Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Kong, Lingli; Liu, Jianfang; Zhang, Meng; Lu, Zhuoyang; Xue, Han; Ren, Amy; Liu, Jiankang; Li, Jinping; Ling, Wai Li; Ren, Gang

doi:10.1038/s41467-023-41266-x

Title: Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Journal Article · 13 September 2023 · Nature Communications

DOI: https://doi.org/10.1038/s41467-023-41266-x · OSTI ID:1999868

;

; Lu, Zhuoyang; Xue, Han; Ren, Amy;

; Li, Jinping;

;

Abstract Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: NONE; AC02-05CH11231

OSTI ID:: 1999868

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 14; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

References (51)

UCSF Chimera?A visualization system for exploratory research and analysis Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Journal of Computational Chemistry, Vol. 25, Issue 13 https://doi.org/10.1002/jcc.20084	journal	January 2004
Advances in Graphene‐Based Liquid Cell Electron Microscopy: Working Principles, Opportunities, and Challenges Ghodsi, Seyed Mohammadreza; Megaridis, Constantine M.; Shahbazian‐Yassar, Reza Small Methods, Vol. 3, Issue 5 https://doi.org/10.1002/smtd.201900026	journal	January 2019
Computer Visualization of Three-Dimensional Image Data Using IMOD Kremer, James R.; Mastronarde, David N.; McIntosh, J. Richard Journal of Structural Biology, Vol. 116, Issue 1 https://doi.org/10.1006/jsbi.1996.0013	journal	January 1996
EMAN: Semiautomated Software for High-Resolution Single-Particle Reconstructions Ludtke, Steven J.; Baldwin, Philip R.; Chiu, Wah Journal of Structural Biology, Vol. 128, Issue 1 https://doi.org/10.1006/jsbi.1999.4174	journal	December 1999
Production and Neurotropism of Lentivirus Vectors Pseudotyped with Lyssavirus Envelope Glycoproteins Desmaris, Nathalie; Bosch, Assumpcio; Salaün, Christine Molecular Therapy, Vol. 4, Issue 2 https://doi.org/10.1006/mthe.2001.0431	journal	August 2001
High-voltage electron microscopy of wet whole cancer and normal cells Parsons, D. F.; Matricardi, V. R.; Subjeck, J. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 290 https://doi.org/10.1016/0005-2736(72)90056-9	journal	December 1972
Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure–Activity relationships for 1-[N-(Methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidin-1-yl)butanes Finke, Paul E.; Oates, Bryan; Mills, Sander G. Bioorganic & Medicinal Chemistry Letters, Vol. 11, Issue 18 https://doi.org/10.1016/S0960-894X(01)00492-9	journal	September 2001
Cryo-electron microscopy for structural analysis of dynamic biological macromolecules Murata, Kazuyoshi; Wolf, Matthias Biochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1862, Issue 2 https://doi.org/10.1016/j.bbagen.2017.07.020	journal	February 2018
The Current Revolution in Cryo-EM Egelman, Edward H. Biophysical Journal, Vol. 110, Issue 5 https://doi.org/10.1016/j.bpj.2016.02.001	journal	March 2016
A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen Moffat, Jason; Grueneberg, Dorre A.; Yang, Xiaoping Cell, Vol. 124, Issue 6 https://doi.org/10.1016/j.cell.2006.01.040	journal	March 2006
Probing electrochemical surface/interfacial reactions with liquid cell transmission electron microscopy: a challenge or an opportunity? Prabhudev, Sagar; Guay, Daniel Current Opinion in Electrochemistry, Vol. 23 https://doi.org/10.1016/j.coelec.2020.05.001	journal	October 2020
Near-atomic resolution cryo-EM for molecular virology Hryc, Corey F.; Chen, Dong-Hua; Chiu, Wah Current Opinion in Virology, Vol. 1, Issue 2 https://doi.org/10.1016/j.coviro.2011.05.019	journal	August 2011
Three-dimensional Structure of HIV-1 Virus-like Particles by Electron Cryotomography Benjamin, Jordan; Ganser-Pornillos, Barbie K.; Tivol, William F. Journal of Molecular Biology, Vol. 346, Issue 2 https://doi.org/10.1016/j.jmb.2004.11.064	journal	February 2005
Seeing GroEL at 6 Å Resolution by Single Particle Electron Cryomicroscopy Ludtke, Steven J.; Chen, Dong-Hua; Song, Jiu-Li Structure, Vol. 12, Issue 7 https://doi.org/10.1016/j.str.2004.05.006	journal	July 2004
CTF determination and correction in electron cryotomography Fernández, J. J.; Li, S.; Crowther, R. A. Ultramicroscopy, Vol. 106, Issue 7 https://doi.org/10.1016/j.ultramic.2006.02.004	journal	May 2006
Live Cell Electron Microscopy Is Probably Impossible de Jonge, Niels; Peckys, Diana B. ACS Nano, Vol. 10, Issue 10 https://doi.org/10.1021/acsnano.6b02809	journal	October 2016
Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody Kwong, Peter D.; Wyatt, Richard; Robinson, James Nature, Vol. 393, Issue 6686 https://doi.org/10.1038/31405	journal	June 1998
The crystal structure of the bacterial chaperonln GroEL at 2.8 Å Braig, Kerstin; Otwinowski, Zbyszek; Hegde, Rashmi Nature, Vol. 371, Issue 6498 https://doi.org/10.1038/371578a0	journal	October 1994
Dynamic microscopy of nanoscale cluster growth at the solid–liquid interface Williamson, M. J.; Tromp, R. M.; Vereecken, P. M. Nature Materials, Vol. 2, Issue 8 https://doi.org/10.1038/nmat944	journal	July 2003
Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM Li, Xueming; Mooney, Paul; Zheng, Shawn Nature Methods, Vol. 10, Issue 6 https://doi.org/10.1038/nmeth.2472	journal	May 2013
Electron microscopy of specimens in liquid de Jonge, Niels; Ross, Frances M. Nature Nanotechnology, Vol. 6, Issue 11, p. 695-704 https://doi.org/10.1038/nnano.2011.161	journal	October 2011
Viruses as vaccine vectors for infectious diseases and cancer Draper, Simon J.; Heeney, Jonathan L. Nature Reviews Microbiology, Vol. 8, Issue 1 https://doi.org/10.1038/nrmicro2240	journal	December 2009
Virus entry: molecular mechanisms and biomedical applications Dimitrov, Dimiter S. Nature Reviews Microbiology, Vol. 2, Issue 2 https://doi.org/10.1038/nrmicro817	journal	February 2004
Light microscopy of proteins in their ultrastructural context M’Saad, Ons; Bewersdorf, Joerg Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-17523-8	journal	July 2020
Mechanisms of SARS-CoV-2 entry into cells Jackson, Cody B.; Farzan, Michael; Chen, Bing Nature Reviews Molecular Cell Biology, Vol. 23, Issue 1 https://doi.org/10.1038/s41580-021-00418-x	journal	October 2021
Reducing effects of particle adsorption to the air–water interface in cryo-EM Noble, Alex J.; Wei, Hui; Dandey, Venkata P. Nature Methods, Vol. 15, Issue 10 https://doi.org/10.1038/s41592-018-0139-3	journal	September 2018
Real-time observation of dynamic structure of liquid-vapor interface at nanometer resolution in electron irradiated sodium chloride crystals Ren, Amy; Lu, David; Wong, Edward Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-65274-9	journal	May 2020
Fully Mechanically Controlled Automated Electron Microscopic Tomography Liu, Jinxin; Li, Hongchang; Zhang, Lei Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep29231	journal	July 2016
Atomically precise graphene nanoribbons: interplay of structural and electronic properties Houtsma, R. S. Koen; de la Rie, Joris; Stöhr, Meike Chemical Society Reviews, Vol. 50, Issue 11 https://doi.org/10.1039/D0CS01541E	journal	January 2021
Viral detection by electron microscopy: past, present and future Roingeard, Philippe Biology of the Cell, Vol. 100, Issue 8 https://doi.org/10.1042/BC20070173	journal	August 2008
Electron microscopy of whole cells in liquid with nanometer resolution Jonge, N. d.; Peckys, D. B.; Kremers, G. J. Proceedings of the National Academy of Sciences, Vol. 106, Issue 7 https://doi.org/10.1073/pnas.0809567106	journal	January 2009
Extracellular vesicles and viruses: Are they close relatives? Nolte-‘t Hoen, Esther; Cremer, Tom; Gallo, Robert C. Proceedings of the National Academy of Sciences, Vol. 113, Issue 33 https://doi.org/10.1073/pnas.1605146113	journal	July 2016
Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector. Karacostas, V.; Nagashima, K.; Gonda, M. A. Proceedings of the National Academy of Sciences, Vol. 86, Issue 22 https://doi.org/10.1073/pnas.86.22.8964	journal	November 1989
Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2 Schubert, Ulrich; Ott, David E.; Chertova, Elena N. Proceedings of the National Academy of Sciences, Vol. 97, Issue 24 https://doi.org/10.1073/pnas.97.24.13057	journal	November 2000
Robust Parameterization of Elastic and Absorptive Electron Atomic Scattering Factors Peng, L. M.; Ren, G.; Dudarev, S. L. Acta Crystallographica Section A Foundations of Crystallography, Vol. 52, Issue 2 https://doi.org/10.1107/S0108767395014371	journal	March 1996
A Closed cell for Electron Microscopy Abrams, I. M.; Mcbain, J. W. Science, Vol. 100, Issue 2595 https://doi.org/10.1126/science.100.2595.273	journal	September 1944
High-Resolution EM of Colloidal Nanocrystal Growth Using Graphene Liquid Cells Yuk, Jong Min; Park, Jungwon; Ercius, Peter Science, Vol. 336, Issue 6077 https://doi.org/10.1126/science.1217654	journal	April 2012
Real-Time Imaging of Pt3Fe Nanorod Growth in Solution Liao, H. -G.; Cui, L.; Whitelam, S. Science, Vol. 336, Issue 6084 https://doi.org/10.1126/science.1219185	journal	May 2012
Direction-Specific Interactions Control Crystal Growth by Oriented Attachment Li, D.; Nielsen, M. H.; Lee, J. R. I. Science, Vol. 336, Issue 6084 https://doi.org/10.1126/science.1219643	journal	May 2012
Electron Diffraction of Wet Proteins: Catalase Matricardi, V. R.; Moretz, R. C.; Parsons, D. F. Science, Vol. 177, Issue 4045 https://doi.org/10.1126/science.177.4045.268	journal	July 1972
Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M. Journal of Virology, Vol. 89, Issue 17 https://doi.org/10.1128/JVI.00844-15	journal	September 2015
Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells Strauss, Joshua D.; Hammonds, Jason E.; Yi, Hong Journal of Virology, Vol. 90, Issue 3 https://doi.org/10.1128/JVI.01880-15	journal	November 2015
Virus Entry, Assembly, Budding, and Membrane Rafts Chazal, Nathalie; Gerlier, Denis Microbiology and Molecular Biology Reviews, Vol. 67, Issue 2 https://doi.org/10.1128/MMBR.67.2.226-237.2003	journal	June 2003
Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications Yi, Hong; Strauss, Joshua D.; Ke, Zunlong Journal of Histochemistry & Cytochemistry, Vol. 63, Issue 10 https://doi.org/10.1369/0022155415593323	journal	June 2015
IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure Zhang, Lei; Ren, Gang PLoS ONE, Vol. 7, Issue 1 https://doi.org/10.1371/journal.pone.0030249	journal	January 2012
Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy Zhang, Ying; Huang, Tao; Jorgens, Danielle M. PLOS ONE, Vol. 12, Issue 5 https://doi.org/10.1371/journal.pone.0176839	journal	May 2017
Altering the Tropism of Lentiviral Vectors through Pseudotyping Cronin, James; Zhang, Xian-Yang; Reiser, Jakob Current Gene Therapy, Vol. 5, Issue 4 https://doi.org/10.2174/1566523054546224	journal	August 2005
Effect of Electron Energy on the Radiation Chemistry of Liquid Water Pimblott, Simon M.; LaVerne, Jay A. Radiation Research, Vol. 150, Issue 2 https://doi.org/10.2307/3579851	journal	August 1998
Cryo-electron tomography related radiation-damage parameters for individual-molecule 3D structure determination Xue, Han; Zhang, Meng; Liu, Jianfang Frontiers in Chemistry, Vol. 10 https://doi.org/10.3389/fchem.2022.889203	journal	August 2022
In situ TEM of Biological Assemblies in Liquid Dukes, Madeline J.; Gilmore, Brian L.; Tanner, Justin R. Journal of Visualized Experiments, Issue 82 https://doi.org/10.3791/50936	journal	December 2013
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy Rames, Matthew; Yu, Yadong; Ren, Gang Journal of Visualized Experiments, Issue 90 https://doi.org/10.3791/51087	journal	January 2014

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