From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy
2010
Background. In cell biology, the study of proteins and organelles requires the combination of 4 different imaging approaches, from live recordings with light microscopy (LM) to electron 5 microscopy (EM). 6 Methodology. To correlate dynamic events in adherent cells with both ultrastructural and 3D 7 information, we developed a method for cultured cells that combines confocal time-lapse 8 images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture 9 substrate, we created coordinates that were conserved at every step of the sample 10 preparation and visualization processes. Specifically designed for cryo-fixation, this method 11 allowed a fast freezing of dynamic events within seconds and their ultrastructural 12 characterization. We provide examples of the dynamic oligomerization of GFP-tagged 13 myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the 14 ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. 15 Conclusion. Accessible and versatile, we show that this approach is efficient to routinely 16 correlate functional and dynamic LM with high resolution morphology by EM, with immuno- 17 EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with 18 scanning-EM. 19
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