A Photostable, pH-Invariant Fluorescein Derivative for Single-Molecule Microscopy

We herein report the comprehensive characterization of the spectral and single-photon fluorescence properties of a recently synthesized fluorescein derivative and its biotinylated analog. The fluorophore displays significant increases in photostability compared to the known fluorescein label fluorescein isothiocyanate (FITC), as well as superb pH independence. This fluorescein variant has two readily accessible functional groups (aniline NH2 and phenol OH) that can be activated or blocked independently and can serve, for instance, as a fluorescent bridge between two different recognition motifs. Excellent single-photon counting fluorescence data demonstrates that it is also a particularly appropriate probe for single-molecule studies of biological interactions.