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  • Time-resolved cryogenic electron tomography for the study of transient cellular processes. Molecular biology of the cell Yoniles, J., Summers, J. A., Zielinski, K. A., Antolini, C., Panjalingam, M., Lisova, S., Moss, F. R., Perna, M. A., Kupitz, C., Hunter, M. S., Pollack, L., Wakatsuki, S., Dahlberg, P. D. 2024: mbcE24010042


    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. [Media: see text].

    View details for DOI 10.1091/mbc.E24-01-0042

    View details for PubMedID 38717434