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FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist |
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| Authors: | Valentina Baena Ryan Conrad Patrick Friday Ella Fitzgerald Taeeun Kim John Bernbaum Heather Berensmann Adam Harned Kunio Nagashima Kedar Narayan |
| Affiliation: | 1.Center for Molecular Microscopy, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; (V.B.); (R.C.); (P.F.); (E.F.); (T.K.); (H.B.); (A.H.); (K.N.);2.Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA;3.National Institute of Allergy and Infectious Diseases, Division of Clinical Research, Integrated Research Facility at Fort Detrick (IRF-Frederick), Frederick, MD 21702, USA; |
| Abstract: | The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures. |
| Keywords: | FIB-SEM volumeEM vEM SARS-CoV-2 virological synapse segmentation |