Single-Molecule Fluorescence Defines a New Dye Chemistry

Century-old fluorescent dyes have regained life in the wave of super-resolution microscopy. The achievement of single-molecule resolution transcends conventional ensemble averaging, imposing specific photophysical requirements on fluorescent dyes at a millisecond temporal resolution and ∼1 nm single-molecule spatial precision. This review aims to synthesize the fundamental principle governing single-molecule fluorescence in terms of single-molecule photon flux, switching kinetics, and the structure–function correlations. It emphasizes the role of structural modulation down to the atomic level for tuning single-molecule fluorescence characteristics to enhance imaging resolutions and proposes a taxonomy of landmark structural modifications for regulating single-molecule fluorescence. The analysis would provide guidelines for ongoing dye engineering efforts to develop next-generation dyes.