Fluorene-based fluorescent probes with high two-photon action cross-sections for biological multiphoton imaging applications.

Two-photon fluorescence microscopy is a powerful tool for the study of dynamic cellular processes and live-cell imaging. Many commercially available fluorescent probes have been used in multiphoton-based imaging studies despite exhibiting relatively low two-photon absorption cross-section values in the tunability range of ultrafast Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with low fluorescence quantum yield and/or photoinstability (i.e., photobleaching) on exposure to the high peak power and photon density provided by the ultrafast laser source. To address the demand for better performing dyes, we prepare fluorophores tailored for multiphoton imaging. These fluorophores are based on the fluorene ring system, known to exhibit high fluorescence quantum yield (>0.7) and high photostability. Furthermore, an amine-reactive fluorescent probe for the covalent attachment onto amine-containing biomolecules is also prepared. Epi-fluorescence and two-photon fluorescence microscopy images of H9c2 rat cardiomyoblasts stained with an efficient two-photon absorbing fluorene fluorophore is demonstrated. Additionally, single-photon spectral characteristics of the amine-reactive fluorophore, as well as the two-photon absorption cross sections of its model adduct in solution, and spectral characterization of a bovine serum albumin (BSA) as a model bioconjugate are presented.