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Lysosomal pH plays an essential role in mediating various biological processes such as immunization, cell metabolism and enzyme activity. Herein, by utilizing the fluorescence resonance energy transfer (FRET) strategy, a two-photon (TP) ratiometric fluorescent probe (NpLys-pH) has been developed for tracking of lysosomal pH changes in living cells, tissues, and zebrafish. NpLys-pH was constructed by conjugating a pH turn-on TP fluorophore 1 (D-π-A-structured naphthalene derivative) with a pH turn-off naphthalimide fluorophore 2 via a non-conjugated linker. Meanwhile, NpLys-pH has two potential pH response sites that modulate the fluorescence signal by ICT and PET, respectively. The FRET process exists between the TP fluorophore 1 and naphthalimide fluorophore 2. In addition, NpLys-pH respond to pH rapidly and reversibly with high selectivity and sensitivity and has been applied for tracking lysosomal pH changes in living cells, tissues and zebrafish. We expect that the new probe present here can prompt the development of a wide variety of TP ratiometric fluorescent probes which can find application in detecting other important analytes in biological systems.

Bisulfite (HSO3−) and formaldehyde (FA) are hazardous environmental pollutants that pose serious risks to human health. Herein, we report for the first time a novel TP-FRET-based TP-ratiometric reversible fluorescence sensor NPXH, which is based on combining TP naphthalimide fluorophore with a deep red-emitting xanthene fluorophore via a nonconjugated linker, for reversible detection of bisulfite/formaldehyde. In presence of HSO3−, NPXH shows a significant ratio enhancement (∼6-fold) in I535/I630, ultra-fast response (less than 8 s) and super low detection limit (7.48 nM). In addition, the fluorescence signal of NPXH is easily and rapidly recovered by FA within 1 min. More impressively, NPXH has shown excellent detection capability in food and water samples and has been successfully applied to HSO3−/FA detection in liver tissue, zebrafish and mice.

Superoxide anion radical (O 2 − ), the “primary” reactive oxygen species (ROS) in living systems, is linked to a variety of physiological and pathological processes. Therefore, developing an effective strategy to monitor the fluctuation of O 2 − in biological systems is of great importance. This paper describes a new turn-on two-photon fluorescent probe for endogenous O 2 − detection and imaging, which was rationally designed and synthesized via a non-redox strategy. In the presence of O 2 − , the probe exhibited notable fluorescence enhancement (∼235-fold) with a low detection limit down to 1 nM, indicating a high signal-to-background ratio and excellent sensitivity. In addition, short response time, good biocompatibility, low cytotoxicity, long-term stability against light illumination, specificity to O 2 − over general reductants, and pH stability were demonstrated, indicating that the requirements for cellular O 2 − determination are met. Furthermore, the probe was successfully applied in two-photon fluorescence imaging of endogenous O 2 − in living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ∼150 μm, illustrating the promising potential for practical applications in complex biosystems and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of O 2 − .

Thiophenols are widely utilized for producing pesticides, polymers and pharmaceuticals. Studies have shown that prolonged exposure of human to thiolphenol can result in serious health problems such as significant damage of the central nervous system. Herein, we have developed a novel turn-on two-photon fluorescent probe TP-ASH for selective and sensitive detection of thiophenols. We first constructed a new two-photon dye by connecting a coumarin fluorophore and a naphthalene fluorophore through 2,5- disubstituted-1,3,4-oxadiazole ring to afford d -π-A-π- d -structure. Fluorescent studies showed that this new dye possess large two-photon active absorption across-section (250 GM) and high fluorescence quantum yield (0.85). Encouraged by the results, we proceeded to conjugate this novel dye to a thiophenol recognition moiety, 4-dinitrobenzene-ether (DNB). The resulting probe displayed high selectivity towards thiophenols over their similar analogues, aliphatic thiols. The probe also exhibited high sensitivity, with a detection limit of 6.7 nM and a fluorescence enhancement of 163-fold. In addition, we have successfully applied the probe for two-photon imaging studies of thiphenols in living cells and mice tissues. We envision that the probe will add useful tools in detecting thiophenols in environmental samples and biological systems.

Calcium ion (Ca2+) has proven to be the second messenger that is directly related to diverse normal biochemical functions and pathological processes. Thus, understanding the critical functions and dynamics of endogenous Ca2+ at cellular and animal levels is highly demanded. However, reliable fluorescence detecting and imaging strategies are usually challenging to achieve with high sensitivity, low background signal, and high imaging resolution for Ca2+ detecting and imaging analysis in vivo. Inspired by supramolecular self-assembly strategy, we herein report a novel supramolecular near-infrared (NIR) fluorescent nanosensor (NIRCD-Ca) for Ca2+ detecting and imaging analysis in vitro and in vivo with three parts: a guest NIR-fluorophore was labeled an adamantane moiety, a host supramolecular equipped with β-CD polymers, and a calcium ion chelating site of O,O'-Bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (BAPTA) as a fluorescence quenching moiety by photoinduce electron transfer effect (PET-effect). In the present work, NIRCD-Ca exhibits excellent water solubility and biocompatibility, very low background fluorescence with a significant ∼18-fold fluorescence intensity enhancement in the presence of Ca2+, superb resolution for mice imaging by NIR-emission, as well as extreme low detection line as low as 9.7 nM. All these features are favorable for detecting of Ca2+ in complex biological samples and applying for NIR imaging of Ca2+ in mouse model directly with satisfactory sensitivity, suggesting that the NIRCD-Ca is an ideal effective nanosensor for Ca2+ detecting and imaging analysis in biosystems. Furthermore, the nanosensor NIRCD-Ca has potentiality to become an essential detection technique for the investigation of signaling pathways involving Ca2+ in biosystems.

A naphthalene-based two-photon fluorescent probe for thiophenols has been reported in this work. The probe can be applied to the quantification of thiophenols with a linear range covering from 2.0 × 10 −8 to 7.0 × 10 −6 mol L −1 . It exhibited a high selectivity and excellent sensitivity with a detection limit of 9.6 nM. Moreover, it was successfully used for practical detection of thiophenol in water samples with a good recovery, and two-photon imaging of thiophenol in live cells and tissues at a depth of 40–155 μm.

Activation of cancer cell apoptosis and understanding its working mechanism play a critical role in formulating effective cancer treatment strategies. Herein, we report the design, synthesis, and biological evaluations of a selenium-containing fluorescent apoptotic agent MPSE. MPSE enabled the tracking and imaging of cancer cell apoptosis process. Its emission at 550 nm turned off in the presence of thioredoxin reductase (TrxR) in mitochondria, by forming a non-emissive compound MP. This emission “turn-off” phenomenon allows the monitoring of reactive oxygen species (ROS) induced mitochondria damages. As dysfunctional mitochondria were removed by lysosomes, MP entered lysosomes and its protonation led to bright emissions peaked at 475 nm. The new emission enables the tracking of lysosome activation during the apoptosis process. With the help of MPSE, along with other biological techniques, we have revealed three signal transduction pathways that activate cancer cell apoptosis and inhibit tumor cell proliferation. Our results showed that MPSE was highly useful in elucidating the working mechanism of theranostic-based optical apoptotic agents. We hope that understanding this mechanism will greatly facilitate the development of new cancer treatments.

Detection and imaging of hydroxyl radical (•OH) with high sensitivity and selectivity in biological systems is of great importance in evaluating the role of •OH plays in the physiological and pathological processes. Herein, a novel mitochondrion-targeting turn-on fluorescent probe RThy , has been developed to detect •OH in living cells and zebrafish. Probe’s turn on mechanism is based on the cleavage of azo-bond by •OH. In RThy sensing system, possessing two groups: a lipophilic cation as a targeting-group, and an azo group acts not only as a quenching-group for tunable light absorption but also as recognizing-group of •OH. In the absence of •OH, almost non-fluorescence emission at 550 nm was observed. However, after reaction with •OH, bright fluorescence emission at 550 nm gradually increased with the continuous addition of •OH, displaying ˜50 folds fluorescence intensity enhancements and a quite low detection limit of 8 nM. The selectivity test showed RThy exhibits excellent selectivity toward •OH among all the ROS/RNS species tested. Furthermore, the probe has been successfully employed for imaging •OH in living cells and zebrafish, thus demonstrating RThy as a useful tool for studying •OH in practical sample application, and providing powerful theoretical basis and technical support for investigation of the interaction •OH with physiological and pathology.