Improving cryoEM and cryoFIB performance through amelioration of mechanical stress in vitreous ice.

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Project Summary/Abstract Stress introduced during vitrification has been proposed as a likely cause of two experimentally-observed methodological challenges: loss of resolution in cryo-electron microscopy (cryoEM) and attrition of lamellae milled in cryo-focused ion beam micromachining (cryoFIB). CryoEM is limited by a loss of information when the first few electrons strike, and mounting evidence points to beam-catalyzed relaxation of the built-up stress as the cause. A limitation of cryoFIB is that milled specimens break or snap during milling because of stress, but the source of this stress has not been shown. When specimens are prepared by plunge-freezing, water expands while the specimen support contracts. Additives that disrupt the open hydrogen-bonding network of water change its thermal expansivity. We will use volume-modulating excipients to change the thermal expansion coefficient of cryoEM and cryoFIB samples and eliminate mechanical stress from this source. Rheological properties of ice suggest that thermal annealing of cryoEM grids will allow solvent to flow and alleviate stress. We will warm cryoEM grids without devitrifying them to relieve stress. Therefore, our goal is to test whether volume-modulating excipients or thermal annealing will solve the problem of beam-induced motion and improve performance by 1. increasing resolution in cryoEM and 2. increasing yield in cryoFIB.