Dissertation Title: Ultrastructural membrane expansion microscopy

Abstract: 

Neuroscientists have long studied the complex neural circuits by examining their ultrastructure and molecular features. Electron microscopy (EM) has greatly advanced our fundamental understanding of neurobiology by revealing ultrastructural features through dense labeling of membranous structures while fluorescence microscopy (FM) allowed neuroscientists to identify and study specific biomolecules of interest. However, it would be ideal if such imaging could be achieved with FM (i.e., conventional light microscope), allowing anyone to identify and localize biomolecules in the detailed ultrastructural context of neural circuits. In this study, we report a novel membrane probe and modified expansion microscopy (ExM) protocol aimed at achieving conventional light microscope images that are comparable to low-resolution EM, enabling the visualization of ultrastructural features in thick mice brain tissue sections with molecular contrast, and pointing the way towards the possibility of tracing and reconstructing the neural circuitries. We demonstrate the ability of this novel strategy, which we call ultrastructure membrane expansion microscopy (umExM), to show ultrastructural features that were previously observable with EM using light microscopy and the localization biomolecules in the ultrastructural context with a resolution of ~60 nm. Combining umExM with existing fluorescence fluctuation imaging methods (i.e., SRRF), we achieved a resolution of ~30 nm. umExM may enable the routine use of ultrastructure imaging in neurobiology.

Committee members: 

Edward S. Boyden, Ph.D.
Y. Eva Tan Professor in Neurotechnology, Howard Hughes Medical Institute, McGovern Institute Professor, Departments of Brain and Cognitive Science, Media Arts and Sciences, and Biological Engineering
Massachusetts Institute of Technology

Sebastian Seung, Ph.D.
Evnin Professor in Neuroscience
Professor, Department of Computer Science and Princeton Neuroscience Institute
Princeton University

Jeremy M. Baskin, Ph.D.
Nancy and Peter Meinig Family Investigator in the Life Sciences Associate Professor, Department of Chemistry and Chemical Biology and Weill Institute for Cell and Molecular Biology
Cornell University