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Electron Microscopic Tomography of Whole, Frozen-Hydrated Rat-Liver Mitochondria at 400 kv

Published online by Cambridge University Press:  02 July 2020

C.A. Mannella ,
C.-E Hsieh  and
M. Marko
C.A. Mannella
Affiliation:
Biological Microscopy and Image Reconstruction Resource, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509 Department of Biomedical Sciences, School of Public Health, University at Albany (SUNY)
C.-E Hsieh
Affiliation:
Biological Microscopy and Image Reconstruction Resource, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
M. Marko
Affiliation:
Biological Microscopy and Image Reconstruction Resource, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY12201-0509
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Extract

Electron microscopic tomography is providing important new insights about the internal structure of the mitochondrion. In particular, the infoldings of the mitochondrial inner membrane (cristae), which are usually rendered as lamelliform baffles, are revealed to have considerable tubular nature. Rather than opening wide to the peripheral compartment (between the inner and outer membranes), the cristae connect to the outside and to each other through narrow (20-30 nm) tubular segments, which can be hundreds of nanometers long. This suggests that diffusion of ions, metabolites and proteins between the intracristal and intermembrane spaces may be restricted.

The earlier tomographic reconstructions were done on conventionally prepared, plastic-embedded specimens, which raises the usual concerns about structural preservation. More recently, we have undertaken tomography of isolated rat-liver mitochondria that have been embedded in vitreous ice (by plunge-freezing in iso-osmotic buffer without chemical fixatives or stains). These frozen hydrated specimens are imaged with a JEOL 4000FX equipped with a Gatan cryo-transfer holder and a Tietz automated data collection system, with a Ik × Ik CCD. For 3D reconstructions, images were recorded at a dose of 5 e−Å2 at 2° increments over the range +/− 60°.

Type
Cryotechniques, Immunocytochemistry, and Electron Microscopy I. Molecular Approach
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 416 - 417
Copyright
Copyright © Microscopy Society of America

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References

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5. This research is funded by NSF grant MCB-9506113 and utilizes the facilities of the Biological Microscopy and Image Reconstruction Resource which is funded by NIH grant RR01219 and NSF grant BIR-9219043.Google Scholar