Reactive two-photon fluorescent probes for biological imaging

Two-photon fluorescence microscopy is a prominent tool in biological imaging analysis. Many commercially available fluorescent dyes currently being used have sufficed for multiphoton based imaging of biological samples. While measured two-photon cross-sections (in Goppert Meyer, GM units) of some of the dyes are available, many exhibit relatively low two-photon cross-section values in the tunability range of Ti:sapphire lasers commonly used in multiphoton microscopy imaging. For example, Bodipy FL exhibits a maximum GM unit of 18 at 925 nm, compared to a range of 2-4 GM units from 775 - 875 nm. Furthermore, available fluorophores may be plagued with either low fluorescence quantum yield and/or the additional problem of rapid photobleaching upon exposure to the high peak power provided by the fs laser source. In order to address the demand for better performing dyes for two-photon based imaging, we have prepared a new series of reactive fluorophores tailored for multiphoton imaging. These fluorophores are based upon the fluorene ring system, known to exhibit high fluorescence quantum yields, typically > 0.7, and possess high photostability. They have been functionalized with various moieties to act, e.g., as efficient amine-reactive fluorescent probes for the covalent attachment onto amine-containing biomolecules. Single-photon spectral characteristics, as well as measured two-photon cross sections of a reactive fluorophore and its model conjugate in solution, as well as spectral characterizations of a bovine serum albumin (BSA) conjugate are presented.