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 approximately 82 or approximately 12 K, respectively, and the results were compared with particular interest in the doses (10-200 e-/A2) and resolutions (3-8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at approximately 12K through the first several tens of e-/A2, 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-/A2 showed better results at approximately 82 K than approximately 12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.
Iancu
Cristina V
CV
Division of Biology, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125, USA.
Wright
Elizabeth R
ER
Heymann
J Bernard
JB
Jensen
Grant J
GJ
D020285:Cryoelectron Microscopy / Q000379:methods*; D015925:Cryopreservation / Q000379:methods*; D006371:Helium / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry*; D014054:Tomography / Q000379:methods*
D006371:Helium; D009584:Nitrogen