Abstract
Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.
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Acknowledgements
We thank A. Teitelboim and G. Bellapadrona for preparation of samples. U2OS cells were a kind gift from B. Geiger (Weizmann Institute of Science). M.E. thanks P. Christie (University of Texas, Houston Medical School) for collaboration on Agrobacterium structures, which was supported by the US Israel Binational Agricultural Research and Development Fund. Electron microscopy was performed at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science. This work was additionally supported by the Gerhardt M.J. Schmidt Minerva Center for Supramolecular Architecture. The lab of M.E. has benefited from the historical generosity of the Harold Perlman family.
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S.G.W., L.H. and M.E. designed the experiments. S.G.W. and L.H. performed experiments. S.G.W., L.H. and M.E. analyzed the data. L.H. performed the simulation calculations. S.G.W., L.H. and M.E. wrote the manuscript.
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Wolf, S., Houben, L. & Elbaum, M. Cryo-scanning transmission electron tomography of vitrified cells. Nat Methods 11, 423–428 (2014). https://doi.org/10.1038/nmeth.2842
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DOI: https://doi.org/10.1038/nmeth.2842