From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy.

Coralie Spiegelhalter (1) , Valérie Tosch (1) , Didier Hentsch (1) , Marc Koch (1) , Pascal Kessler (1) , Yannick Schwab (1) , Jocelyn Laporte (1, 2)

1 IGBMC - Institut de Génétique et de Biologie Moléculaire et Cellulaire
2 Collège de France - Chaire Génétique Humaine

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.

Domaines: Biologie moléculaire; Biochimie, Biologie Moléculaire

Coralie Spiegelhalter, Valérie Tosch, Didier Hentsch, Marc Koch, Pascal Kessler, et al.. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy.. PLoS ONE, 2010, 5 (2), pp.e9014. ⟨10.1371/journal.pone.0009014⟩. ⟨hal-00452375v2⟩