Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography.
Cryo-electron tomography (cryo-ET) allows the visualization of supramolecular architecture in cells preserved in a close-to-physiological state. In order to supplement the structural information obtained by cryo-ET with the functional state of the molecules involved based on fluorescent labeling we developed a method of correlating light microscopy and cryo-ET. This method is suitable for investigating complicated cellular landscapes such as mature neurons grown in culture. It has the advantage that a correlation is obtained without exposing a feature of interest to additional electron irradiation, and that it does not rely on visual recognition of features. Different modes of correlation are presented here: a feature identified on a light microscopy image is used to guide the cryo-ET investigation, and cryo-tomograms are correlated to light microscopy images. Cryo-tomograms of a neuronal synapse and of an isolated presynaptic terminal are shown as examples of the correlative method. The correlation method presented here can be expected to provide new insights into the structure-function relationship of supramolecular organization in neurons.
Lucić
Vladan
V
Max-Planck Institute for Biochemistry, Department of Structural Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Kossel
Albrecht H
AH
Yang
Ting
T
Bonhoeffer
Tobias
T
Baumeister
Wolfgang
W
Sartori
Anna
A
D000818:Animals; D002478:Cells, Cultured; D020285:Cryoelectron Microscopy / Q000379:methods*; D004705:Endocytosis; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008027:Light; D008853:Microscopy / Q000379:methods*; D015233:Models, Statistical; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014054:Tomography / Q000379:methods*
D005456:Fluorescent Dyes