UCLA

Since its advent in 2013, Microcrystal Electron Diffrac9on (MicroED) has revolu9onized structural determina9on across a spectrum of samples, encompassing small organic molecules to proteins. MicroED closed a crucial gap between the determina9on of structures from 2D crystals and 3D crystals, facilita9ng the study of small 3D crystals that are too small for analysis by X-ray diffraction. The structures of molecules that were previously intractable using other exis9ng structural methodologies have been finally determined using MicroED. As MicroED is an emerging cryogenic electron microscopy (cryo-EM) technique, forging an entirely new branch in the field of structural biology, it is still under active development. The early success of MicroED was largely enabled by technological advancements introduced in cryo-EM, including high performance cameras and state-of-the-art electron microscopes. More recently, the implementation of focused ion beam (FIB) milling has opened many new avenues for MicroED. Crystal samples of any kind can be thinned to a suitable thickness for MicroED using a FIB mill. It is now possible to access crystals that are submerged in surrounding media or otherwise too large for the transmission of electrons. This work seeks to further develop the application of FIB milling in the MicroED workflow to solve critical issues related to sample preparation. The innovative methodologies that were designed and demonstrated in this thesis have accelerated MicroED development and will allow structural biologists to determine challenging molecular structures more rapidly.