ELECTRON TOMOGRAPHY OF ICE-EMBEDDED PROKARYOTIC CELLS

Whole cells of archaea were embedded in vitreous ice by plunge freezin g and investigated by automated energy-filtered electron tomography at 120 kV. The embedded cells were between 300 and 750 nm thick, and the ir structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images, The dose was kept within tolerable l imits. A resolution of 20 nm allowed visualization of the individual s talks of the S-layer of Pyrobaculum aerophilum cells, which had underg one partial lysis, in three dimensions. The attainable resolution for low-dose electron tomography under different experimental conditions w as theoretically investigated in terms of the specimen thickness. To o btain 2-nm resolution at 120 kV (300 kV), the specimen must not be thi cker than 100 nm (150 nm). For a resolution of 10 nm, the maximum thic kness is 450 nm (700 nm). An accelerating voltage of 300 kV is advanta geous, mainly for specimens thicker than 100 nm. Experimental investig ations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.