Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells


Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM.

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Figure 1: Flowchart of the steps for CLEM of mammalian cells.
Figure 2: Use of fLM to determine cross-sectional cell thickness and cell permissivity to RSV.
Figure 3: Carbon evaporation onto gold Finder EM grids.
Figure 4: Representative light microscopy image of the ideal cell density present on an EM grid.
Figure 5: Gatan Cryoplunge3 system setup for plunge-freezing.
Figure 6: Leica EM Cryo CLEM system.
Figure 7: Cryo-fluorescence microscopy grid map of HIV-1 virus-like particles tethered to HT1080 cells collected using the Leica LASX software.
Figure 8: Cryo-TEM map of grid with coordinates imposed from Leica LASX software.
Figure 9: Entire cryo-CLEM imaging workflow with an HIV-1 Gag-tetherin specimen.
Figure 10: Cryo-CLEM imaging of retroviral endocytosis and fusion.
Figure 11: Montage maps provide cellular context for cryo-ET data.
Figure 12: Quantitative segmentation data of HIV-1 particles with tetherin.
Figure 13: Examples of poor-quality grids for cryo-CLEM imaging.


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We thank the Robert P. Apkarian Integrated Electron Microscopy Core of Emory University for microscopy services and support. This work was supported in part by grants from Emory University, Children's Healthcare of Atlanta, and the Georgia Research Alliance to E.R.W.; a grant from the Center for AIDS Research at Emory University (P30 AI050409); a grant from the James B. Pendleton Charitable Trust to E.R.W. and P.W.S.; public health service grants R01GM104540, R21AI101775, and R01GM104540-03S1 from the NIH to E.R.W.; NSF grant 0923395 to E.R.W.; public health service grant R01GM114561 from the NIH to E.R.W. and P.J.S.; public health service grant R01AI058828 from the NIH to P.W.S.; public health service grants R01GM054787 and R01AI053668 from the NIH to G.B.M.; public health service grant R01GM094198 from the NIH to P.J.S.; and public health service grant F32GM112517 from the NIH to J.D.S. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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C.M.H. and E.R.W. wrote the manuscript. C.M.H., J.D.S., Z.K., R.S.D., J.E.H., E.A., T.M.D., M.M., G.B.M., P.J.S., P.W.S., and E.R.W. designed and performed the experiments, and edited the manuscript. R.E.S. and F.L. processed and analyzed data. All authors read and approved the manuscript.

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Hampton, C., Strauss, J., Ke, Z. et al. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12, 150–167 (2017).

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