Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria

A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed on the basis of a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylate-functionalized merocyanine probe shows not only a mitochondria-targeting property but also highly selective detection and monitoring of Cys over other biothiols such as homocysteine (Hcy) and glutathione (GSH) and hydrogen sulfide (H₂S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F₅₁₈/F₄₅₂), which are linearly proportional to Cys concentrations in the range of 0.5-40μM. More importantly, the probe and its released fluorophore, merocyanine, exhibit strong two-photon excited fluorescence (TPEF) with two-photon action cross-section (σ_max) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively, which is highly desirable for high contrast and brightness ratiometric two-photon fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150μm by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.