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


METHODS
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.


CONCLUSIONS
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.
Imaging Centre, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Illkirch, France.
Coralie
C
Spiegelhalter
Valérie
V
Tosch
Didier
D
Hentsch
Marc
M
Koch
Pascal
P
Kessler
Yannick
Y
Schwab
Jocelyn
J
Laporte
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D055032:Electron Microscope Tomography / Q000379:methods*; D005615:Freezing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D056004:Molecular Dynamics Simulation; D011312:Pressure; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D015203:Reproducibility of Results
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C408986:MTMR2 protein, human; C427064:MTMR4 protein, human; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C099588:myotubularin