Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts

Abstract

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 3-20nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and real-time monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics.

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

“…Particle encapsulation using protein scaffolds (Kedersha & Rome, 1986) or synthetic DNA structures (T. G. Martin et al, 2016) has also been proposed for avoiding air-water interface and preferred orientation issues. Lastly, decreasing the time between sample application and freezing in order to outrun air-water interface adsorption altogether might be possible with further technological development (Arnold et al, 2017;Feng et al, 2017;Frank, 2017;Jain et al, 2012). The time it takes for a particle to diffuse to an air-water interface, to diffuse across the air-water interface, and for subsequent bulk particles to adsorb to the resulting viscoelastic protein network film might be on the order of tens of milliseconds or greater, which appears to be largely dependent on protein surface hydrophobicity, protein concentration, and protein structure.…”

Automated cryo-EM sample preparation by pin-printing and jet vitrification

“…To improve sample deposition on EM grids, several developments have been presented, including droplet-based methods [20][21][22][23][24] , e.g. on nanowire grids 25 , and using capillaries followed by sample thinning by controlled water evaporation 26 . We sought a way to obtain sample thickness layers suitable for cryo-EM without the need for blotting, nanowire grids, and/or extra water evaporation steps.…”

Microscale Fluid Behavior during Cryo-EM Sample Blotting

“…As a result, one might have to prepare and screen a number of grids before one is found for which the ice thickness is suitable for high-resolution data collection over an acceptable fraction of the area. This lack of reliability has led recent efforts to investigate entirely new ways to make uniformly thin films, including spraying small-volume droplets of samples onto continuous carbon films (2,3) or onto self-wicking grids (4,5), and dip-pen or equivalent writing of thin liquid films onto EM grids (6,7). However, more information is needed about how the standard method fails in order to guide development of alternative approaches.…”