Journal
PLOS ONE
Volume 5, Issue 2, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0009014
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Funding
- Institut National de la Sante et de la Recherche Medicale (INSERM)
- Centre National de la Recherche Scientifique (CNRS)
- University of Strasbourg (UdS)
- College de France
- Association Francaise contre les Myopathies (AFM)
- Fondation Recherche Medicale [FRM DEQ20071210538]
- Agence Nationale de la Recherche [ANR-07-BLAN-0065-03, ANR-08-GENOPAT-005]
- E-rare program
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Background: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion: Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.
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