Design of Emission Ratiometric Metal-Ion Sensors with Enhanced Two-Photon Cross Section and Brightness

摘要

Two-photon excitation fluorescence microscopy (TPEM) has rapidly evolved into a widely used tool in biological and biomedical research. Compared to traditional fluorescence microscopy, TPEM offers intrinsic 3D resolution combined with reduced phototoxicity, increased specimen penetration, and negligible background fluorescence. At present, most fluorophores used as labels or sensor platforms in TPEM have been adopted from linear microscopy and are not optimized for two-photon excitation. Notably, the fluorescence brightness (ηδ), defined by the product of TPA cross section (δ) and emission quantum yield (η), is typically low due to a modest δ. The development of new TPEM-optimized fluorophores is particularly vital in the context of biological metal-ion sensing since most of currently available ratiometric sensors, including the widely used dyes fura-2 and indo-1, exhibit a low brightness that decreases even further upon cation binding. In addition, the majority of ratiometric metal-ion sensors offer only a large shift of the excitation peak but not emission energy. If only a single two-photon excitation source is on hand, such sensors are not suitable for dynamic ratiometric TPEM imaging with temporal resolution. In this communication, we address these problems with a molecular design approach that yields both an increase in δ and a shift of the peak emission energy upon metal-ion binding in a polar environment.