UC Berkeley

Recent advances in fluorescence microscopes, such as super-resolution microscopy and fluorescence lifetime imaging microscopy (FLIM), have expanded our capabilities to decipher biological systems. However, the full potential of these technologies remains limited by the molecular tools available to label and track biological components. While genetically encoded fluorescent proteins and self-labeling enzymes have become standard, they often impose significant cellular perturbations.Small-molecule fluorophores and labeling strategies offer an alternative—one that bypass the need for genetic manipulation. Recent innovations in bioorthogonal chemistry have expanded the capabilities of small-molecule probes, enabling site-specific labeling and incorporation of extremely photostable dyes. This dissertation explores the development and application of chemically driven imaging tools that leverage the technologies that modern fluorescence microscopy has to offer.Chapter One provides a comprehensive overview of small-molecule probes designed to visualize organelle dynamics, highlighting how these tools can be applied across microscopy modalities and biological systems. Chapter Two presents the design and implementation of MAO-SiR, a novel high-density environmentally sensitive (HIDE) probe specifically developed for the inner mitochondrial membrane. MAO-SiR enables prolonged super-resolution imaging of mitochondrial dynamics, addressing a critical need for stability and spatial fidelity in live-cell imaging. Chapter Three describes a new FLIMbased multiplexing platform that employs bioorthogonal chemistry to control fluorescence lifetime through ligation strategies. This approach enables simultaneous imaging of three organelles using a single dye, without genetic manipulation, expanding the capabilities of FLIM multiplexing.Together, these chapters demonstrate how small-molecule tools can overcome the limitations of genetically encoded systems, providing new avenues for precise, noninvasive, and scalable live-cell imaging.