A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (...

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Bibliographic Details
Published in:	Advanced biology Vol. 7; no. 10; p. e2200202
Main Authors:	Hinton, Jr, Antentor, Katti, Prasanna, Christensen, Trace A, Mungai, Margaret, Shao, Jianqiang, Zhang, Liang, Trushin, Sergey, Alghanem, Ahmad, Jaspersen, Adam, Geroux, Rachel E, Neikirk, Kit, Biete, Michelle, Lopez, Edgar Garza, Shao, Bryanna, Vue, Zer, Vang, Larry, Beasley, Heather K, Marshall, Andrea G, Stephens, Dominique, Damo, Steven, Ponce, Jessica, Bleck, Christopher K E, Hicsasmaz, Innes, Murray, Sandra A, Edmonds, Ranthony A C, Dajles, Andres, Koo, Young Do, Bacevac, Serif, Salisbury, Jeffrey L, Pereira, Renata O, Glancy, Brian, Trushina, Eugenia, Abel, E Dale
Format:	Journal Article
Language:	English
Published:	Germany 01-10-2023
Subjects:	Animals Cells, Cultured Mice Microscopy, Electron, Scanning Mitochondria - metabolism Mitochondrial Membranes - metabolism mitochondria-endoplasmic reticulum communication focused ion beam SEM serial-section TEM automated serial block-face SEM mitochondrial morphology mitochondrial dynamics
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Summary:	Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author Contributions A.H.J., P.K., and T.A.C. contributed equally to this work. Conceptualization: T.A.C., E.T., K.N., E.G.L., Z.V., P.K., B.G., R.O.P., E.D.A., and A.H.J.; Methodology: P.K., L.V., J.S., S.T., M.M., M.B., S.A.M., J.L., T.A.C., J.L.S., E.T., B.G., E.D.A., and A.H.J.; Software: A.H.J., P.K., T.A.C., J.L.S., R.O.P., B.G., E.T., and E.D.A.; Validation: P.K., T.A.C., M.M., L.Z., S.T., L.V., T.A.C., J.L.S., J.S., R.O.P., E.D.A., and A.H.J.; Formal analysis, E.G.L., Z.V., P.K., K.N., M.M., L.V., J.S., T.A.C., J.L.S., R.O.P., E.D.A., and A.H.J.; Investigation: E.G.L., Z.V., P.K., L.V., J.S., H.K.B., K.N., A.G.M., T.A.R., T.A.C., J.L.S., B.G., R.O.P., E.D.A., and A.H.J.; Resources: E.G.L., Z.V., P.K., S.A.M., T.A.C., J.L.S., R.O.P., E.D.A., E.T., and A.H.J.; Data curation: E.G.L., Z.V., P.K., L.Z., K.N., H.K.B., D.S., A.G.M., T.A.R., R.O.P., E.D.A., E.T., and A.H.J.; Writing: P.K., E.G.L., Z.V., D.S., I.H., J.P., R.E.G., L.Z., P.K., K.N., M.B., A.J., A.A., H.K.B., A.G.M., T.A.C., M.B., S.A.M., B.G., T.A.C., J.L.S., R.O.P., E.D.A., E.T., and A.H.J.; Visualization: P.K., T.A.C., A.A., S.A.M., R.O.P., S.D. R.A.C.E., C.K.E.B, J.P., J.L.S., E.T., B.G., E.D.A., and A.H.J.; Supervision: P.K., T.A.C., S.A.M., R.O.P., S.D., J.L.S., E.T., B.G., E.D.A., and A.H.J.; Project administration: P.K., T.A.C., S.A.M., R.O.P., S.D., J.L.S., E.T., B.G., E.D.A., and A.H.J.; Funding acquisition: B.G., E.T., and E.D.A. All authors contributed to data interpretation, manuscript editing, and submission. All authors have read and agreed to the published version of the manuscript.
ISSN:	2701-0198 2701-0198
DOI:	10.1002/adbi.202200202