Abstract
The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has long been hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-defined images to be recorded. A new “G2 Polara” microscope from FEI Company was used to image various biological samples cooled by either liquid nitrogen or liquid helium to ∼82 or ∼12 K, respectively, and the results were compared with particular interest in the doses (10–200 e −/Å 2) and resolutions (3–8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at ∼12 K through the first several tens of e −/Å 2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no difference in the preservation of medium-resolution (3–5 nm) structural detail at the two temperatures. Tomographic reconstructions produced with total doses between 10 and 350 e −/Å 2 showed better results at ∼82 K than ∼12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.
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