Fluorescent probes, as noninvasive tools for visualizing the metabolism of biomolecules, hold great potential to explore their physiological and pathological processes. For cysteine (Cys), however, none of the reported fluorescent probes could image the metabolic processes in living cells. To achieve this goal, we developed a coumarin derivative based on rational design of the dual recognition sites for Cys and its metabolite, SO2. The probe displayed distinct two channels with turn-on fluorescent emission toward Cys and SO2, which were successfully applied for imaging both A549 cells and zebrafish. Further, with reversible fluorescent responses toward Cys, the probe could image the enzymatic conversion of Cys to SO2 in living A549 cells in a ratiometric manner. The present work reports the first probe to image the endogenous generated SO2 without incubation of the SO2 donors.

The first two-photon probe, Lyso-OC, was proposed for use in monitoring cell autophagy by detection of the change in the lysosomal polarity during the membrane fusion process of autophagy. The Lyso-OC probe exhibited desirable optical properties and a detection signal that was selective to the polarity change. More importantly, Lyso-OC displayed real-time monitoring of autophagy in living cells.

The development of biocompatible two-photon fluorophores with a large absorption cross-section is challenging, despite the presence of theoretical guidelines. By rendering asymmetric PRODAN dye centrosymmetric, we designed and synthesized a novel class of two-photon fluorophores (TPZ). Their photophysical properties were investigated and their imaging potentials in cells, tissues and zebrafish were showcased.

A new two-photon fluorescent probe (MCN) for viscosity imaging was developed based on a 6-substituted quinoline framework. MCN showed an excellent “off–on” fluorescence response (ca. 90-fold enhancement) with viscosity increasing in the glycerol–water viscosity system. MCN showed great sensitivity to viscosity (R2 = 0.98, x = 0.65), which gave rise to cell imaging for micro-viscosity or real-time cell imaging during apoptosis with low cytotoxicity under two-photon excitation (λex = 800 nm). Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with MCN revealed that the intracellular average viscosity value was 73.45 ± 21.55 cP in cytosol. Imaging in living tissue slices indicated that MCN can work in deep tissue (∼130 μM) under two-photon excitation. Moreover, MCN also showed the capacity for in vivo imaging viscosity in zebrafish with obvious fluorescence emission.

•The first two-photon fluorescent probe for Cu (II) /PPi in aqueous solution was developed.•The probe exhibited large two-photon absorption cross sections at 740 nm.•The probe can detect PPi released from DNA amplification after polymerase chain reactions.•The probe was applied to image PPi in deep living tissues and zebrafish under TPM.The first two-photon fluorescent probe (PC) for selectively detecting biological Cu (Ⅱ) and pyrophosphate (PPi) has been developed based on 7-substituted coumarin in this study. The probe presented excellent selective two-photon “on-off-on” detection signal for Cu(II) /PPi in aqueous solution. The two-photon detection ensemble (PCCu) can detect PPi released from DNA amplification after the polymerase chain reactions (PCR). The probe showed low cytotoxicity and good biocompatibility, and therefore can be applied for imaging Cu(II)/PPi in living cells under two-photon excitation. Furthermore, the ensemble probe (PCCu) was also used to image PPi in deep living rat tissues (~100 µm) and in 5-days old zebrafish.A two-photon fluorescent probe for biological Cu (Ⅱ) and PPi detection in aqueous solution and in vivo has been developed.Download high-res image (369KB)Download full-size image

Mitochondria and lysosomes are essential cellular organelles in most eukaryotic cells by playing the physiological roles to support the normal functions of cells, as well as the life of the whole body. To date, small-molecule fluorescent probes have been considered as one of the vital tools for monitoring and visualizing multiple biological analytes. This review summarized the recent advances in small-molecule two-photon fluorescent probes for metal ions, reactive oxygen species (ROS) and reactive sulfur species (RSS), and changes inside micro-environment (e.g., pH, viscosity and polarity) in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.Download high-res image (140KB)Download full-size image

AuPt alloy nanoparticles (NPs) were facilely synthesized with oleylamine as the stabilizing ligand and characterized by high-resolution transmission electron microscopy, powder X-ray diffraction, inductively coupled plasma-atomic emission spectrometer analysis, and so on. In addition, the AuPt alloys supported by the nano CeO2 exhibit high selectivity and efficiency in hydrogenation of benzylidene acetone under ambient temperature and pressure. By analyzing the catalytic performance over the NPs with different Au:Pt compositions, we found that the TONPt values (based on the number of Pt atoms) vary in the same trend with the change of conversion. Despite that gold itself shows no catalytic activity, the improved conversion and TONPt with the alloy catalysts clearly show the promotion effect of gold on the catalytic activity of the platinum. The inactive metal significantly improves the catalytic activity of active metal, which shows that the AuPt alloy exhibits an interesting synergistic effect.

A two-photon fluorescent probe (DMPCA) for the detection of bisulfite anion in water was developed based on twisted-intramolecular-charge-transfer (TICT) mechanism. Fluorescence intensity of DMPCA increased dramatically (65 fold) upon the addition of HSO3−. The two-photon absorption cross-section of DMPCA was found to be 725 GM at 700 nm upon the addition of HSO3−. The fluorescent probe showed an excellent selectivity for bisulfite anion over other anions. The nucleophilic addition reaction mechanism with aldehyde was confirmed with NMR and MALDI-TOF. The TICT detection mechanism was confirmed by viscosity-dependent fluorescence spectra and time resolved fluorescence spectra. Moreover, DMPCA was successfully applied to the detection of bisulfite anion in HeLa cells with low cytotoxicity under two-photon excitation.

Two water-soluble carbazole-based two-photon fluorescent (TPF) probes (HCH and HCM) for ClO− detection were developed with rapid response, high selectivity and large two-photon absorption cross sections, which utilized the oxidative dehydrogenation of oxime to the nitrile oxide. It is found that the alkyl substituent groups in the cationic pyridinium moieties could affect the one-/two-photon luminescent properties of probes, and HCH was more suitable for ClO− detection than HCM thanks to the better stability of fluorescence, larger fluorescence enhancement folds and two-photon absorption cross sections for HCHCNO, the corresponding product of HCH toward ClO−. Bio-imaging study revealed that HCH could be used to detect ClO− in living cells under two-photon excitation, and co-staining experiments of HCH and MitoTracker Red FM (co-localization coefficient: 0.90) established that HCH was predominantly present in mitochondria.

A two-photon fluorescent probe (NQ) for cysteine (Cys) and homocysteine (Hcy) was developed based on twisted-intramolecular-charge-transfer (TICT) mechanism. NQ showed excellent “OFF–ON” fluorescence (at 540 nm) detection signal on addition of Cys/Hcy with high selectivity. The detection mechanism was successfully proved by theoretical calculations, viscosity dependent fluorescence and time resolved fluorescence spectra. NQ exhibits large two-photon absorption (TPA) cross-section with Cys/Hcy (580 and 710 GM) at nearly 800 nm, respectively. Cell-culture results indicated that NQ was cell-permeable and could be used to detect Cys/Hcy with low cytotoxicity under two-photon excitation in living cells. To our best knowledge, NQ is the first TICT based two-photon fluorescent probe for Cys/Hcy.A TICT based two-photon fluorescent probe was developed to detect cysteine (Cys) and homocysteine (Hcy) with high selectivity.

A novel two-photon fluorescent probe (HQ) for hypochlorite (ClO−) has been developed based on specific ClO−-promoted oxidation of a CN bond. The probe shows linear fluorescence responses to ClO− with 23.5-fold enhancement. Two-photon fluorescence detection signals of HQ to ClO− were observed with high selectivity and sensitivity. The good biocompatibility guaranteed the use of HQ to detect intracellular ClO− under two-photon excitation. Moreover, HQ has been successfully applied to monitor the endogenously produced hypochlorite in living cells (BV-2) stimulated by lipopolysaccharide (LPS). HQ provides a new ClO− detection agent for the discovery of pharmacological microglia modulators by high-throughput screening.

Two novel water-soluble coumarin-based compounds (OC7, NC7) were designed and synthesized as two-photon fluorescent probes for biological Mg2+ detection. The compounds feature a β-keto acid as a high selective binding site for Mg2+ and the coumarin framework as the two-photon fluorophore. OC7 and NC7 show significant “off-on” detecting signals (9.05-fold and 23.8-fold fluorescence enhancement) and lower detection limits compared with previous reported two-photon fluorescent probes for Mg2+. Moreover, OC7–Mg2+ and NC7–Mg2+ exhibit large two-photon absorption cross sections (340 and 615 GM) at the near-infrared wavelengths (740 and 860 nm), which indicates that the probes are very suitable for detection of Mg2+ in vivo. Both OC7 and NC7 are pH-insensitive and of low cytotoxicity and can be applied to image intracellular Mg2+ under two-photon microscopy (TPM). Our results provide a strategy to modify the coumarin fluorophore to get better two-photon fluorescent properties. And the results also suggest that electronic density of β-keto acid plays a very important role in the recognition of Mg2+.

•A novel Zn2+ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd2+ detection was reported.•The HQZn exhibited good two-photon properties and high selectivity for Cd2+ (especially over Zn2+).•The HQZn could be used for detecting intracellular Cd2+ with low cytotoxicity.A novel Zn2+ complex as the ratiometric two-photon fluorescent probe (HQZn) for Cd2+ detection was synthesized. Fluorescence emission spectra of the probe showed a large red-shift (75 nm) and obvious enhancement of fluorescent intensity upon the addition of Cd2+. HQZn shows high selectivity for Cd2+ over other metal ions, and can eliminate the interference of Zn2+ during Cd2+ detection. Maldi-TOF MS spectra indicated that the response of HQZn to Cd2+ was caused by central metal displacement. Cell cytotoxicity and bio-imaging studies revealed that HQZn was cell-permeable and it could be used to detect intracellular Cd2+ with low cytotoxicity under two-photon excitation.A novel Zn2+ complex as the ratiometic two-photon fluorescent probe (HQZn) for Cd2+ detection was synthesized.

A water-soluble “turn-on” fluorescent probe (RD1) for Fe3+ based on rhodamine B was designed and synthesized. The fluorescent probe showed “turn-on” fluorescent and colorimetric responses to Fe3+ with a high selectivity in water containing less than 1% organic cosolvent. Furthermore, bioimaging investigations indicated that the new probe was cell permeable and suitable for monitoring intracellular Fe3+ in living cells by confocal microscopy with low cytotoxicity.

A water-soluble fluorescent probe for Hg2+ based on a rhodamine B derivative was designed and synthesized. The new probe showed reversible colorimetric and fluorescent response to Hg2+ in a fully aqueous solution. The probe exhibited real-time detection of Hg2+ with high selectivity in media containing less than 1% organic cosolvent. Furthermore, bioimaging studies indicated that the new probe was cell permeable and suitable for the real-time imaging of Hg2+ in living cells by confocal microscopy.

A new quinoline based fluorescent probe (DQ) for Zn2+ and Cd2+ has been designed and synthesized. The new probe shows quite different fluorescence response to Zn2+ and Cd2+ in aqueous solution. IR and NMR spectra demonstrated that the different response is caused by amide tautomerization of the probe molecule when coordinating with Zn2+ and Cd2+. The dissociation constants of Zn2+ and Cd2+ complex were detected to be 3.36 and 30.62 nM, respectively. Other metal ions have no effect on the fluorescence spectrum of the probe. Thus the new probe can detect Zn2+ and Cd2+ in water with high selectivity and sensitivity.

A naked-eye turn-on fluorescent Fe3+ probe (RQ6) was developed by linking a new conjugated quinoline fluorescent group to the rhodamine platform. The probe can detect Fe3+ with high selectivity over other metal ions. Bioimaging studies indicated that RQ6 was cell permeable and suitable for detecting Fe3+ in the living cells by confocal microscopy.

A new two-photon fluorescent probe (MCN) for viscosity imaging was developed based on a 6-substituted quinoline framework. MCN showed an excellent “off–on” fluorescence response (ca. 90-fold enhancement) with viscosity increasing in the glycerol–water viscosity system. MCN showed great sensitivity to viscosity (R2 = 0.98, x = 0.65), which gave rise to cell imaging for micro-viscosity or real-time cell imaging during apoptosis with low cytotoxicity under two-photon excitation (λex = 800 nm). Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with MCN revealed that the intracellular average viscosity value was 73.45 ± 21.55 cP in cytosol. Imaging in living tissue slices indicated that MCN can work in deep tissue (∼130 μM) under two-photon excitation. Moreover, MCN also showed the capacity for in vivo imaging viscosity in zebrafish with obvious fluorescence emission.

The first two-photon probe, Lyso-OC, was proposed for use in monitoring cell autophagy by detection of the change in the lysosomal polarity during the membrane fusion process of autophagy. The Lyso-OC probe exhibited desirable optical properties and a detection signal that was selective to the polarity change. More importantly, Lyso-OC displayed real-time monitoring of autophagy in living cells.

A novel two-photon fluorescent probe (HQ) for hypochlorite (ClO−) has been developed based on specific ClO−-promoted oxidation of a CN bond. The probe shows linear fluorescence responses to ClO− with 23.5-fold enhancement. Two-photon fluorescence detection signals of HQ to ClO− were observed with high selectivity and sensitivity. The good biocompatibility guaranteed the use of HQ to detect intracellular ClO− under two-photon excitation. Moreover, HQ has been successfully applied to monitor the endogenously produced hypochlorite in living cells (BV-2) stimulated by lipopolysaccharide (LPS). HQ provides a new ClO− detection agent for the discovery of pharmacological microglia modulators by high-throughput screening.

A water-soluble “turn-on” fluorescent probe (RD1) for Fe3+ based on rhodamine B was designed and synthesized. The fluorescent probe showed “turn-on” fluorescent and colorimetric responses to Fe3+ with a high selectivity in water containing less than 1% organic cosolvent. Furthermore, bioimaging investigations indicated that the new probe was cell permeable and suitable for monitoring intracellular Fe3+ in living cells by confocal microscopy with low cytotoxicity.

A water-soluble fluorescent probe for Hg2+ based on a rhodamine B derivative was designed and synthesized. The new probe showed reversible colorimetric and fluorescent response to Hg2+ in a fully aqueous solution. The probe exhibited real-time detection of Hg2+ with high selectivity in media containing less than 1% organic cosolvent. Furthermore, bioimaging studies indicated that the new probe was cell permeable and suitable for the real-time imaging of Hg2+ in living cells by confocal microscopy.