Chapter 2 Correlated light and electron microscopy/electron tomography of mitochondria in situ.
Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of "virtual" 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts.
National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California, USA.
Guy A
GA
Perkins
Mei G
MG
Sun
Terrence G
TG
Frey
D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008853:Microscopy / Q000379:methods*; D008928:Mitochondria / Q000648:ultrastructure*; D014054:Tomography / Q000379:methods*