The OFF–ON and ON–OFF type pH probes based on rosamine were designed by using the relative electron densities between pyronine and various linked heterocycles. Probe 1a with an indole–pyronine skeleton gave an OFF–ON pH response (pKa = 1.41) with decreasing pH, and the relative fluorescence intensity increased 15-fold, while probe 1b with an imidazole–pyronine skeleton did not give an ON–OFF response to different pH values. When pyronine was connected with a quinolinyl group, i.e., probes 1c–d, the red emission (around 575–800 nm) gave a monotonous ON–OFF pH response (pKa = 3.26 and 2.62, respectively) with decreasing pH. The relative fluorescence intensities decreased 263- and 46-fold, respectively. Changes in the electron donating abilities of the nitrogen containing heterocycles were used to explain variations in PET processes within the probes, and their pH-dependent PET mechanisms were verified using time-dependent density functional theory calculations. Confocal fluorescence imaging was also used to evaluate the potential biomedical application of probes 1a–d. Ultimately, probe 1d with an appropriate pKa value and good biocompatibility showed lysosome targeting ability.

•pH probes for lysosome detection in normal cells.•Differentiation of normal cells from cancer cells by lysosome-biomarker.•The PET mechanism promoted by fluorophore based reactions (FBR-PET).In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5–6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a−c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF−ON response at 580–800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a−c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells.

In this paper, phenoxazinium was used as a fluorophore for the design of pH probes by the photoinduced electron transfer (PET) mechanism. Phenoxazinium with an aliphatic morpholinyl group (probe 1a) gave increased emission at 665 nm with pH ranging from 7.4 to 4.4; meanwhile, the other one with an aromatic diethylaminophenyl group (probe 1b) gave nearly OFF–ON emission at 679 nm with pH ranging from 7.4 to 4.2. They both were reversible pH probes with good selectivity. Their optical properties, especially the PET mechanism, were illustrated by (TD)DFT theory. Fluorescence confocal imaging of probes 1a–b and a typical phenoxazinium dye (Oxazine 1) was also performed, and the results indicated that probes 1a–b are lysosome-targetable biomarkers.

The lysosome-targetable OFF–ON type pH sensor that does not emit at pH = 4.0 is adopted for the selective detection of cancer cells, and the acidity difference of lysosomes in cancer and normal cells is verified. Three pH probes based on Darrow Red derivatives were designed and prepared that were demonstrated to be lysosome-specific biomarkers with inducible emission at 580–850 nm by the comparable in cellular imaging assays using HeLa, KB, and V79 cells. Of these, a pyridineium-2-yl Darrow Red analogue with a pKa of 2.4 was found to be a lysosome tracker for cancer cells, it is a unique pH sensor for the optical identification and distinction of cancer cells from normal cells and has potential application as a fluorescent biomaker of cancer cells in in vitro assays.

Two dibenzoxanthenium derivatives bearing a 3,4-diaminophenyl group, compounds 1a and 1b, have been prepared and evaluated as near-infrared probes for the detection of nitric oxide. Compound 1a gave a 3-fold emission enhancement towards nitric oxide at 681 nm, whereas compound 1b exhibited OFF–ON emission behavior towards nitric oxide, resulting in a 35-fold increase in the fluorescence intensity with an emission maximum of 750 nm being approved through (TD)DFT calculations. Probe 1b showed independent emission in the presence of common cations, amino acids, reactive oxygen and nitrogen species; furthermore, it can be used for the in cellulo imaging of nitric oxide under acidic and neutral conditions.

Long wavelength chromophores are of interest for material and biological applications. Six hemicyanine dyes (1a–f) constructed with oxazolo-pyridinium, indolium, benzooxazolium, or benzothiazolium groups and terminal anilino groups with multiple methylenes were prepared. They all exhibited fluorescence from 600 to 850 nm with emission maxima in the near-infrared region. The Stokes shifts became larger in more polar solvents, likely as a result of the greater dipole moments and geometry relaxations in the excited states as suggested by (TD)DFT calculations. The hemicyanine dye with the benzothiazolium group (1d) exhibited the best photostability and thermal stability among those tested. The potential application of 1d as a nucleic acid-staining fluorophore was evaluated. Fluorescent responses were observed from 575 to 850 nm when 1d was titrated with commercially available DNA and RNA. Dye 1d was also used in the selective fluorescent staining of RNA in live HeLa, KB and V79 cells. The results indicated that 1d can be used as a near-infrared fluorescent probe for nucleolus imaging in live cells.

Compared to the nitrogen-reaction based pH optical responsive compounds, oxygen-reaction related pH sensors have attracted less attention. In this paper, hemicyanine based pH probes are designed by establishing the equilibrium between phenolate and phenol, and their reversible absorption and emission responses towards pH are evaluated. The indolium–phenol based tetramethylene hemicyanine (1a) has colorimetric responses at 455 and 578 nm due to the protonating and deprotonating processes; its emission spectra shows ratiometric changes at 594 and 654 nm with large Stokes shifts under acidic (139 nm) and basic conditions (76 nm). The bromide substituent of the hemicyanine (1b) has a lower pKa value compared with unsubstituted hemicyanine (1a), which suggests that adjustable pKa can be achieved by the modification of electron withdrawing groups. The theoretical calculations based on the density functional theory (DFT) were also used to explain the optical properties. Moreover, the in cellulo fluorescence imaging shows that the hemicyanine (1a) can be used for the detection of intracellular pH levels.

•Synthesis and optical properties of trimethine cyanine with terminal amino groups are reported.•Optical film of trimethine cyanine with long alkyl chain was obtained by spin-coating.•Strong third-order nonlinear reverse saturable absorption and nonlinear refraction were found.Organic molecules with charge flow abilities gain much attention in third-order nonlinear optical materials. To evaluate the hypothesis of terminal donor and/or acceptor groups participating organic resonance structures for third-order nonlinear optical materials, conjugated double bonds structures with terminal amino groups are selected in this paper. The trimethine cyanine dye with quinolone skeleton (3a) was synthesized, it shows strong reverse saturable absorption and nonlinear refraction in dimethyl formamide solution at 532 nm. Then, its derivative compound (3b) with long alkyl chains was synthesized in order to improve the film forming performance, the spinning coating thin film exhibits strong reverse saturable absorption with the third-order nonlinear susceptibilities χI(3)=3.42×10−8esu and χ(3)=3.23×10−9esu under nanosecond and picosecond laser beams respectively. The results validate that the organic compounds with terminal donor and/or acceptor groups participating resonance structures have potential value for the design of the third-order nonlinear optical materials.

•Third-order NLO properties of the phenoxazinium doped PMMA films were obtained.•Reverse saturated absorption changes to saturated absorption by the different percentage of phenoxazinium in the PMMA films.•Different states of films were obtained.The third-order nonlinear optical properties of spin coating poly(methyl methacrylate) films doped with 3,7-bis(diethylamino)phenoxazin-5-ium chloride (1) are reported. The third-order nonlinear optical properties were tested by Z-scan technology with nanosecond laser beam at 532 nm. The third-order nonlinear absorptions change from reverse saturation absorption to saturation absorption with increasing percentage of the phenoxazinium dye in the poly(methyl methacrylate) film. The different forms of the spin coating films were obtained by atomic force microscopy and transmission electron microscope, and the different aggregation phenomenon of the phenoxazinium in the films can explain the adjustable third-order nonlinear absorption phenomena. Moreover, large third-order nonlinear optical susceptibilities (up to 10−7 esu) and high second hyperpolarizabilities (up to 10−28 esu) are found with these films. The results indicate that the phenoxazinium dye is the potential material for the third-order nonlinear optical application.

Long wavelength chromophores are of interest for material and biological applications. Six hemicyanine dyes (1a–f) constructed with oxazolo-pyridinium, indolium, benzooxazolium, or benzothiazolium groups and terminal anilino groups with multiple methylenes were prepared. They all exhibited fluorescence from 600 to 850 nm with emission maxima in the near-infrared region. The Stokes shifts became larger in more polar solvents, likely as a result of the greater dipole moments and geometry relaxations in the excited states as suggested by (TD)DFT calculations. The hemicyanine dye with the benzothiazolium group (1d) exhibited the best photostability and thermal stability among those tested. The potential application of 1d as a nucleic acid-staining fluorophore was evaluated. Fluorescent responses were observed from 575 to 850 nm when 1d was titrated with commercially available DNA and RNA. Dye 1d was also used in the selective fluorescent staining of RNA in live HeLa, KB and V79 cells. The results indicated that 1d can be used as a near-infrared fluorescent probe for nucleolus imaging in live cells.

Two dibenzoxanthenium derivatives bearing a 3,4-diaminophenyl group, compounds 1a and 1b, have been prepared and evaluated as near-infrared probes for the detection of nitric oxide. Compound 1a gave a 3-fold emission enhancement towards nitric oxide at 681 nm, whereas compound 1b exhibited OFF–ON emission behavior towards nitric oxide, resulting in a 35-fold increase in the fluorescence intensity with an emission maximum of 750 nm being approved through (TD)DFT calculations. Probe 1b showed independent emission in the presence of common cations, amino acids, reactive oxygen and nitrogen species; furthermore, it can be used for the in cellulo imaging of nitric oxide under acidic and neutral conditions.