Developing Uniform, Photon-Emitting Nanoprobes for Multi-Color Electron Microscopy

Abstract

Studying cellular processes relies on an understanding of subcellular protein interactions, often facilitated by cellular features. Unfortunately, there are no well-established methods for nanoscale imaging of these features alongside proteins. Existing bioimaging methods require complicated correlation methods or have complex sample preparation protocols. Electron microscopy (EM) has the highest resolution of all microscopy methods and can reveal cellular morphology with a nanoscale resolution. However, the resulting images are grayscale and do not label specific proteins. Inspired by probes in fluorescence imaging, this thesis developed a library of nine multi-colored nanoprobes for EM to permit protein visualization. The nanoprobes were composed of rare-earth elements that emit detectable photons of different wavelengths when excited by an electron beam. Photon emissions were spatially correlated with a simultaneously collected high-resolution EM image, producing a colorized EM image that labels tagged proteins with different colors. Each nanoprobe generated detectable photons with a high signal-to-noise ratio. They were small and uniformly shaped, making them ideal for bioimaging. Up to seven probes were simultaneously in a sample to demonstrate their distinct colors. This thesis illustrates, for the first time to our knowledge, multicolor EM, unlocking the potential to visualize proteins in their cellular context.