2’,7’-Difluorofluorescein

2’,7’-Difluorofluorescein (CAS No.: 195136-58-4) is a fluorinated fluorescein derivative widely employed as a pH-sensitive fluorescent probe in biochemical and cell-based research, characterized by a pKa of approximately 4.7 and robust photostability under experimental conditions. Upon excitation at 488 nm, it exhibits pronounced pH-dependent fluorescence intensity associated with the formation of dianionic species, while reduced pH sensitivity at 450 nm excitation enables ratiometric analysis, facilitating accurate quantification of pH in the acidic to near-neutral range of approximately 2–7. The compound undergoes dynamic excited-state proton transfer among multiple protonation states, including rapid deprotonation of its cationic form, providing a mechanistic basis for its sensitivity to microenvironmental pH changes. This probe is particularly valuable for investigating intracellular and extracellular pH dynamics, including endosomal and lysosomal acidification, as well as for measuring pH in submicron aerosol particles and other confined systems. In addition to its use as a standalone imaging agent, it serves as a fluorescent label for proteins, a component in calcium indicator systems, and an anisotropy probe for studying molecular interactions. Its applications span cell biology, signal transduction research involving pH-sensitive pathways, and fluorescence-based assay development, where experimental concentrations are typically optimized according to assay design, often within low micromolar ranges in vitro.