Abstract
Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
Highlights
Sample preparation of frozen-hydrated biological macromolecules continues to present a major challenge for routine structure determination by three-d imensional cryogenic transmission electron microscopy
The nanochannels are enclosed by ultrathin membranes made from silicon-rich nitride (SiNx) that allow for transmission of the electron beam when a thin film containing the sample is encapsulated in the space between them
While the precise origin for this clustering needs further investigation, we suggest that different thickness and geometry of the confined ice layer in the subregions for each of the multishot positions may lead to preferential directionality of specimen movement, possibly combined with microlensing effects resulting from adjacent exposures that induce the build-u p of semi-s tatic charge on the non- conductive SiNx membrane
Summary
Sample preparation of frozen-hydrated biological macromolecules continues to present a major challenge for routine structure determination by three-d imensional cryogenic transmission electron microscopy (cryo-E M).
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