Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.
Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.
Sartori
Anna
A
Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, 82152 Martinsried, Germany. sartori@biochem.mpg.de
Gatz
Rudolf
R
Beck
Florian
F
Rigort
Alexander
A
Baumeister
Wolfgang
W
Plitzko
Juergen M
JM
D000818:Animals; D001331:Automation; D003196:Computer Graphics; D020285:Cryoelectron Microscopy / Q000379:methods*; D004583:Electrons; D004867:Equipment Design; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D012984:Software; D014054:Tomography / Q000379:methods*; D014584:User-Computer Interface