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.

Oxazinium derivatives have recently played an important role in bioanalysis attributing to the distinguished properties, thus a detailed study of the structure-property relationship is especially significant. Herein, pH-sensitive optical properties of Nile Blue (1a), N-monoalkyl-Nile Blue (1b) and Azure A (1c) have been carried out in extreme acid and base conditions. Dyes 1a and 1c showed colorimetric changes by the protonation of nitrogen atom in strong acidic condition (pH < 2.0), and dyes 1a − c exhibited colorimetric changes by equilibrium between amino and imide groups in very strong basic case (pH > 7.6). Besides, their fluorescent properties were closed to ON − OFF and OFF − ON emissions at 640–820 nm under strong acidic and basic conditions. Moreover, the absorption and emission properties were reversible, and there were no remarkable optical intensity changes of dyes 1a − c under subacidic and neutral solutions (pH = 3.0–7.0). The (TD) DFT calculations were used to optimize the most stable structures of their corresponding protonated and deprotonated forms, and their absorption and emission properties were also explained. Their fluorescent properties nearly ON-OFF and OFF − ON in strong acidic and basic conditions at near-infrared region will give the possible application in pH detection for extreme conditions.

•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.

•One step synthesis of diaminodibenzoxanthenium skeleton.•The PET type pH probes with lower pKa.•Long wavelength emission in near-infrared region.The fluorescent pH probes, 3,11-diamino-7-(4-(diethylamino)-phenyl)- dibenzo[c,h]xanthen-14-ium trifluoromethanesulfonate (1a) and 3,11-bis-(diethylamino)-7-(4-(diethylamino)phenyl)dibenzo[c,h]xanthen-14-ium trifluoromethanesulfonate (1b), were reported as pH probes worked in strong acidic conditions. Probes 1a–b were prepared by one-step condensation with 4-diethylaminobenzaldehyde and aminonaphthalen-1-ol derivatives, their absorption maxima gave slight changes between neutral and acidic conditions, and they exhibited pH-dependent near-infrared OFF–ON emission in the range of pH 2.0–5.0 with increasing fluorescent intensities of 272 and 114 folds, the calculated pKa values were 3.28 and 3.24 for probe 1a and probe 1b, respectively. The probes also showed excellent selectivity toward proton over other interferents in the selectivity and competition assays. The photoinduced electron transfer effect from diethylaminophenyl group to dibenzoxanthenium skeleton was proposed and further verified by theoretical calculation. The work indicates that dibenzoxanthenium would be a near-infrared fluorophore for probe design.

•Hemicyanine dyes with a quaternary ammonium group.•NIR emission, large Stokes shift, water soluble, and large fluorescent enhancement towards nucleic acid.•Detection of RNA in fixed HeLa cells.Two hemicyanine dyes (1a–b) linked with tetraalkyl ammonium group were prepared, and they were evaluated as near-infrared probes for the detection of nucleic acid. Fluorescent responses were observed from 643 to 850 nm when probes 1a–b was titrated with commercially available DNA and RNA. The dimethylaminophenyl containing hemicyanine (1a) gave a 32.8-fold and 33.5-fold emission enhancement towards DNA and RNA, while the dimethylaminonaphthalen containing hemicyanine (1b) gave a 33.0-fold and 38.8-fold emission enhancement respectively. They also show large fluorescence Stokes shifts. The fluorescence of 1a–b distinguishes clear nucleoli staining accompanied by faint cytoplasm in cellular environment, and nucleic acid digest test of ribonuclease indicates their markedly higher affinity for RNA. These results prove that probes 1a–b are very attractive staining reagents for visualizing RNA in fixed cells.

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.

•Phenothiazinium containing optical films•Strong third-order nonlinear optical (NLO) absorption•Large third-order NLO susceptibilitiesThe third-order nonlinear optical properties of poly(methyl methacrylate) films doped with charge flowable 3,7-di(piperidinyl)phenothiazin-5-ium chloride, which tested by Z-scan method with nanosecond laser beam at 532 nm, are reported. Large third-order nonlinear optical susceptibilities (up to 10− 7 esu) and high second hyperpolarizabilities (up to 10− 27 esu) are found. The third-order nonlinear absorptions change from reverse saturated absorptions to saturated absorptions with different percentage of the phenothiazinium dye in the poly(methyl methacrylate) films, which can be explained by the accumulation phenomenon of the phenothiazinium. The results suggest that the phenothiazinium salt is a promising material for third order non-linear applications.

A cyanobenzo[a]phenoxazine-based pH probe with pKa = 5.0 exhibits OFF–ON emission at 625–850 nm upon excitation at 600 nm in aqueous buffers. The in cellulo imaging experiments with HeLa cells indicate that the probe can serve as a lysosome-specific probe under red light excitation (633 nm) with near infrared emission (650–790 nm).

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.

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.

A new probe based on the squaraine skeleton with a 2,4-dinitrobenzenesulfonyl unit is reported as a fluorescent probe for biothiol. It shows turn-on properties with red emission towards biothiols in acetonitrile–PBS buffer (1:9, v/v) solution, and its properties of rapid response, high selectivity and high sensitivity make it a potential probe with real applications. Moreover, the in vitro assays show that the probe can be used in fluorescence imaging for the detection of intracellular biothiol levels.

A series of unsymmetric pentamethine cyanine dyes featuring benzoxazolyl and benzothiazolyl groups were synthesized, the structures were characterized by 1H NMR, UV–vis absorption spectroscopy and mass spectrometry. The dyes with different anions such as chloride, tetraphenylborate and tetra(4-fluorophenyl)borate were also obtained and their solubility can be changed by the choice of the anion part. The third-order nonlinear optical properties were measured by the Z-scan technique with a picosecond laser beam at 532 nm in DMF solution. The third-order nonlinear optical susceptibilities χ(3) and the second-order hyperpolarizabilities γ' were obtained. The results reveal that these dyes exhibit strong reverse saturable absorption and nonlinear refraction.Graphical abstractHighlights► A series of unsymmetric pentamethine cyanine dyes were synthesized. ► Dyes with B(C6H4F)4− anion part possess favorable solubility. ► The third-order nonlinear optical (NLO) properties of dyes were reported.

•The molecular electrostatic potential (ESP) properties were used.•The chalcone compounds were used for the WORM type device.•The molecular geometries were optimized by the addition of a changeable electric field in the x direction.•The structure–property relationship was discussed.The molecular electrostatic potential (ESP) properties were used for the explanation of organic electric memory ability. Several chalcone compounds, owning a negative ESP region locates at the oxygen atom, were selected in this paper to validate the selection of compounds for organic memory materials. The synthesis, characterization, fabrication of the organic memory devices and the electrical properties for them were reported, and they were shown as WORM (write once read many times) type memory devices. The molecular geometries were optimized by the addition of a changeable electric field in the x direction inside the molecules using FF-DFT (Finite Field-Density Functionary Theory) method. The relationship between ESP of the molecules under different electric field and the property was discussed, and the mechanisms associated with the memory effect were also elucidated from DFT calculation results.Figure optionsDownload full-size imageDownload as PowerPoint slide

Six symmetrical and unsymmetrical trimethine cyanine dyes bearing benzothiazolyl and benzoxazolyl groups were synthesized. Their linear optical properties were studied. It was found that the variation of hetero atoms play an important role in the spectral properties of the dyes. Sequential replacement of the oxygen atom by the sulfur atom resulted in a change in shade from yellow to purple. In order to understand the relationship between the molecular structures and the spectral properties of these dyes, theoretical calculations were made using the Gaussian programme. Key parameters related to absorption and emission spectra were reported and discussed.Highlights► Symmetrical and unsymmetrical trimethine cyanine dyes were synthesized. ► The absorption and emission bands were regularly changed by the variation of the heteroatom from oxygen to sulfur. ► DFT and TD-DFT calculations were used to explain the spectral properties.

A cyanobenzo[a]phenoxazine-based pH probe with pKa = 5.0 exhibits OFF–ON emission at 625–850 nm upon excitation at 600 nm in aqueous buffers. The in cellulo imaging experiments with HeLa cells indicate that the probe can serve as a lysosome-specific probe under red light excitation (633 nm) with near infrared emission (650–790 nm).

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.

A new probe based on the squaraine skeleton with a 2,4-dinitrobenzenesulfonyl unit is reported as a fluorescent probe for biothiol. It shows turn-on properties with red emission towards biothiols in acetonitrile–PBS buffer (1:9, v/v) solution, and its properties of rapid response, high selectivity and high sensitivity make it a potential probe with real applications. Moreover, the in vitro assays show that the probe can be used in fluorescence imaging for the detection of intracellular biothiol levels.

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.