Fluorescence provides a mechanism for achieving contrast in biological imaging that enables investigations of molecular structure, dynamics, and function at high spatial and temporal resolution. Small-molecule organic fluorophores have proven essential for such efforts and are widely used in advanced applications such as single-molecule and super-resolution microscopy. Yet, organic fluorophores, like all fluorescent species, exhibit instability in their emission characteristics, including blinking and photobleaching that limit their utility and performance. To overcome this limitation and to push the limit of biological imaging, we develop self-healing organic fluorophores, wherein the triplet state is intramolecularly quenched by a covalently attached stabilizer, exhibit markedly improved photostability for biological imaging in vitro and in living cells. Chapter 1 is a general introduction to fluorophores used for biological imaging and the photophysical framework for fluorophore stability. Chapter 2 overviews the methods used in this study. Chapter 3 is focused on the triplet-state-quenching mechanism of photostabilization in self-healing fluorophore. Chapter 4 discusses the generalization of this self-healing approach to organic fluorophores in different colors and structural categories. Chapter 5 presents that self-healing fluorophores improve single-molecule imaging in living cells. Chapter 6 presents a quantitative model for self-healing fluorophore photostability, leading to enhanced photostability that are not attainable with previous photo-protection approaches. Chapter 7 addresses the future directions.