Development of Multiplexed Imaging Strategies for Fluorescence and Electron Microscopy

Abstract

Microscopy in biological research often relies on complementary analysis across multiple imaging modalities to reveal complex molecular contents and their spatial organization. Selecting the most appropriate method for a given experiment requires balancing and assessing the need for imaging speed, molecular specificity, accessibility, and resolution. In general, optical microscopy offers strong molecular specificity and multiplexed target detection, whereas electron microscopy provides superior spatial resolution. This dissertation explores two challenges in improving multiplexed imaging strategies across fluorescence microscopy and electron microscopy. Chapter 1 introduces the general principles of microscopy, conventional strategies for multiplexed imaging, and the limitations of these approaches. Chapter 2 addresses the fundamental challenge of improving single-shot spectral multiplexing and presents a polymer dot (Pdot)-based strategy for achieving 11-color imaging through a single round of labeling, excitation, and acquisition. Chapter 3 examines current limitations in implementing labeling strategies within cryogenic electron microscopy (cryo-EM) workflows and introduces a live-cell delivery approach using the mild detergent saponin to deliver exogenous probes prior to cryofixation while potentially retaining ultrastructure. Together, this dissertation discusses new approaches for supporting biological research through powerful and accessible multiplexed imaging strategies.