Direct deposition on pre-cooled grids: a new approach to sub-millisecond cryoEM grid preparation

There has been a rapid expansion in new approaches to sample preparation for single-particle cryoEM. One drive has been around speeding up the process both for the application of time-resolved studies and in outrunning some of the negative effects which may come from the sample interacting with the air-water interface, such as degradation and preferred orientation. Here, we set out to significantly improve the speed of grid preparation by removing the plunging stage and directly spraying onto pre-cooled grids which are situated in the cryogen, allowing us to produce grids where two samples are mixed and frozen on the grid in less than 1 ms. We have previously shown a preliminary proof of principle with this new approach and here we provide a detailed description of the approach taken and the methodology behind it. This approach allows us to move beyond the millisecond timescale to open up more systems to time-resolved EM and potentially improve samples.