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

By Joseph Yoniles, Jacob A. Summers, Kara Zielinski1, Cali Antolini, Mayura Panjalingam, Stella Lisova, Frank R. Moss, Maximus Aldo Di Perna, Christopher Kupitz2, Mark S. Hunter2, Lois Pollack3, Soichi Wakatsuki, Peter D. Dahlberg

1. Center for Free-Electron Laser Science 2. SLAC National Accelerator Laboratory 3. Cornell University

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Category

BioXFEL Publications

Published on

Type

journal-article

Author

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

Citation

Yoniles, J., Summers, J. A., Zielinski, K. A., Antolini, C., Panjalingam, M., Lisova, S., Moss, F. R., Di Perna, M. A., Kupitz, C., Hunter, M. S., Pollack, L., Wakatsuki, S., & Dahlberg, P. D. (2024). Time-resolved cryogenic electron tomography for the study of transient cellular processes. Molecular Biology of the Cell, 35(7). https://doi.org/10.1091/mbc.e24-01-0042

Abstract

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer 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 before 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 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

DOI

http://dx.doi.org/10.1091/mbc.e24-01-0042

Funding

NSF-STC Biology with X-ray Lasers (NSF-1231306)