Organic Fluorophores for Single-Molecule Fluorescence Imaging: Strategies, Challenges, and Perspectives

Single-molecule fluorescence techniques have been listed as the important tools in biophysics, chemical biology and biomedical research, enabling the resolution of molecular heterogeneity, transient states, and nanoscale biological organization that are hidden in ensemble measurements. In these experiments, organic small-molecule fluorophores are widely used as labels for single-molecule Förster resonance energy transfer (smFRET), single-molecule tracking, single-molecule localization microscopy (SMLM), and stimulated emission depletion (STED) microscopy. Their performance, however, is limited by a finite photon budget, blinking, photobleaching, and the local environment. This review discusses recent strategies to improve the photostability and emission stability of organic fluorophores under the single-molecule conditions, including dye scaffold and substituent engineering, self-healing dyes, oxygen-scavenging systems, redox buffer systems, photostabilizing additives, microenvironmental regulation, and illumination optimization. Building on this framework, this review further examines the applicability limits of these strategies in complex biological systems, as well as remaining challenges in live-cell compatibility, near-infrared (NIR) fluorophore design, standardized performance evaluation, and data-driven optimization of experimental conditions.