Thesis Defense – Joseph Kim

Abstract: Fundamental understanding of neurons, the cellular unit of the central and peripheral nervous system, have been advanced by the advent of new microscopy techniques. In the past 20 years, cryo-electron tomography (cryo-ET) has emerged as a potent electron microscopy methodology to study neurons at nanometer or sub-nanometer resolution. By growing primary neuronal cultures on electron microscopy (EM) grids and rapidly freezing them in liquid ethane, they will be preserved in a near-native, hydrated state amenable for cryo-electron microscopy (cryo-EM). Tilted images (tilt series) can be acquired on thin cellular regions to reconstruct into a 3D volume known as a tomogram for 3D visualization. Such tomograms have provided new neurobiological insights such as cytoskeletal architecture and receptor organization at the postsynaptic membrane. However, neuronal cryo-ET is still a nascent field that can benefit from improvements in technique and new applications. In my work, I will describe my efforts in developing new cryo-ET technologies for neuronal cell biology such as micropatterning EM grids and montage tilt series acquisition. I will then detail applying these technologies to establish a new neuronal model while simultaneously discovering difficulties in sample preparation. Finally, I will present preliminary results on utilizing cryo-ET and auxiliary techniques for new ultrastructural insights on neuritogenesis and axon/dendrite distinction.