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30-Jun-2026
Single particle cryo-EM (SPA) and cryogenic electron tomography (cryo-ET) are cryo-EM techniques that allow the structural characterisation of proteins and macromolecular assemblies either in vitro following purification, or in situ. Preparation of samples for each technique requires their vitrification, where samples are rapidly frozen in glass-like vitreous ice to preserve their integrity and ensure they are amenable to imaging by electron microscopy.
Optimisation of the vitrification process represents a major bottleneck in the preparation of samples for cryo-EM analysis. For SPA, a prerequisite for high quality data collection is the formation of ice that is not so thick that noise is introduced to the analysis, but is thick enough to support the particles in sufficient orientations for 3D reconstruction. Unfortunately for researchers in the field, there are several parameters that contribute to ice thickness upon vitrification, and challenging samples can require numerous optimisation steps. Furthermore, the process for assessing ice thickness requires the use of costly, highly sophisticated cryo-EM microscopes not amenable to use in high throughput screens.
EasyGrid, developed by Gergely Papp and his team of researchers at EMBL Grenoble, presents a solution to this bottleneck by integrating grid preparation and the assessment of ice thickness into a modular high-throughput platform compatible with both SPA and cryo-ET. For SPA applications, empty grids are exposed to an atmospheric plasma treatment module to enhance their hydrophilicity, with the sample then applied by an automated picoliter drop dispenser that can deliver droplets as small as 50 pL to multiple sites across the grid. A subsequent module utilises a pressure wave generator to ensure the spreading of SPA samples (or the removal of media from cells destined for cryo-ET) before the grids are vitrified by colliding ethane jets. This form of vitrification is more rapid than the commonly used plunge-freezing method, and further reduces the chance of crystalline ice formation, especially in thicker cryo-ET samples. The grids are kept in liquid nitrogen vapour to prevent ice contamination and stored in grid boxes labelled using a proprietary radio frequency tag compatible with cryogenic temperatures to enable their identification.
The final and perhaps most novel module is the EasyGrid Control module, where ice thickness can be measured using digital holographic microscopy. In this module a measurement laser beam undergoes a phase shift due to the difference in refractive index of the sample compared to surrounding nitrogen gas. It is then combined with a reference beam to produce a hologram that can be analysed to enable sub-wavelength thickness measurements across each grid. Importantly, measurements made using this technique correlate with measurements made using cryo-electron microscopes when screening the grids.
Overview of the EasyGrid Modular workflow for SPA and CryoET samples. From Gemin, O. et al.
In collaboration with researchers at the EMBL Heidelberg Instruct centre, SPA and cryo-ET datasets were collected to test the performance of the platform. They found that grids prepared using EasyGrid yielded high resolution 3D reconstructions of various proteins (e.g. Apoferritin to 1.94Å, KR2 rhodopsin to 2.3 Å, and guanidinase to 3 Å), whilst cryo-ET samples exhibited sevenfold improvement in the vitrification quality achieved within the nuclei of large human cells versus with traditional plunge-freezing methods.
Going forward, the use of high throughput platforms such as EasyGrid will enable researchers to more rapidly progress to cryo-EM data collection by expediting the grid preparation process. In addition to this, EasyGrid also circumvents some of the costs associated with screening grids using expensive cryo-EM microscopes prior to data collection and will allow researchers to make use of vital funding more effectively.
A video demonstration of how EasyGrid works, courtesy of EMBL.
Read the full paper in Nature Methods here.
