New Near-Infrared Fluorescent Probes with Single-Photon Anti-Stokes-Shift Fluorescence for Sensitive Determination of pH Variances in Lysosomes with a Double-Checked Capability

Two near-infrared luminescent probes with Stokes-shift and single-photon anti-Stokes-shift fluorescence properties for sensitive determination of pH variance in lysosomes have been synthesized. A morpholine residue in probe A, which serves as a targeting group for lysosomes in viable cells, was attached to the fluorophores via a spirolactam moiety, while a mannose residue was ligated to probe B, resulting in an increased biocompatibility and solubility in water. Probes A and B contain closed spirolactam moieties, and show no Stokes-shift or anti-Stokes-shift fluorescence under neutral or alkali conditions. However, the probes incrementally react to pH variance from 7.22 to 2.76 with measurable increases in both Stokes-shift and anti-Stokes-shift fluorescence at 699 and 693 nm under 645 and 800 nm excitation, respectively. This acid-activated fluorescence is produced by the breaking of the probe spirolactam moiety, which greatly increased the overall p-conjugation in the probes. These probes possess upconversion near-infrared fluorescence imaging advantages including minimum cellular photo-damage, increased tissue penetration, and minimum biological fluorescence background. They display an excellent photostability with a low dye photobleaching and show a good biocompatibility. They are selective and capable of detecting pH variances in lysosomes at excitation with two different wavelengths, i.e., 645 and 800 nm.