Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures.
The use of fully automated data collection methods for electron tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self-consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post-cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by pre-irradiation. We believe that the accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Furthermore neither the choice of embedding resin nor microwave post-curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal pre-irradiation coupled with low-temperature automated data collection greatly reduces shrinkage and should result in high-quality data for three-dimensional reconstructions.
Braunfeld
M B
MB
Howard Hughes Medical Institute, University of California at San Francisco 94143-0448.
Koster
A J
AJ
Sedat
J W
JW
Agard
D A
DA
D015925:Cryopreservation; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods*; D016613:Plastic Embedding; D014054:Tomography / Q000379:methods*