HBT-Cy 1 has been previously reported as a highly selective fluorescent probe for lysosome visualization in live cells. To further investigate the role of the structural components of HBT-Cy in lysosome selectivity, cyanine based fluorescent probe series (2–5) have been synthesized in good yields by connecting benzothiazolium cyanine (Cy) with 2-hydroxyphenylbenzothiazole (HBT) via a meta phenylene ring. Probes 2–5 exhibited exceptional photophysical properties including bright red-emission (λ_em ≈ 630–650 nm), a large Stokes shift (Δλ > 130 nm) and high fluorescence quantum yields (ϕ_fl ≈ 0.1–0.5). Probes 2, 3, and 5 exhibited exceptional selectivity towards cellular lysosomes in NHLF and MO3.13 cells. Our further study revealed that the phenyl benzothiazolium cyanine component (6) was the lysosome directing group in the HBT-Cy probe structure. The attachment of the hydroxyphenyl benzothiazole (HBT) component to the HBT-Cy probe structure has significantly improved its photophysical properties. Lysosome probes 2, 3 and 5 exhibited excellent biocompatibility, quick staining, bright red fluorescence, and wash-free application for live cell imaging. These probes further exhibited excellent characteristics for bioimaging experiments including a non-alkalinizing nature, high biocompatibility, high photostability and long-term imaging ability (>4 hours).

Molecular probes based on the excited-state intramolecular proton-transfer (ESIPT) mechanism have emerged to be attractive candidates for various applications. Although the steady-state fluorescence mechanisms of these ESIPT-based probes have been reported extensively, less information is available about the fluorescence lifetime characteristics of newly developed NIR-emitting dyes. In this study, four NIR-emitting ESIPT dyes with different cyanine terminal groups were investigated to evaluate their fluorescence lifetime characteristics in a polar aprotic solvent such as CH₂Cl₂. By using the time-correlated single-photon counting (TCSPC) method, these ESIPT-based dyes revealed a two-component exponential decay (τ₁ and τ₂) in about 2–4 nanoseconds (ns). These two components could be related to the excited keto tautomers. With the aid of model compounds (5 and 6) and low-temperature fluorescence spectroscopy (at −189 ℃), this study identified the intramolecular charge transfer (ICT) as one of the major factors that influenced the τ values. The results of this study also revealed that both fluorescence lifetimes and fractional contributions of each component were significantly affected by the probe structures.

A near-infrared (NIR) emitting (λ_em = 700–900 nm) molecular probe exhibits excellent selectivity towards mitochondria for live-cell imaging.