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.