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PLoS One. 2010 Feb 3;5(2):e9014. doi: 10.1371/journal.pone.0009014.

From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.

Author information

1
Imaging Centre, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Illkirch, France.

Abstract

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.

PMID:
20140253
PMCID:
PMC2815783
DOI:
10.1371/journal.pone.0009014
[Indexed for MEDLINE]
Free PMC Article

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