Correlative Cyo-fluorescence and cryo-TEM of samples embedded in vitreous ice
Bacterial cells, organelles or macromolecular complexes labeled with either exogenous fluorescent probes or genetically encoded markers such as GFP are plunge frozen on an EM grid using the FEI Vitrobot.
Grids are then viewed at liquid nitrogen temperature on the Olympus IX81 fluorescence microscope equipped with an FEI cryogenic stage and a sensitive CCD camera. The frozen grid is then transferred to the cryo-TEM (FEI Tecnai G2 F20 FE-TEM) and regions of interest that were identified in fluorescence micrographs are imaged at high resolution.
FEI cryo-fluorescence stage on the Olympus microscope. The inset shows a cryo-fluorescnece image of E. coli cells expressing S105-GFP fusion protein (Dewey, J., Savva, C., White, R., Vitha, S., Holzenburg, A. and Young, R.: Micron Scale Holes Terminate the Phage Infection Cycle. Proc. Natl. Acad. Sci. USA, 107, 2219-2223, 2010)
Cryo-fluorescence and cryo-TEM image of the same area of the grid. The arrows point to a single E. coli cell expressing S105-GFP fusion protein.
The large hole in E. coli plasma membrane caused by S105, the lambda-holin, has been captured in vivo by cryo-EM tomography. The red line indicates the size of the hole. Inset: cryofluorescence image of the same cell revealing the accumulation of S105-GFP. The location of the fluorescent patch corresponds to the membrane hole in the EM micrograph (arrows). Scale bar in the EM image bar corresponds to 500 nm.