Methods for Transmission Electron Microscopy and Electron Tomography

     Cells of Synechocystis sp. PCC 6803 are cryofixed for electron microscopy by high pressure freezing [Figure 1, Figure 2, Figure 3, Figure 4, Figure 5] or by plunge freezing into liquid nitrogen-cooled propane [Figure 6] followed by freeze substitution (Gilkey and Staehelin, 1986). The cells were embedded in Epoxy resin, which when polymerized are sectioned using an ultra microtome. For standard transmission electron microscopy, sections of approximately 60 nm are cut [Figure7A, Figure 7B], labeled with uranyl acetate and Reynold's lead citrate and then viewed using a Philips CM12 transmission electron microscope at 80 KV.

     For electron tomography sections of 100-300 nm were cut and viewed using either a high voltage electron microscope or an intermediate voltage electron microscope. To produce tomograms (3-D computer reconstructions of the cell), images were taken at every 1.5° at a range of tilt angles (usually -60° to +60°) [Figure 8, Tilting sample (movie)]; a specialized tilting specimen stage was required for this purpose. Using the IMOD software program developed at the University of Colorado, Boulder (Kremer et al., 1996), these images were annotated [Figure 9], aligned and merged to generate a 3-D model. The primary advantage of electron tomography is the increased resolution in the z-axis from approximately 60 nm of the thin sections for transmission electron microscopy to around 2-4 nm with the computer-produced sections (O'Toole et al., 2002). [Tomogram look-through (movie)]

 


References

  • Gilkey, J.C. and Staehelin, L.A. (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure.
    J. Elecron microsc. Tech. 3: 177-210

  • Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116: 71-76

  • O'Toole, E.T., Winey, M. J., McIntosh, J.R., Mastronarde, D.N. (2002) Electron Tomography of Yeast Cells. Meth. Enzymol. 351: 81-95

 

 

 

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