Highly Multiplexed Fluorescence Microscopy with Spectrally Tunable Semiconducting Polymer Dots

Abstract

The need for advanced imaging tools has grown rapidly with the increasing complexity of biological questions. To understand how cells behave and interact in near physiological conditions, in either thin tissue slices or across whole tissues, researchers must be able to map a wide range of molecular targets across space and scale. Traditional fluorescence imaging is limited to around five targets for a single sample. Commonly used probes often suffer from spectral overlap, narrow Stokes shifts, and susceptibility to photobleaching. This bottleneck restricts multiplexing and narrows the biological insight that can be gained from a single experiment. Beyond that, deep tissue imaging poses additional challenges. Light scattering and absorption, especially in thick or uncleared samples, can significantly degrade resolution and signal quality. Together, these limitations reduce both the depth and dimensionality of the information that can be extracted from complex specimens. As a result, questions involving spatial organization, tissue-scale signaling, or multicellular interactions often remain only partially answered. In this dissertation, I explore new strategies to push the boundaries of multiplexed fluorescence imaging, both in thin sections and in intact, cleared tissues. Chapter 1 introduces the core principles of fluorescence microscopy and highlights the constraints posed by conventional labeling tools in high-plex settings. Chapter 2 presents a new approach based on spectrally tunable semiconducting polymer dots (Pdots), which achieve large Stokes shifts and high brightness, enabling simultaneous detection of over 20 targets without specialized hardware or iterative cycles. Building on this foundation, Chapter 3 extends this capability into three dimensions through the integration of polymer-dot labeling with light sheet fluorescence microscopy and tissue clearing techniques. With further optimization on this system, I aim to build a fast, highly-multiplexed and scalable volumetric imaging platform, suitable for generating rich molecular and structural maps of biological systems of thick or whole organs.