Ionic Liquids in Simplifying Cryogenic Electron Microscopy

The goal of this thesis was to simplify the sample preparation process for cryogenic electron microscopy (cryo-EM), clearing the way for the imaging of larger biomolecules and further expansion of the field. Various protic ionic liquids (PILs) were chosen for synthesis according to their pH and other physical properties. After several failed synthesizes, one PIL, cholinium dihydrogen phosphate, was chosen for further testing. This solution was put through a series of vitrification tests in order to understand its crystallization limits. Once limits were understood, cholinium dihydrogen phosphate was combined with ribosomal proteins and viewed under a transmission electron microscope to collect negative stain images. After adjusting the ratio of PIL to buffer and the concentration of ribosomes, images of whole intact ribosomes were captured. Samples were then placed in an EM grid, manually dipped in liquid nitrogen, and viewed using the the cryo-EM. These grids revealed ice too thick to properly image, an issue that was not solved by using a more aggressive blotting technique. Although the sample preparation process was not simplified, progress was made towards doing so and further testing using different techniques may result in success.