Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

Joost Snijder; Andrew J. Borst; Annie Dosey; Alexandra C. Walls; Anika Burrell; Vijay S. Reddy; Justin M. Kollman; David Veesler

Here, we present a simple and cost-effective method that builds on the standard plunge freezing procedure to overcome the aforementioned shortcomings. Although previous reports mentioned the possibility of multiple sample applications to increase particle density in suspended vitreous ice (Cheng et al. 2015) , this has never been described in detail to the best of our knowledge. We systematically tested this method on various samples and consistently observed that it greatly improved particle density in thin films of suspended vitreous ice, including cases in which ramping up the concentration failed to do so. We believe this procedure can benefit the exponentially growing numbers of single particle cryoEM practitioners who will inevitably run into samples that prove reluctant to evenly spread over holey cryoEM grids.