Rapid quantification of the effects of blotting for correlation of light and cryo-light microscopy images.
Recent technical developments allowed the accurate correlation of fluorescently labelled organelles in living cells to cryo-electron micrographs. We aimed at expanding this approach to Plasmodium berghei sporozoites, the motile forms of a rodent malaria parasite, which can be imaged by cryo-electron tomography in toto without the need for sectioning. Sporozoites are crescent shaped eukaryotic cells that move on flat supports including EM grids in a circular, unidirectional manner. While sporozoites can be visualized with fluorescent light and cryo-light microscopy prior to tomography, few motile sporozoites remained on the grid after blotting excess liquid impairing a complete correlation from light microscopy to cryo-electron tomography. Comparison with cells showing different adhesion strengths demonstrated that the ratio of cells remaining on the grid can be rapidly determined, but that the integrity of the cells has to be carefully monitored as the blotting applies high physical stress to the cells. We demonstrate a quick technique to assess not only feasibility of direct correlation without fixation but also the damage caused by blotting.
Lepper
S
S
Department of Parasitology, Hygiene Institute, University of Heidelberg Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.
Merkel
M
M
Sartori
A
A
Cyrklaff
M
M
Frischknecht
F
F
D020285:Cryoelectron Microscopy / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010962:Plasmodium berghei / Q000648:ultrastructure*