A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography

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−/Å2) 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−/Å2, 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−/Å2 showed better results at ∼82 K than ∼12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.