Correlative Light–Electron Microscopy as a Tool to Study In Vivo Dynamics and Ultrastructure of Intracellular Structures (original) (raw)
Abstract
Correlative light–electron microscopy (CLEM) is a very effective technique that combines live-cell imaging and immuno-electron microscopy for ultrastructural morphological characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the user to follow the movements and/or behavior of intracellular structures in a live cell and to fix it at the moment of interest. The subsequent immuno-electron microscopy processing can then reveal the three-dimensional architecture of the same structure, together with precise recognition of the GFP-labeled protein. The process resembles the taking of a high-resolution snapshot of an interesting live scene. Considering that CLEM is a very useful but technically demanding and time-consuming technique, accurate protocols will be helpful to simplify the work of scientists who are willing to apply this method for their own purposes. Here, we present a detailed protocol that describes all of the “tricks” and know-hows involved in carrying out the crucial steps of a CLEM experiment.
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Acknowledgements
We would like to thank C.P. Berrie for critical reading of the manuscript. This work was supported by Telethon grant GTF08001.
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Authors and Affiliations
- Institute of Protein Biochemistry, Naples, Italy
Elena V. Polishchuk & Alberto Luini - Telethon Institute of Genetics and Medicine, Naples, Italy
Roman S. Polishchuk & Alberto Luini
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- Elena V. Polishchuk
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Correspondence toAlberto Luini .
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Editors and Affiliations
- College of Medicine, Professor, Department of Pathology, University of Vermont, Beaumont Ave 89, Burlington, 05405-1742, Vermont, USA
Douglas J. Taatjes - , Abt. Zell- und Molekularpathologie, Universität Zürich, Schmelzbergstr. 12, Zürich, 8091, Switzerland
Jürgen Roth
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Polishchuk, E.V., Polishchuk, R.S., Luini, A. (2012). Correlative Light–Electron Microscopy as a Tool to Study In Vivo Dynamics and Ultrastructure of Intracellular Structures. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4\_20
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- DOI: https://doi.org/10.1007/978-1-62703-056-4\_20
- Published: 24 August 2012
- Publisher Name: Humana Press, Totowa, NJ
- Print ISBN: 978-1-62703-055-7
- Online ISBN: 978-1-62703-056-4
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