Simons Electron Microscopy Center, New York Structural Biology Center 1 article published in JoVE Biology Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton William C. Budell1, Luis Allegri2, Venkat Dandey1, Clinton S. Potter1, Bridget Carragher1 1The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, 2piTree Software The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.