Time-resolved cryogenic electron tomography for the study of transient cellular processes

Yoniles, Joseph; Summers, Jacob A.; Zielinski, Kara A.; Antolini, Cali; Panjalingam, Mayura; Lisova, Stella; Moss, Frank R.; Di Perna, Maximus Aldo; Kupitz, Christopher; Hunter, Mark S.; Pollack, Lois; Wakatsuki, Soichi; Dahlberg, Peter D.

doi:10.1091/mbc.e24-01-0042

Title: Time-resolved cryogenic electron tomography for the study of transient cellular processes

Journal Article · 08 May 2024 · Molecular Biology of the Cell

DOI: https://doi.org/10.1091/mbc.e24-01-0042 · OSTI ID:2349323

Yoniles, Joseph ^[1]; Summers, Jacob A. ^[2]; Zielinski, Kara A. ^[3]; Antolini, Cali ^[4]; Panjalingam, Mayura ^[5]; Lisova, Stella ^[4]; Moss, Frank R. ^[4]; Di Perna, Maximus Aldo ^[2]; Kupitz, Christopher ^[4]; Hunter, Mark S. ^[4]; Pollack, Lois ^[3]; Wakatsuki, Soichi ^[6]; Dahlberg, Peter D. ^[4]

Stanford Univ., CA (United States); SLAC
Stanford Univ., CA (United States)
Cornell Univ., Ithaca, NY (United States)
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Department of Chemistry, New York University, New York, New York, USA
Stanford Univ., CA (United States); SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second prior to freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Laboratory Directed Research and Development (LDRD) Program; National Institutes of Health (NIH); National Science Foundation (NSF)

Contributing Organization:: CryoEM & Bioimaging Research

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 2349323

Journal Information:: Molecular Biology of the Cell, Journal Name: Molecular Biology of the Cell Journal Issue: 7 Vol. 35; ISSN 1059-1524

Publisher:: American Society for Cell BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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