Perhaps surprisingly though, the core design of fenestrated EM grid supports has remained largely unchanged. These developments have increased the speed of sample preparation and reduced the required sample volume by orders of magnitude. The inefficiency of current cryo-EM sample preparation approaches to yield reproducible results has led major efforts to explore different ways to make uniformly thin films, including the development of self-wicking grids ( Wei et al., 2018), spraying small-volume droplets onto holey support films ( Feng et al., 2017 Rubinstein et al., 2019 Kontziampasis et al., 2019) and direct thin film dispensing onto cryo-EM grids ( Jain et al., 2012 Arnold et al., 2017 Ravelli et al., 2020). Even if such conditions are found, additional problems encountered in practice include preferential orientation of particles ( Tan et al., 2017 Noble et al., 2018), preferential interaction of particles with the support film ( Meyerson et al., 2014) and partial denaturation of particles due to exposure to the large air-water interface ( Donaldson et al., 1980 Glaeser and Han, 2017 Glaeser, 2018 Noble et al., 2018 D’Imprima et al., 2019). Frequently, time-consuming parameter optimisation is required to find conditions suitable for high-resolution imaging, and these conditions are typically different for each sample. A recognised shortcoming of this method is that a reproducibly thin and consistently uniform ice layer can often not be achieved ( Carragher et al., 2019 Armstrong et al., 2020). The prevailing method for making thin specimens suitable for cryo-EM has not changed substantially since its introduction several decades ago: a filter paper is pressed against one or both sides of a fenestrated support film spanning a metallic mesh grid across which a droplet of the biological sample is suspended, and then rapidly plunged into a liquid cryogen ( Adrian et al., 1984). Within this layer, ideally not much thicker than the particle itself, the particles must be partitioned in random orientations.

To obtain a three-dimensional structure, a thin film of an aqueous solution containing the protein of interest must be cooled so rapidly that water molecules do not crystallise but form amorphous ice.

Sample preparation of frozen-hydrated biological macromolecules continues to present a major challenge for routine structure determination by three-dimensional cryogenic transmission electron microscopy (cryo-EM). 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. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. 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 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. 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. Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules.

Global parameters in comsol 5.1