Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography.
Electron tomography is currently the only method that allows the direct three-dimensional visualization of macromolecules in an unperturbed cellular context. In principle, tomography should enable the identification and localization of the major macromolecular complexes within intact bacteria, embedded in amorphous ice. In an effort to optimize conditions for recording data that would bring us close to the theoretical limits, we present here a comparison of the dose tolerance of Caulobacter crescentus cells embedded in amorphous ice at liquid helium versus liquid nitrogen temperature. The inner and outer cell membranes, and the periodic structure of the S-layer of this Gram-negative bacterium provide ideal features to monitor changes in contrast and order as a function of dose. The loss of order in the S-layer occurs at comparable doses at helium and nitrogen temperatures. Macroscopic bubbling within the cell and the plastic support develops at both temperatures, but more slowly at helium temperature. The texture of the bubbles is finer in initial stages at helium temperature, giving an impression of contrast reversal in some parts of the specimen. Bubbles evolve differently in different organelles, presumably a consequence of their different chemical composition and mechanical properties. Finally, the amorphous ice "flows" at helium temperature, causing changes in the relative positions of markers within the specimen and distorting the cells. We conclude that for cryo-electron tomography of whole cells liquid nitrogen temperature provides better overall data quality.
Comolli
Luis R
LR
Life Science Division, Lawrence Berkeley National Laboratory, CA, USA.
Downing
Kenneth H
KH
D000818:Animals; D016935:Caulobacter crescentus / Q000166:cytology / Q000648:ultrastructure*; D002462:Cell Membrane / Q000528:radiation effects / Q000648:ultrastructure; D003080:Cold Temperature; D020285:Cryoelectron Microscopy / Q000379:methods*; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D005583:Fourier Analysis; D006371:Helium; D008297:Male; D008870:Microtubules / Q000528:radiation effects / Q000648:ultrastructure; D009584:Nitrogen; D013094:Spermatozoa / Q000737:chemistry; D047270:Strongylocentrotus purpuratus; D013696:Temperature; D014054:Tomography / Q000379:methods*
D006371:Helium; D009584:Nitrogen