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

Area of Science:

• Life Sciences
• Biophysics
• Microscopy

Background:

• Cryo-electron tomography (cryo-ET) offers high-resolution imaging of biological samples in near-native states.
• Time-resolved studies are crucial for understanding dynamic cellular processes but have been limited in in situ applications.
• Existing cryo-ET methods primarily focus on static snapshots rather than dynamic events.

Purpose of the Study:

• To develop a novel freezing apparatus for time-resolved cryo-electron tomography (cryo-ET) studies conducted in situ.
• To enable the capture of rapid cellular structural changes occurring within milliseconds to seconds.
• To expand the temporal resolution of cryo-ET for dynamic biological investigations.

Main Methods:

• A new freezing device was engineered to mix cellular samples with stimulants before rapid plunge-freezing in liquid ethane.
• The apparatus allows precise control over reaction times, ranging from milliseconds to over a second, before vitrification.
• The method was validated using the bacterium *Caulobacter crescentus* exposed to an acidic buffer.

Main Results:

• The Time-Resolved cryo-ET (TR-cryo-ET) approach successfully visualized structural alterations in *Caulobacter crescentus* within 1.5 seconds.
• Observed changes included surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement.
• The study demonstrated the feasibility of capturing dynamic cellular events at high resolution.

Conclusions:

• The developed TR-cryo-ET method introduces a subsecond temporal dimension to cryo-ET.
• This technique significantly enhances the capability to visualize induced structural changes at molecular, organelle, and cellular levels.
• TR-cryo-ET opens new avenues for studying rapid biological processes in their native cellular environment.