New pH-activatable ratiometric fluorescent probes have been developed that have a coumarin structure attached to benzothiazole or indole units with one or two conjugated bonds. They have optical responses in acidic conditions. Probes with benzothiazole (1) and indole (2, 3) units have opposite responses to changes in pH, in terms of shifts in absorption and emission. Quantum chemical calculations with the B3LYP exchange functional provide insights into the differences in optical properties between 1 and 2/3. Probe 3 (7-diethylamino-3-[4-(3,3-dimethyl-3H-indol-2-yl)-buta-1,3-dienyl]-chromen-2-one) displayed a significant 122 nm red shift of the emission maximum; the new peak is located in the near-infrared region. In vitro assays demonstrate that probe 3 can be used in quantitative fluorescence imaging for the detection of intracellular pH distributions. In addition to the large Stokes shift, it has rapid responsivity, excellent cell permeability, and high selectivity. This study reveals that the conjugation of coumarin with benzothiazole or indole to form an intramolecular charge transfer (ICT) platform is an effective method to construct new fluorescent pH probes for acidic microenvironments.

Several benzo[a]phenoxazine derivatives containing substituted N-aromatic groups are evaluated for their pH-dependent absorption and emission properties. Among the compounds exhibiting optical responses under near-neutral and subacid pH conditions, benzo[a]phenoxazine derivatives with an electron-withdrawing aromatic group attached to nitrogen of the imino group show potential application as near-infrared pH sensors. Three water-soluble pH probes based on benzo[a]phenoxazine with different pyridinium structures are designed and synthesized. Their reversible pH-dependent emissions in buffer solution containing 0.1% dimethyl sulfoxide (DMSO) locate in 625-850 nm with the fluorescent enhancement of 8.2-40.1 times, and their calculated pKa values are 2.7, 5.8, and 7.1, respectively. A composite probe containing the three benzo[a]phenoxazines shows a linear pH-emission relationship in the range of pH 1.9-8.0. Real-time detection of intracellular pH using an in vitro assay with HeLa cells is also reported.

Six mitochondria/lysosomes self-targetable and viscosity-sensitive dyes (1a–1f) were developed via simple structure modification on cyanine-derived dyes. They all showed remarkable OFF–ON fluorescent response to viscosity in the near-infrared region (652–690 nm) and exhibited good linear relationship with solution viscosity. The transient absorption spectra were used to evaluate the excited-state lifetime of dye 1a in different viscosity environments. Furthermore, cellular imaging assays indicated that different derivatives (1a–1f) with the same chromophore core exhibited different organelle-targeting abilities. Among them, dyes 1a–1c could sense lysosomal viscosity fluctuations while dyes 1d–1f could be applied in mitochondrial viscosity detections.

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A benzo[a]phenoxazinium-based chemosensor bearing an N,N-di(2-picolyl)ethylenediamine unit was successfully synthesized. It is a long-wave emission and fully water-soluble fluorescent sensor with good membrane permeability for the selective detection of Zn(2+).