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Journal articles on the topic 'Cryogenic electron tomography'

Author: Grafiati

Published: 25 May 2024

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1

Zickert, Gustav, and Simon Maretzke. "Cryogenic electron tomography reconstructions from phaseless data." Inverse Problems 34, no. 12 (October 4, 2018): 124001. http://dx.doi.org/10.1088/1361-6420/aade22.

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2

Carlson, David B., Jeff Gelb, Vadim Palshin, and James E. Evans. "Laboratory-Based Cryogenic Soft X-Ray Tomography with Correlative Cryo-Light and Electron Microscopy." Microscopy and Microanalysis 19, no. 1 (January 18, 2013): 22–29. http://dx.doi.org/10.1017/s1431927612013827.

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AbstractHere we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 μm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

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3

Ong, Quy, Ting Mao, Neda Iranpour Anaraki, Łukasz Richter, Carla Malinverni, Xufeng Xu, Francesca Olgiati, et al. "Cryogenic electron tomography to determine thermodynamic quantities for nanoparticle dispersions." Materials Horizons 9, no. 1 (2022): 303–11. http://dx.doi.org/10.1039/d1mh01461g.

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4

Yipyintum, Chetarpa, Ji Yeong Lee, Jin-Yoo Suh, and Boonrat Lohwongwatana. "Hydride formation mechanisms in Zr-containing amorphous alloys during sample preparation and atom probe tomography." Materials Testing 65, no. 3 (March 1, 2023): 431–37. http://dx.doi.org/10.1515/mt-2022-0452.

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Abstract Hydride formation in Zr-containing amorphous alloys as an experimental artifact was investigated utilizing atom probe tomography, transmission electron microscopy, and focused ion beam with normal and cryogenic conditions. The amount of hydrogen existing in the atom probe specimens decreased significantly by utilizing focused ion beam milling under cryogenic condition. Also, the formation of hydride was confirmed by diffraction pattern analysis in the remaining tip of the specimen after the atom probe tomography experiment. With those collected pieces of evidence, sources of hydrogen in the atom probe tomography were discussed.

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5

Chang, Yi-Wei, Songye Chen, Elitza I. Tocheva, Anke Treuner-Lange, Stephanie Löbach, Lotte Søgaard-Andersen, and Grant J. Jensen. "Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography." Nature Methods 11, no. 7 (May 11, 2014): 737–39. http://dx.doi.org/10.1038/nmeth.2961.

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6

Dahlberg, Peter D., Saumya Saurabh, Jiarui Wang, Annina M. Sartor, Wah Chiu, Lucy Shapiro, and William E. Moerner. "Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution." Biophysical Journal 118, no. 3 (February 2020): 20a—21a. http://dx.doi.org/10.1016/j.bpj.2019.11.293.

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7

Frischknecht, Freddy, and Marek Cyrklaff. "Imaging Motile Pathogens with Light Microscopy and Cryogenic Electron Tomography." Microscopy Today 17, no. 6 (November 2009): 30–35. http://dx.doi.org/10.1017/s1551929509991027.

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The most prominent vector-transmitted diseases in the first and third world are Lyme disease and malaria, respectively. In both cases the transmitted agents are introduced into the skin from where they eventually disseminate into the blood using active motility. We are interested in deciphering the molecular mechanisms underlying the motility of these pathogens and how they relate to the ultrastructure of the pathogens. Here we provide an overview of the microscopy techniques that we use to achieve these goals.

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8

Kudryashev, Mikhail, Simone Lepper, Wolfgang Baumeister, Marek Cyrklaff, and Friedrich Frischknecht. "Geometric constrains for detecting short actin filaments by cryogenic electron tomography." PMC Biophysics 3, no. 1 (2010): 6. http://dx.doi.org/10.1186/1757-5036-3-6.

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9

Yoniles, Joseph. "Time-resolved cryogenic electron tomography with mix-and-spray microfluidic devices." Biophysical Journal 123, no. 3 (February 2024): 419a. http://dx.doi.org/10.1016/j.bpj.2023.11.2552.

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10

Löbling, Tina I., Johannes S. Haataja, Christopher V. Synatschke, Felix H. Schacher, Melanie Müller, Andreas Hanisch, André H. Gröschel, and Axel H. E. Müller. "Hidden Structural Features of Multicompartment Micelles Revealed by Cryogenic Transmission Electron Tomography." ACS Nano 8, no. 11 (September 17, 2014): 11330–40. http://dx.doi.org/10.1021/nn504197y.

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11

Moussavi, Farshid, Geremy Heitz, Fernando Amat, Luis R. Comolli, Daphne Koller, and Mark Horowitz. "3D segmentation of cell boundaries from whole cell cryogenic electron tomography volumes." Journal of Structural Biology 170, no. 1 (April 2010): 134–45. http://dx.doi.org/10.1016/j.jsb.2009.12.015.

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12

Kudryashev, Mikhail, Marek Cyrklaff, Björn Alex, Leandro Lemgruber, Wolfgang Baumeister, Reinhard Wallich, and Friedrich Frischknecht. "Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography." Cellular Microbiology 13, no. 5 (January 30, 2011): 731–41. http://dx.doi.org/10.1111/j.1462-5822.2011.01571.x.

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13

Leng, Boxun, Zhengzhong Shao, Paul H. H. Bomans, Laura J. Brylka, Nico A. J. M. Sommerdijk, Gijsbertus de With, and Weihua Ming. "Cryogenic electron tomography reveals the template effect of chitosan in biomimetic silicification." Chemical Communications 46, no. 10 (2010): 1703. http://dx.doi.org/10.1039/b922670b.

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14

Dahlberg, Peter D., and W. E. Moerner. "Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale." Annual Review of Physical Chemistry 72, no. 1 (April 20, 2021): 253–78. http://dx.doi.org/10.1146/annurev-physchem-090319-051546.

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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.

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15

Depelteau, Jamie S., Gert Koning, Wen Yang, and Ariane Briegel. "An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy." Microscopy and Microanalysis 26, no. 3 (April 14, 2020): 413–18. http://dx.doi.org/10.1017/s1431927620001385.

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AbstractVisualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

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16

Antolini, Cali. "Development of sample preparation methods for time-resolved cryogenic electron tomography and micro crystal electron diffraction." Biophysical Journal 123, no. 3 (February 2024): 420a. http://dx.doi.org/10.1016/j.bpj.2023.11.2555.

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17

Dahlberg, Peter D., Davis Perez, Corey W. Hecksel, Wah Chiu, and W. E. Moerner. "Metallic support films reduce optical heating in cryogenic correlative light and electron tomography." Journal of Structural Biology 214, no. 4 (December 2022): 107901. http://dx.doi.org/10.1016/j.jsb.2022.107901.

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18

Dahlberg, Peter D., Davis Perez, Zhaoming Su, Wah Chiu, and W. E. Moerner. "Cryogenic Correlative Single‐Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials." Angewandte Chemie 132, no. 36 (May 11, 2020): 15772–78. http://dx.doi.org/10.1002/ange.202002856.

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19

Dahlberg, Peter D., Davis Perez, Zhaoming Su, Wah Chiu, and W. E. Moerner. "Cryogenic Correlative Single‐Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials." Angewandte Chemie International Edition 59, no. 36 (May 11, 2020): 15642–48. http://dx.doi.org/10.1002/anie.202002856.

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20

Boltje, Daan, Jacob Hoogenboom, Arjen Jakobi, Grant Jensen, Caspar Jonker, Abraham Koster, Mart Last, et al. "ENZEL - A cryogenic, retrofittable, coincident fluorescence, electron, and ion beam solution for the cryo-electron tomography workflow." Microscopy and Microanalysis 27, S1 (July 30, 2021): 3228–29. http://dx.doi.org/10.1017/s1431927621011120.

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21

Jiang, Lin, Yang He, Chongmin Wang, Cedric Bouchet-Marquis, Lee Pullan, Brandon Van Leer, Liu Zhao, et al. "Cryogenic Electron Microscopy Combined with Energy-Dispersive X-ray Spectroscopy Tomography for Materials Science." Microscopy and Microanalysis 28, S1 (July 22, 2022): 328–30. http://dx.doi.org/10.1017/s1431927622002082.

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22

Li, Xiangyan, Bing Han, Xuming Yang, Zhipeng Deng, Yucheng Zou, Xiaobo Shi, Liping Wang, Yusheng Zhao, Sudong Wu, and Meng Gu. "Three-dimensional visualization of lithium metal anode via low-dose cryogenic electron microscopy tomography." iScience 24, no. 12 (December 2021): 103418. http://dx.doi.org/10.1016/j.isci.2021.103418.

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23

Zhou, Zhongwu, Kunpeng Li, Rui Yan, Guimei Yu, Christopher J. Gilpin, Wen Jiang, and Joseph M. K. Irudayaraj. "The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography." Nanoscale 11, no. 29 (2019): 13783–89. http://dx.doi.org/10.1039/c9nr02042j.

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We show that the extracted native chromatin presents a bifurcated hierarchical structure. The transitional 30 nm chromatin branching structure is the penultimate structure presenting 30 nm chromatin fiber unwinding into multiple nucleosomal arrays. Some nucleosomal arrays form the helix ribbon structure, while others randomly twist. Our work provides structural insights on the regulation of eukaryotic transcription.

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24

Jiang, Xi, Jing Sun, Ronald N. Zuckermann, and Nitash P. Balsara. "Structure-dependent Conducting Properties of Phosphonated Polypeptoid Electrolyte Membranes Revealed by Cryogenic Electron Tomography." Microscopy and Microanalysis 25, S2 (August 2019): 1822–23. http://dx.doi.org/10.1017/s143192761900984x.

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25

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 (July 30, 2021): 3234–35. http://dx.doi.org/10.1017/s1431927621011144.

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26

Berlepsch, Hans v., Christoph Böttcher, Katja Skrabania, and André Laschewsky. "Complex domain architecture of multicompartment micelles from a linear ABC triblock copolymer revealed by cryogenic electron tomography." Chemical Communications, no. 17 (2009): 2290. http://dx.doi.org/10.1039/b903658j.

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27

An, Qi, Chunyang Hong, and Haitao Wen. "Fracture Patterns of Rocks Observed under Cryogenic Conditions Using Cryo-Scanning Electron Microscopy." Processes 11, no. 7 (July 7, 2023): 2038. http://dx.doi.org/10.3390/pr11072038.

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Cryogenic fracturing, which uses liquid nitrogen (LN2) as a fracturing fluid, is a waterless fracturing method. However, previous attempts to investigate the fracture morphology of rocks after LN2 quenching have been mainly based on standard scanning electron microscopy (SEM) analysis at room temperature. This can be problematic since thermally-induced fractures created by temperature difference tend to close as a sample warms and thermal stress relaxes. To address this issue, we established a novel approach employing Cryo-scanning electron microscopy (Cryo-SEM) to investigate the fracture patterns induced by liquid nitrogen quenching under cryogenic conditions. This method can achieve in-situ visualization of fractures and pores with a nano-scale resolution at −190 °C. X-ray computed tomography (CT) is also employed to illustrate the fracture distribution inside samples. Cryo-SEM and standard SEM are compared, and statistical assessments are conducted to quantify fracture aperture size and closure scale. The results demonstrate that Cryo-SEM can more accurately preserve native fracture morphology and provide a more accurate means of evaluating fracture scales generated during LN2 quenching, particularly at higher temperature differences between rock and liquid nitrogen. Distinct fracture patterns and fracture width are observed for various rock types (i.e., coal, sandstone, shale, granite) by using these methods. More prominently, the maximum fracture width of coal, sandstone, shale and granite were 89.17 µm, 1.29 µm, 0.028 µm and 2.12 µm when the temperature difference between LN2 and rock samples were 296 °C. LN2 is shown to exhibit superior fracturing efficiency on coal and granite, characterized by complex fracture networks with branched fractures. This research contributes to our understanding of liquid nitrogen fracturing mechanisms and may offer effective approaches for unconventional reservoirs stimulation.

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28

Elad, Nadav, Giuliano Bellapadrona, Lothar Houben, Irit Sagi, and Michael Elbaum. "Detection of isolated protein-bound metal ions by single-particle cryo-STEM." Proceedings of the National Academy of Sciences 114, no. 42 (October 2, 2017): 11139–44. http://dx.doi.org/10.1073/pnas.1708609114.

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Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.

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29

Klumpe, Sven, Jakub Kuba, Oda H. Schioetz, Philipp S. Erdmann, Alexander Rigort, and Jürgen M. Plitzko. "Recent Advances in Gas Injection System-Free Cryo-FIB Lift-Out Transfer for Cryo-Electron Tomography of Multicellular Organisms and Tissues." Microscopy Today 30, no. 1 (January 2022): 42–47. http://dx.doi.org/10.1017/s1551929521001528.

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Abstract:Cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environment at molecular resolution. Most cells and all tissues, however, are too thick to be imaged directly by transmission electron microscopy (TEM). Overcoming this limitation requires the production of thin biological sections called lamellae. The procedure to obtain lamellae of cells, either seeded or grown directly on electron microscopy grids, requires cryo-focused ion beam (cryo-FIB) milling to thin the samples. This method faces an additional challenge when dealing with tissues and multicellular organisms, as these samples must be high-pressure frozen, which embeds the sample in a thick layer of ice. Nonetheless, lamellae can still be prepared from such samples by extracting a small volume and transferring it to a receiver grid for lamella preparation, a process called lift-out. Here, we describe the available workflows to produce lamellae by lift-out at cryogenic conditions and recent developments in gas injection system (GIS)-free approaches to the lift-out transfer. These advances expand the applications of cryo-ET, enabling the investigation of tissues and whole organisms in situ at molecular resolution.

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30

Carter, Stephen D., João I. Mamede, Thomas J. Hope, and Grant J. Jensen. "Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo." Proceedings of the National Academy of Sciences 117, no. 47 (November 5, 2020): 29702–11. http://dx.doi.org/10.1073/pnas.1920323117.

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Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.

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31

Taraska, Justin, and Kem Sochacki. "Imaging the structure of the plasma membrane with platinum replica and cryogenic electron microscopy and tomography of unroofed cells." Microscopy and Microanalysis 27, S1 (July 30, 2021): 1894–95. http://dx.doi.org/10.1017/s1431927621006899.

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32

Klimov, Paul V., Abram L. Falk, David J. Christle, Viatcheslav V. Dobrovitski, and David D. Awschalom. "Quantum entanglement at ambient conditions in a macroscopic solid-state spin ensemble." Science Advances 1, no. 10 (November 2015): e1501015. http://dx.doi.org/10.1126/sciadv.1501015.

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Entanglement is a key resource for quantum computers, quantum-communication networks, and high-precision sensors. Macroscopic spin ensembles have been historically important in the development of quantum algorithms for these prospective technologies and remain strong candidates for implementing them today. This strength derives from their long-lived quantum coherence, strong signal, and ability to couple collectively to external degrees of freedom. Nonetheless, preparing ensembles of genuinely entangled spin states has required high magnetic fields and cryogenic temperatures or photochemical reactions. We demonstrate that entanglement can be realized in solid-state spin ensembles at ambient conditions. We use hybrid registers comprising of electron-nuclear spin pairs that are localized at color-center defects in a commercial SiC wafer. We optically initialize 103 identical registers in a 40-μm3 volume (with 0.95−0.07+0.05 fidelity) and deterministically prepare them into the maximally entangled Bell states (with 0.88 ± 0.07 fidelity). To verify entanglement, we develop a register-specific quantum-state tomography protocol. The entanglement of a macroscopic solid-state spin ensemble at ambient conditions represents an important step toward practical quantum technology.

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33

Van Veen, Dave, Jesús G. Galaz-Montoya, Liyue Shen, Philip Baldwin, Akshay S. Chaudhari, Dmitry Lyumkis, Michael F. Schmid, Wah Chiu, and John Pauly. "Missing Wedge Completion via Unsupervised Learning with Coordinate Networks." International Journal of Molecular Sciences 25, no. 10 (May 17, 2024): 5473. http://dx.doi.org/10.3390/ijms25105473.

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Cryogenic electron tomography (cryoET) is a powerful tool in structural biology, enabling detailed 3D imaging of biological specimens at a resolution of nanometers. Despite its potential, cryoET faces challenges such as the missing wedge problem, which limits reconstruction quality due to incomplete data collection angles. Recently, supervised deep learning methods leveraging convolutional neural networks (CNNs) have considerably addressed this issue; however, their pretraining requirements render them susceptible to inaccuracies and artifacts, particularly when representative training data is scarce. To overcome these limitations, we introduce a proof-of-concept unsupervised learning approach using coordinate networks (CNs) that optimizes network weights directly against input projections. This eliminates the need for pretraining, reducing reconstruction runtime by 3–20× compared to supervised methods. Our in silico results show improved shape completion and reduction of missing wedge artifacts, assessed through several voxel-based image quality metrics in real space and a novel directional Fourier Shell Correlation (FSC) metric. Our study illuminates benefits and considerations of both supervised and unsupervised approaches, guiding the development of improved reconstruction strategies.

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34

Abramovich, Juliana. "Novel SARS-CoV-2 VLP system and structural characterization by cryogenic electron tomography reveals orf3A and orf7A accessory proteins as critical for assembly." Biophysical Journal 123, no. 3 (February 2024): 419a—420a. http://dx.doi.org/10.1016/j.bpj.2023.11.2554.

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35

DESBOIS, G., J. L. URAI, F. PÉREZ-WILLARD, Z. RADI, S. OFFERN, I. BURKART, P. A. KUKLA, and U. WOLLENBERG. "Argon broad ion beam tomography in a cryogenic scanning electron microscope: a novel tool for the investigation of representative microstructures in sedimentary rocks containing pore fluid." Journal of Microscopy 249, no. 3 (January 16, 2013): 215–35. http://dx.doi.org/10.1111/jmi.12011.

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36

Douglas, James O., Ayman El-Zoka, Michele Conroy, Finn Giuliani, and Baptiste Gault. "Development of Site Specific Cryogenic Specimen Preparation and Transfer of Frozen Liquids for Complementary High-Resolution Analysis by Scanning Transmission Electron Microscopy and Atom Probe Tomography." Microscopy and Microanalysis 29, Supplement_1 (July 22, 2023): 1700–1701. http://dx.doi.org/10.1093/micmic/ozad067.876.

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37

Draganova, Elizabeth B., Hui Wang, Melanie Wu, Shiqing Liao, Amber Vu, Gonzalo L. Gonzalez-Del Pino, Z. Hong Zhou, Richard J. Roller, and Ekaterina E. Heldwein. "The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice." PLOS Pathogens 20, no. 1 (January 16, 2024): e1011936. http://dx.doi.org/10.1371/journal.ppat.1011936.

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Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress–budding at the inner nuclear membrane–is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.

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38

Cullen, David A., Haoran Yu, Michael J. Zachman, Jaehyung Park, Nancy N. Kariuki, Leiming Hu, Rangachary Mukundan, K. C. Neyerlin, and Deborah J. Myers. "Automating Correlative Electron Microscopy for Heavy Duty Fuel Cell Development." ECS Meeting Abstracts MA2022-02, no. 39 (October 9, 2022): 1444. http://dx.doi.org/10.1149/ma2022-02391444mtgabs.

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The efficiency and durability of proton exchange membrane fuel cells has come to the forefront of research and development efforts as sustainable transportation applications shift from passenger to heavy duty vehicles. Complementary research expertise and advanced instrumentation are required to accelerate materials discovery and degradation mitigation of fuel cell components, with significant attention on catalysts, ionomers and membranes. The scanning transmission electron microscope (STEM) is among the key characterization techniques for accelerating materials and device development, providing key insights into atomic scale structure. Automating STEM data acquisition and analysis enables large data sets with improved statistical relevance and throughput will expand the impact of this method in the rapid materials and accelerated stress test development. We will show how the latest instrument developments in automation and high speed detectors enable new insights into catalyst structure and electrode layer degradation. This includes correlative, multimodal STEM techniques, such as aberration-corrected imaging, secondary electron detection, energy dispersive X-ray spectroscopy, cryogenic microscopy and 4D-STEM, to build a more comprehensive view of the effect of particle size and composition on surface strain and durability. These results will be correlated with information obtained from X-ray scattering techniques and fuel cell performance testing to understand the type and degree of degradation occurring during accelerated stress tests under development for heavy duty applications. The prospect of automated electron tomography coupled with identical location (IL)-STEM to provide detailed three-dimensional information on catalyst-support interactions will also be discussed. This material is based on work performed by the Million Mile Fuel Cell Truck (M2FCT) Consortium, technology managers Greg Kleen and Dimitrios Papageorgopoulus, which is supported by the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Hydrogen and Fuel Cell Technologies Office. Electron microscopy research was supported by the Center for Nanophase Materials Sciences (CNMS), which is a US Department of Energy, Office of Science User Facility at Oak Ridge National Laboratory. The X-ray scattering experiments were performed at beamline 9-ID-C at the Advanced Photon Source (APS) at Argonne National Laboratory (ANL). Use of the APS, an Office of Science user facility operated by ANL, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AS02-06CH11357

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39

Qian, Hui. "Major Factors Influencing the Size Distribution Analysis of Cellulose Nanocrystals Imaged in Transmission Electron Microscopy." Polymers 13, no. 19 (September 28, 2021): 3318. http://dx.doi.org/10.3390/polym13193318.

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Size distributions of cellulose nanocrystals (CNCs), extracted from softwood pulp via strong sulfuric acid hydrolysis, exhibit large variability when analyzed from transmission electron microscopy (TEM) images. In this article, the causes of this variability are studied and discussed. In order to obtain results comparable with those reported, a reference material of CNCs (CNCD-1) was used to evaluate size distribution. CNC TEM specimens were prepared as-stained and dried with a rapid-flushing staining method or hydrated and embedded in vitreous ice with the plunge-freezing method. Several sets of bright-field TEM (BF-TEM), annular dark-field scanning TEM (ADF-STEM) and cryogenic-TEM (cryo-TEM) images were acquired for size distribution analysis to study the contributing factors. The rapid-flushing staining method was found to be the most effective for contrast enhancement of CNCs, not only revealing the helical structure of single CNCs but also resolving the laterally jointed CNCs. During TEM specimen preparation, CNCs were fractionated on TEM grids driven by the coffee-ring effect, as observed from contrast variation of CNCs with a stain-depth gradient. From the edge to the center of the TEM grids, the width of CNCs increases, while the aspect ratio (length to width) decreases. This fractionated dispersion of CNCs suggests that images taken near the center of a droplet would give a larger mean width. In addition to particle fractionation driven by the coffee-ring effect, the arrangement and orientation of CNC particles on the substrate significantly affect the size measurement when CNC aggregation cannot be resolved in images. The coexistence of asymmetric cross-section CNC particles introduces a large variation in size measurement, as TEM images of CNCs are mixed projections of the width and height of particles. As a demonstration of how this contributes to inflated size measurement, twisted CNC particles, rectangular cross-section particles and end-to-end jointed CNCs were revealed in reconstructed three-dimensional (3D) micrographs by electron tomography (ET).

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de Sautu, Marilina, Tobias Herrmann, Gustavo Scanavachi, Simon Jenni, and Stephen C. Harrison. "The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2+." PLOS Pathogens 20, no. 4 (April 4, 2024): e1011750. http://dx.doi.org/10.1371/journal.ppat.1011750.

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Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

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Schmatz, Joyce, Jop Klaver, Mingze Jiang, and Janos L. Urai. "Nanoscale Morphology of Brine/Oil/Mineral Contacts in Connected Pores of Carbonate Reservoirs: Insights on Wettability From Cryo-BIB-SEM." SPE Journal 22, no. 05 (February 6, 2017): 1374–84. http://dx.doi.org/10.2118/180049-pa.

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Summary We used broad-ion-beam slope cutting in combination with scanning electron microscopy undertaken under cryogenic conditions (Cryo-BIB-SEM) to study mineral/oil/brine contacts in reservoir carbonates. This direct-imaging method allows pore-scale investigation of in-situ fluids and their distributions down to the nanometer scale. In this study, we compare two types of carbonate reservoirs: a fine-grained Lixhe limestone (Belgium) and a coarse-grained limestone from the Maastricht area (The Netherlands). In both rock types, we first quantify the porosity with BIB-SEM and derive the spatially resolved pore connectivity of the rock from BIB-SEM on Wood's Metal (WM) injected subsamples. In the second step, subsamples were saturated with the oil analog n-hexadecane, and then flooded with NaCl brine and MgSO4 brine, respectively, to study the effect of the brine chemistry on the microscopic fluid distribution. Cryo-BIB-SEM in combination with high-resolution energy-dispersive-spectroscopy (EDS) imaging and automated image analysis on the saturated samples allowed for a quantification of the oil-droplet size, the lengths of carbonate/oil interfaces, and the 2D contact angle of carbonate with brine and oil. Our results show that these features (e.g., interface length, contact angles, effect of asperities) are present on the scale of a few tens of nanometers to a few micrometers, which is in agreement with numerous theoretical and experimental studies. Lixhe limestone showed relatively less carbonate/oil contacts despite a larger oil fraction in the MgSO4-brine-flooded sample compared with the sample flooded with NaCl brine, indicating a more-hydrophilic nature of the carbonate surface in this experiment. This feasibility study showed that the technique permits the testing of predictions on the morphology and dynamics of contact lines in relation to the mineral properties, which is not possible with other imaging methods, such as X-ray microcomputed tomography (µ-CT), because of limits in resolution.

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El-Zoka, Ayman A., Se-Ho Kim, Heena Khanchandani, Leigh T. Stephenson, and Baptiste Gault. "(Digital Presentation) Advances in Cryo-Atom Probe Tomography Studies on Formation of Nanoporous Metals By Dealloying." ECS Meeting Abstracts MA2022-01, no. 47 (July 7, 2022): 1983. http://dx.doi.org/10.1149/ma2022-01471983mtgabs.

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The selective removal of a less noble (more chemically active) metal from a mixture of 2–3 metals can yield a bicontinuous, open-pore, 3D nanoporous metal (NPM), that is rich in the more noble metal(s)1. NPMs have been successfully developed by the intelligent use of the conventionally-undesired dealloying corrosion. The excellent properties of NPMs are attributed to the surface area-to-volume ratio, and high curvature of nanoligament surfaces 2,3 . Experimental work on NPMs revealed the formation of uniformly nanoporous structure by dealloying AgAu alloy in nitric acid, a method still widely used until today for making nanoporous gold (NPG). This dealloying process is complex: the selective dissolution of the less-noble element should lead to the creation of surface vacancies or adatoms, which migrate across the surface to form surface roughening features, and thus assisting the migration of the residual more-noble atoms, leading to island growth4. Ex-situ characterization cannot fully explain some intricate details at the dealloying interface and at the surface of the formed nanoligaments. Gaining insight into initial stages of dealloying, and the inherent competition between surface roughening from the dissolution of silver atoms, and surface smoothening from surface diffusion of gold atoms5, can only be done effectively by monitoring the changes occurring at the surfaces of alloys in-situ. Several notable in-situ methods were used to characterize formation of NPMs, such as TEM 6, often limited by the 2D nature of the analysis. Also, synchrotron-based methods such as X-ray nanotomography 7 and neutron scattering 8 were limited by resolution and lack of compositional contrast. APT is a powerful technique that provides 3D characterization9 and near-atomic-scale compositional analysis of materials, and could complement the abovementioned suite of techniques, yet the analysis of nanoporous structure comes with challenges. Aiming to develop a universal method for probing corrosion systems by APT, the concept of embedding frozen solutions in corroded systems was developed and reported in a recent reporting 10 of analyzing NPMs along with frozen water-based solutions. This involved the joint use of an ensemble of equipment and techniques that connect the frozen liquid to the atom probe, including a plasma-focused ion beam (PFIB) where the preparation of APT specimens uses a cryostage, and transfer of the frozen sample through ultra-high-vacuum suitcase11. This paved the way for many advances in characterization of corrosion processes, as the possibility of freezing corrosion reactions for APT (now known as cryo-APT) arises. Here, we further develop cryo-APT to probe into the mechanisms of dealloying in AgAu and how that leads to the formation of NPG using our in-situ approach. Nanoligament structure will be correlated with dealloying conditions by making observations at the solid-liquid interface in 3D. References Newman, R. C. 2.05 - Dealloying. in Shreir’s Corrosion (eds. Cottis, B. et al.) 801–809 (Elsevier, 2010). Zielasek, V. et al. Gold Catalysts: Nanoporous Gold Foams. Angew. Chemie Int. Ed. 45, 8241–8244 (2006). Xue, Y., Markmann, J., Duan, H., Weissmüller, J. & Huber, P. Switchable imbibition in nanoporous gold. Nat. Commun. 5, (2014). Forty, A. J. & Rowlands, G. A possible model for corrosion pitting and tunneling in noble-metal alloys. Philos. Mag. A Phys. Condens. Matter, Struct. Defects Mech. Prop. (1981) doi:10.1080/01418618108239399. Erlebacher, J., Newman, C. & Sieradzki, K. Fundamental physics and chemistry of nanoporosity evolution during dealloying. RSC Nanosci. Nanotechnol. 11–29 (2012) doi:10.1039/9781849735285-00011. Liu, P. et al. Dealloying Kinetics of AgAu Nanoparticles by In Situ Liquid-Cell Scanning Transmission Electron Microscopy. Nano Lett. 20, 1944–1951 (2020). Chen-Wiegart, Y. C. K. et al. In situ imaging of dealloying during nanoporous gold formation by transmission X-ray microscopy. Acta Mater. 61, 1118–1125 (2013). Corcoran, S. G., Wiesler, D. G. & Sieradzki, K. An in Situ Small Angle Neutron Scattering Investigation of Ag0.7Au0.3 Dealloying Under Potential Control. MRS Proc. 451, 93 (1996). Gault, B., Moody, M. P., Cairney, J. M. & Ringer, imon P. Atom Probe Microscopy. vol. 160 (Springer New York, 2012). El-Zoka, A. A. et al. Enabling near-atomic–scale analysis of frozen water. Sci. Adv. 6, eabd6324 (2020). Stephenson, L. T. et al. The LaplacE project: An integrated suite for preparing and transferring atom probe samples under cryogenic and UHV conditions. PLoS One (2018) doi:10.1371/journal.pone.0209211. Figure 1

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Tian, Ze-Nan, Xin-Xin Gao, Tao Xia, and Xiao-Bin Zhang. "Evaluation of Landweber Coupled Least Square Support Vector Regression Algorithm for Electrical Capacitance Tomography for LN2–VN2 Flow." Energies 16, no. 22 (November 20, 2023): 7661. http://dx.doi.org/10.3390/en16227661.

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The electric capacitance tomography (ECT) technique has been widely used in phase distribution reconstruction, while the practical application raised nonideal noise and other errors for cryogenic conditions, requiring a more accurate algorithm. This paper develops a new image reconstruction algorithm for ECT by coupling the traditional Landweber algorithm with the least square support vector regression (LSSVR) for cryogenic fluids. The performance of the algorithm is quantitatively evaluated by comparing the inversion images with the experimental results for both the room temperature working medium with the dielectric constant ratio close to cryogenic fluid and the cryogenic fluid of liquid nitrogen/nitrogen vapor (LN2-VN2). The inversion images based on the conventional LBP and Landweber algorithms are also presented for comparison. The benefits and drawbacks of the developed algorithms are revealed and discussed, according to the results. It is demonstrated that the correlated coefficients of the images based on the developed algorithm reach more than 0.88 and a maximum of 0.975. In addition, the minimum void fraction error of the algorithm is reduced to 0.534%, which indicates the significant optimization of the LSSVR coupled method over the Landweber algorithm.

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Taraska, Justin W. "Genetically encoded shape probes for cryogenic electron tomography." Nature Methods, November 6, 2023. http://dx.doi.org/10.1038/s41592-023-02044-1.

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Lovatt, Megan, Conny Leistner, and René A. W. Frank. "Bridging length scales from molecules to the whole organism by cryoCLEM and cryoET." Faraday Discussions, 2022. http://dx.doi.org/10.1039/d2fd00081d.

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Weis, Felix, Wim J. H. Hagen, Martin Schorb, and Simone Mattei. "Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition." Journal of Visualized Experiments, no. 169 (March 19, 2021). http://dx.doi.org/10.3791/62383.

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Chmielewski, David, Guan-Chin Su, Jason T. Kaelber, Grigore D. Pintilie, Muyuan Chen, Jing Jin, Albert J. Auguste, and Wah Chiu. "Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses." PNAS Nexus, March 7, 2024. http://dx.doi.org/10.1093/pnasnexus/pgae102.

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Abstract Alphaviruses are spherical, enveloped RNA viruses with two-layered icosahedral architecture. The structures of many alphaviruses have been studied using cryogenic electron microscopy (cryo-EM) reconstructions, which impose icosahedral symmetry on the viral particles. Using cryogenic electron tomography (cryo-ET), we revealed a polarized symmetry defect in the icosahedral lattice of Chikungunya virus (CHIKV) in situ, similar to the late budding particles, suggesting the inherent imperfect symmetry originates from the final pinch-off of assembled virions. We further demonstrated this imperfect symmetry also present in in vitro purified CHIKV and Mayaro virus (MAYV), another arthritogenic alphavirus. We employed a subparticle based single particle analysis protocol to circumvent the icosahedral imperfection and boosted the resolution of the structure of the CHIKV to ∼3 Å resolution, which revealed detailed molecular interactions between glycoprotein E1-E2 heterodimers in the transmembrane region and multiple lipid-like pocket factors located in a highly conserved hydrophobic pocket. This complementary use of in situ cryo-ET and single particle cryo-EM approach provides a more precise structural description of near-icosahedral viruses and valuable insights to guide the development of structure-based antiviral therapies against alphaviruses.

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Boltje, Daan B., Jacob P. Hoogenboom, Arjen J. Jakobi, Grant J. Jensen, Caspar TH Jonker, Max J. Kaag, Abraham J. Koster, et al. "A cryogenic, coincident fluorescence, electron and ion beam microscope." eLife 11 (October 28, 2022). http://dx.doi.org/10.7554/elife.82891.

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Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

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Asarnow, Daniel, Vada A. Becker, Daija Bobe, Charlie Dubbledam, Jake D. Johnston, Mykhailo Kopylov, Nathalie R. Lavoie, et al. "Recent advances in infectious disease research using cryo-electron tomography." Frontiers in Molecular Biosciences 10 (January 15, 2024). http://dx.doi.org/10.3389/fmolb.2023.1296941.

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With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

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Zhao, Cuicui, Da Lu, Qian Zhao, Chongjiao Ren, Huangtao Zhang, Jiaqi Zhai, Jiaxin Gou, Shilin Zhu, Yaqi Zhang, and Xinqi Gong. "Computational methods for in situ structural studies with cryogenic electron tomography." Frontiers in Cellular and Infection Microbiology 13 (October 4, 2023). http://dx.doi.org/10.3389/fcimb.2023.1135013.

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Cryo-electron tomography (cryo-ET) plays a critical role in imaging microorganisms in situ in terms of further analyzing the working mechanisms of viruses and drug exploitation, among others. A data processing workflow for cryo-ET has been developed to reconstruct three-dimensional density maps and further build atomic models from a tilt series of two-dimensional projections. Low signal-to-noise ratio (SNR) and missing wedge are two major factors that make the reconstruction procedure challenging. Because only few near-atomic resolution structures have been reconstructed in cryo-ET, there is still much room to design new approaches to improve universal reconstruction resolutions. This review summarizes classical mathematical models and deep learning methods among general reconstruction steps. Moreover, we also discuss current limitations and prospects. This review can provide software and methods for each step of the entire procedure from tilt series by cryo-ET to 3D atomic structures. In addition, it can also help more experts in various fields comprehend a recent research trend in cryo-ET. Furthermore, we hope that more researchers can collaborate in developing computational methods and mathematical models for high-resolution three-dimensional structures from cryo-ET datasets.

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