Over the last several decades, there has been a remarkable growth in the number of fluorescence imaging techniques for studying biological systems. Fluorescent small molecules, in particular, have significant advantages in optical imaging and analytical sensing due to their small size, high sensitivity, and fast response time. Functional fluorescent probes, such as photoactivatable fluorophores, can also provide dynamic information concerning the localization and quantity of biomolecules in living cells. Although numerous fluorescent probes are known, within the context of biological systems, “ideal” fluorescent probes are still in high demand, as properties such as cell permeability or cytotoxicity are often difficult to predict on the basis of rational design and theoretical calculations. Hence, there is still a need for simple fluorogenic scaffolds, which allow the rational design of molecules with predictable photophysical properties and are amenable to concise synthesis for high-throughput combinatorial screening. My work in the Chenoweth and Petersson Lab has focused on developing functional fluorescent probes based on modular scaffolds that exhibit advances over classic and contemporary dyes. In undertaking this work, three functional fluorescent scaffolds have been explored: (1) a quinoline-based highly tunable dye, (2) a diazaxanthilidene-based photoconvertible probe, and (3) an acridone-based fluorescent unnatural amino acid (UAA). Each fluorescent scaffold is rationally designed, efficiently synthesized, and utilized in live cell imaging experiments to demonstrate utility.