Time-resolved cryogenic electron tomography using mix-and-inject microfluidic devices
Category
Published on
Type
journal-article
Author
Peter Dahlberg and Joey Yoniles and Jacob Summers and Kara Zielinski and Cali Antolini and Mayura Panjalingam and Stella Lisova and Frank Moss and Maximus Perna and Christopher Kupitz and Mark Hunter and Lois Pollack and Soichi Wakatsuki
Citation
Dahlberg, P., Yoniles, J., Summers, J., Zielinski, K., Antolini, C., Panjalingam, M., Lisova, S., Moss, F., Perna, M., Kupitz, C., Hunter, M., Pollack, L., & Wakatsuki, S. (2025). Time-resolved cryogenic electron tomography using mix-and-inject microfluidic devices. Structural Dynamics, 12(2_Supplement), A53–A53. https://doi.org/10.1063/4.0000362
Abstract
Cryogenic electron tomography (cryo-ET) is the highest resolution three-dimensional imaging technique applicable to the life sciences, enabling sub-nanometer visualization of rapidly frozen biological specimens preserved in their near native states. The rapid freezing process lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here I will discuss our efforts to develop a freezing apparatus for time-resolved studies in situ. Our device is capable of mixing cellular samples with solution phase stimulants before being sprayed directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions into the device, we can control the reaction time from tens of milliseconds to over a second. In a proof-of-principle demonstration, we apply this approach to the freshwater bacterium Caulobacter crescentus mixed with an acidic buffer. We resolved complex structural changes propagating across the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement. This new approach, time-resolved cryo-ET (TR-cryo-ET) enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis, enabling visualization of inducible molecular-, organelle-, or cellular-level structural changes. I will conclude with a discussion of current efforts to test other cellular samples beyond bacteria and the integration of this sample preparation methods with more involved cryo-ET workflows including FIB milling and correlative light and electron microscopy.
