroautophagy, phagophores establish multiple membrane contact sites (MCSs) with other organelles that are pivotal for proper phagophore assembly and growth. In S. cerevisiae, phagophore contacts have been observed with the vacuole, the ER, and lipid droplets. In situ imaging studies have greatly advanced our understanding of the structure and function of these sites. Here, we discuss how in situ structural methods like cryo-CLEM can give unprecedented insights into MCSs, and how they help to elucidate the structural arrangements of MCSs within cells. We further summarize the current knowledge of the contact sites in autophagy, focusing on autophagosome biogenesis in the model organism S. cerevisiae.

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14

Kim, Hong-Lim, Tae-Ryong Riew, Jieun Park, Youngchun Lee e In-Beom Kim. "Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy". International Journal of Molecular Sciences 22, n. 8 (20 aprile 2021): 4273. http://dx.doi.org/10.3390/ijms22084273.

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Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu’s method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.

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15

Bieber, Anna, Cristina Capitanio, Oda Schiøtz, Marit Smeets, Johannes Fenzke, Philipp Erdmann e Jürgen Plitzko. "Precise 3D-correlative FIB-milling of biological samples using METEOR, an integrated cryo-CLEM imaging system". Microscopy and Microanalysis 27, S1 (30 luglio 2021): 3230–32. http://dx.doi.org/10.1017/s1431927621011132.

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16

Jonker, Caspar, Daan Boltje, Jacob Hoogenboom, Arjen Jakobi, Grant Jensen, Abraham Koster, Mart Last et al. "Fluorescence-guided lamella fabrication with ENZEL, an integrated cryogenic CLEM solution for the cryo-electron tomography workflow". Microscopy and Microanalysis 27, S1 (30 luglio 2021): 3234–35. http://dx.doi.org/10.1017/s1431927621011144.

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17

Kapishnikov, Sergey, Kenneth Fahy, William Fyans, Fergal O’Reilly, Tony McEnroe e Paul Sheridan. "Integration of Laboratory Cryo Soft X-ray Tomography into CLEM Workflows for Multimodal Multiscale Imaging of Bulk Samples". Microscopy and Microanalysis 29, Supplement_1 (22 luglio 2023): 1164. http://dx.doi.org/10.1093/micmic/ozad067.595.

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18

Li, Shuoguo, Gang Ji, Yang Shi, Lasse Hyldgaard Klausen, Tongxin Niu, Shengliu Wang, Xiaojun Huang et al. "High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy". Journal of Structural Biology 201, n. 1 (gennaio 2018): 63–75. http://dx.doi.org/10.1016/j.jsb.2017.11.002.

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19

Li, Shuoguo, Xing Jia, Tongxin Niu, Xiaoyun Zhang, Chen Qi, Wei Xu, Hongyu Deng, Fei Sun e Gang Ji. "HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography". Communications Biology 6, n. 1 (29 aprile 2023). http://dx.doi.org/10.1038/s42003-023-04850-x.

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AbstractCryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

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20

Klumpe, Sven, Herman KH Fung, Sara K. Goetz, Ievgeniia Zagoriy, Bernhard Hampoelz, Xiaojie Zhang, Philipp S. Erdmann et al. "A modular platform for automated cryo-FIB workflows". eLife 10 (24 dicembre 2021). http://dx.doi.org/10.7554/elife.70506.

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Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

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21

DeRosier, David J. "Where in the cell is my protein?" Quarterly Reviews of Biophysics 54 (2021). http://dx.doi.org/10.1017/s003358352100007x.

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Abstract The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

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22

Fuest, Marie, Miroslava Schaffer, Giovanni Marco Nocera, Rodrigo I. Galilea-Kleinsteuber, Jan-Erik Messling, Michael Heymann, Jürgen M. Plitzko e Thomas P. Burg. "In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography". Scientific Reports 9, n. 1 (dicembre 2019). http://dx.doi.org/10.1038/s41598-019-55413-2.

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AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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23

Kobylynska, M., P. C. Hawes e R. A. Fleck. "Multi-scale Correlative Workflows, Challenges and Opportunities for Cryo CLEM". Microscopy and Microanalysis 30, Supplement_1 (luglio 2024). http://dx.doi.org/10.1093/mam/ozae044.1041.

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24

Klein, Steffen, Benedikt H. Wimmer, Sophie L. Winter, Androniki Kolovou, Vibor Laketa e Petr Chlanda. "Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies". Communications Biology 4, n. 1 (29 gennaio 2021). http://dx.doi.org/10.1038/s42003-020-01567-z.

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AbstractLamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

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25

Yang, Suyeon, Machi Takeuchi, Rick R. M. Joosten, John P. M. van Duynhoven, Heiner Friedrich e Johannes Hohlbein. "Adapting cryogenic correlative light and electron microscopy (cryo-CLEM) for food oxidation studies". Food Structure, febbraio 2024, 100365. http://dx.doi.org/10.1016/j.foostr.2024.100365.

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26

Szabo, Gréta V., e Thomas P. Burg. "Time Resolved Cryo‐Correlative Light and Electron Microscopy". Advanced Functional Materials, 16 aprile 2024. http://dx.doi.org/10.1002/adfm.202313705.

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AbstractComplex materials exhibit fascinating features especially in situations far from equilibrium. Thus, methods for investigating structural dynamics with sub‐second time resolution are becoming a question of interest at varying spatial scales. With novel microscopy techniques steadily improving, the temporal and spatial limits of multiple imaging methods are investigated with an emphasis on the important role of correlative imaging and cryo‐fixation. A deep‐dive is taken into cryo‐correlative light and electron microscopy (CLEM) as a starting point for multimodal investigations of ultrastructural dynamics at high spatiotemporal resolution. The focus is on highlighting the different microscopy methods that capture the following key aspects: 1) samples are as close to native state as possible 2) dynamic process information is captured, 3) high structural resolution is enabled. Additionally, the size of samples that can be imaged under these conditions is looked at and approaches not only focusing on single molecules, but larger structures are highlighted.

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27

Fu, Xiaofeng, Jiying Ning, Zhou Zhong, Zandrea Ambrose, Simon Charles Watkins e Peijun Zhang. "AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography". Scientific Reports 9, n. 1 (dicembre 2019). http://dx.doi.org/10.1038/s41598-019-55766-8.

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AbstractCorrelative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

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28

Santarella-Mellwig, Rachel, Uta Haselmann, Nicole L. Schieber, Paul Walther, Yannick Schwab, Claude Antony, Ralf Bartenschlager e Inés Romero-Brey. "Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells". Journal of Visualized Experiments, n. 139 (7 settembre 2018). http://dx.doi.org/10.3791/58154.

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