Long wavelength excitation frequency upconversion xanthene probe for fluorescence detection and imaging of cysteine

A significant limitation of existing fluorescent probes is their broad reactivity toward multiple biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). The structural similarity and overlapping reactivity of these thiol-containing species present a critical challenge for the selective discrimination of individual targets within complex biological environments. To address this limitation, we present M-Cys, a cysteine-selective frequency upconversion luminescence (FUCL) probe engineered from heteroanthracene-based fluorophores. By utilizing anti-Stokes emission, M-Cys enables near-infrared (NIR) excitation at 808 nm while simultaneously emitting NIR light (742 nm), thereby overcoming the limitations associated with conventional Stokes-shifted probes. This innovative design achieves a three-fold enhancement in sensitivity compared to traditional 690-nm excitation, with a detection limit of 23.2 nM, in contrast to 65.9 nM, attributable to reduced background interference and improved signal-to-noise ratios for FUCL. Comprehensive selectivity assays confirm M-Cys's specificity for cysteine over competing biothiols, thereby ensuring reliable biological discrimination. In addition to in vitro experiments, M-Cys significantly enhances high-contrast imaging of exogenous cysteine in live cells and facilitates in vivo FUCL visualization in murine models. This methodology leverages FUCL's deep tissue penetration and minimal phototoxicity. By integrating unprecedented sensitivity with translational applicability, this study establishes a versatile optical platform that advances high-precision probes for clinical diagnostics.