Water-Soluble Red-Fluorescent Dyes for Two-Photon Deep-Tissue Imaging

Abstract To realize in vivo deep two-photon fluorescence microscopy (TPFM), a probe is required that can undergo two-photon excitation and emit in the optical window of tissues (650–1100 nm). In addition, the probe should have high water solubility to avoid harmful organic solvents. 2,5-Dimethoxy-1,4-bis{1-[2-[2-(2-hydroxyethoxy)ethoxy]ethyl]-4-(1,3-butadien-1-yl)pyrinium}benzene iodide (MPBBI-PEG) was designed to meet all these criteria. Its water solubility is 10−4 mol dm−3, which is sufficient to stain biomaterials without organic solvents. The two-photon absorption (TPA) maximum is 960 nm with a cross section of 638 GM (1 GM = 1 × 10−50 cm4 per photon per molecule), and the fluorescence emission maximum is 663 nm. Distinct two-photon microscope images of HEK293 cells stained with aqueous MPBBI-PEG were obtained with less than 2 mW excitation. Thus, MPBBI-PEG is especially suitable for non-invasive TPFM imaging of deep tissues.