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Hydrazine is widely used in industrial and agricultural production, but excessive hydrazine possesses a serious threat to human health and environment. Here two new ratiometric fluorescence probes

The opening of mitochondrial permeability transition pore (mPTP) plays a fundamental role in cell apoptosis regulation, ischemia-reperfusion injury, and neurodegenerative disorders. However, the molecular tools for detecting mPTP open in cellular native status have not been reported yet. Herein, we

Ferroptosis is an unique iron-dependent cell death form and currently has been shown to closely relate with ER. Revealing the viscosity fluctuations of ER during ferroptosis is of great significance not only to monitor the occurrence and development of iron poisoning, but also to deeply understand the biological effects of ER in ferroptosis. Herein, we present an ER-targeting fluorescent probe (PV1) to detect viscosity changes of ER during ferroptosis. PV1 utilized a rotatable C-C bond to connect the two rigid π-systems, and responded viscosity by the regulation of the coplanarity of these two planes. PV1 displayed desirable sensitivity and selectivity to viscosity. The biological imaging results suggested that PV1 mainly distributed at ER in live cells, and the viscosity of ER exhibited an evident increase in the process of erastin-induced ferroptosis. After the simultaneous incubation of cells with erastin and Fer-1 or VE, the viscosity of ER showed no marked change, and it suggested that the erastin-induced ferroptosis could be inhibited by Fer-1 and VE. We expect that the developed probe could provide a feasible and rapid method for the in-depth study of the ferroptosis-based disease treatment and drug design.

Superoxide anion (O

Ferroptosis is characterized by the massive lipid peroxidation, and recently has been demonstrated to be closely associated with lipid droplets (LDs). However, the changes of LDs viscosity during ferroptosis are still unrevealed. Herein, we present the changes of the LDs viscosity during ferroptosis by a novel viscosity-sensitive near-infrared (NIR) fluorescent probe (BDHT). Probe BDHT (2-(benzo[d]thiazol-2-yl)-7-(4-(dimethylamino)phenyl)hepta-2,4,6-trienenitrile, C22H19N3S) showed highly sensitive and selective response to viscosity, mainly distributed in cellular LDs. By means of the real-time and in situ NIR imaging, we discovered that the LDs viscosity showed an obvious increase in HeLa cells during the erastin-induced ferroptosis process, while it displayed nearly no change when the cells were simultaneously treated with ferrostatin-1, which is a common inhibitor of ferroptosis. It is also confirmed that the LDs viscosity increased in several types of the cancer cells of erastin-induced and RSL3-induced ferroptosis. We expect that this new NIR probe could provide an effective approach to rapidly monitor ferroptosis, and these findings could greatly promote the in-depth understanding of the biological effects of LDs during ferroptosis.

Peroxynitrite (ONOO-) is one of reactive oxygen species, and plays a vital role in numeorus physiological and pathological processes. Given that the ONOO- level is closely related with various serious diseases, the in situ and real time detection of endogenous ONOO- is highly important for the in-depth study of its roles in living systems. Herein, we present a new fluorescent probe (RHPN) for the real-time detection of intracellular ONOO-. The probe RHPN consists of a rhodamine analogue and an arylhydrazide group as a response site for ONOO-. In response to ONOO-, the probe RHPN converts to an open-ring form and generates strong fluorescence. Moreover, the probe RHPN was successfully used for the imaging of the endogenous and exogenous ONOO- level changes in living cells.

Currently, fluorescent sensors for the detection of H2O in D2O are still quite rare. Herein, we present a dual-site controlled fluorescent sensor (CF-D2O) for the detection of H2O in D2O. As the H2O content in D2O decreases (namely, the purity of D2O increases), sensor CF-D2O provides two turn-on fluorescence signals. The response mechanism of sensor CF-D2O to H2O is based on the deprotonation of the two hydroxyl groups, as well as the FRET process between coumarin and xanthene units.

Sulfur dioxide (SO2) plays critical biological roles in living systems, and participates in a series of physiological and pathological processes. Aspartate aminotransferase 2 (AAT-2) catalyzes L-cysteine to produce endogenous SO2, which is mainly expressed in the mitochondria of cells. Herein, we present a new mitochondria-targeting and ratiometric fluorescent probe (MNP) for the detection of SO2 in living cells. The probe MNP employed a positively charged benzopyrylium moiety as the response site for SO2, and displayed a selective ratiometric response to SO2 on the basis of a FRET mechanism. Biological experiments demonstrated that the probe MNP was mainly distributed in mitochondria and could be used for the imaging of SO2 in living cells.

Various fluorescence probes for cyanide anions in water solution have been reported, but the investigation for endogenous cyanide recognition in plant tissues and mammalian cells is still limited. Here, a mitochondria-targeted cyanide fluorescent probe Mito-CP with colorimetric and ratiometric response was developed. The detection for cyanide was performed via the nucleophilic addition of cyanide to carbon-carbon double bond between coumarin moiety and pyridine moiety of the probe, which resulted in a color change from red to colorless and fluorescence change from red to blue. The probe showed high sensitivity (0.17 nM) and selectivity for cyanide over other common anion species and biothiols and could detect cyanide in real water samples with quantitative detection. Probe Mito-CP was successfully applied to detect endogenous cyanide in cassava root plant tissues. In addition, colocalization experiment showed that the probe has the property of targeting mitochondria with overlap coefficient being 0.93. A549 and HeLa living cells imaging experiment demonstrated the practical value of the probe in tracing CN− in biological systems. The probe also can detect endogenous cyanide change in mitochondria of rat pheochromocytoma (PC12) cells.

SO2 is recognized as the fourth gas messenger followed by NO, H2S and CO, it plays pivotal roles in many cellular activities and apoptosis in mitochondria and lysosomes. However, current molecular tools for of sensing SO2 in mitochondria and lysosomes respectively, which is not conducive to well defining the functions and relationships between mitochondria- and lysosome-SO2 in regulation of cellular activities. Thus, it is critical to develop powerful tool to detect mitochondria- and lysosome-SO2 simultaneously. Herein, we have rationally designed and synthesized a novel fluorescent probe DML-P based on a dual organelle-targeted groups for imaging SO2 in mitochondria and lysosomes simultaneously for the first time. The novel probe showed well water-solubility, high selectivity and high stability. Furthermore, probe has achieved in tracing endogenous SO2 with two stimulation methods by one- and two-photon modes. Significantly, the probe has succeeded in tracing endogenous SO2 in lysosomes and mitochondria concurrently in living cells. We hope this probe could provide a significative tool for studying the system functions and connections of mitochondria-and lysosomes-SO2.

As an important parameter of intracellular metabolism, pH plays important roles in maintaining normal physiological processes. The abnormal pH could cause disorder of cell function which may cause neurological diseases. Herein, we present two novel ratiometric fluorescent probes to detect pH changes. The probes employed 2-(2′-hydroxyphenyl)benzothiazole as fluorescent platform, and displayed desirable fluorescence response to pH on the basis of excited state intramolecular proton transfer (ESIPT) process. The probe BtyC-1 showed green fluorescence at 546 nm under acidic conditions, while it displayed strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm under alkaline conditions. Biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH and the NH4Cl-induced pH changes in living cells.

Cysteine (Cys) is an important mediator to regulate the redox state of endoplasmic reticulum (ER), and its level is closely related with many ER stress induced serious diseases. Herein, we present an ER-specific fluorescent probe for the ratiometric imaging of cellular Cys for the first time. The probe exhibited desirable selectivity and sensitivity to Cys. Biological imaging experiments demonstrated that the probe possessed an ER-targeting property, showed ratiometric response to Cys in ER, and could be applied for the ratiometric imaging of Cys level changes during H2O2-induced redox imbalance in living cells.

Sulfur dioxide (SO2), as the fourth gas signal molecule, is closely related to many diseases including respiratory diseases, nervous system diseases, cardiovascular diseases and cancers. Herein, we present a novel mitochondria-targeted ratiometric fluorescent probe (CS) for the detection of SO2 in living cells and zebrafishes. The probe CS was constructed on the base of coumarin and benzopyranium fluorophores, and exhibited intense blue and red fluorescence simultaneously. After the response to SO2, the fluorescence at 433 nm enhanced significantly while the fluorescence at 683 nm decreased obviously. The probe CS has a high sensitivity and selectivity to SO2. Moreover, the probe CS has been successfully applied to the detection of mitochondrial SO2 in living cells and zebrafishes.

PDMS is biocompatible, economically viable, transparent, and facile to handle and thus is suitable for fluorescent microscopy and biological research. However, there has been no report about polysiloxane-based fluorescent probes applied in bioimaging. In this report, a two-photon polysiloxane-based reversible luminescent probe (P1) was fabricated for the first time. P1 is a powerful tool for detecting the ClO–/GSH cycle in situ both in live cells and in zebrafish. This work demonstrates the potential of polysiloxane-based fluorescent probes for versatile in vivo or in vitro applications in the future.

SO2 has been recently identified as an essential gas messenger followed by NO, CO and H2S. However, abnormal concentrations of SO2 in our bodies can cause many diseases. Thus, the real-time monitoring of SO2 to well define the generation, physiological and pathological functions of SO2 is urgently needed. In this work, we developed a novel SO2 fluorescent probe on the basis of the conjugation of semi-cyanine and coumarin derivate dyes with superior features, such as near-infrared (NIR) and two-photon dual-mode monitoring, a large Stokes shift (175 nm), ultrafast response towards SO2 (within 10 s), high selectivity and photostability. Furthermore, this probe could sense SO2 by dual colorimetric and fluorescence means. In biological imaging, the probe was able to trace exogenous and endogenous SO2 in living cells, mitochondria, E. coli, zebrafish and mice under an NIR and two-photon dual-mode. These results demonstrated that the probe has strong potential for studying the physiological and pathological functions of SO2in vitro and in vivo.

Cell polarity is one of the important parameters of the biological microenvironment and controls the synthesis of functional proteins. As one of the key links in protein synthesis, the endoplasmic reticulum is highly sensitive to the cellular environment and closely related to cell polarity. Herein, we present an ER-targeting fluorescent probe (NSp) for the detection of cell polarity. The probe NSp used naphthalimide as a fluorescent platform and the p-toluenesulfonamide group as an ER targeting group. The red shift of the probe NSp in different solvents can reach 45 nm, and the displacement value in the system of dioxane and water can reach 50 nm. In addition, biological experiments show that the probe NSp shows a difference in the fluorescence intensity of normal cells and cancer cells, and the differentiation between normal cells and cancer cells is successful under conditions of endoplasmic reticulum stress. Besides, the probe can be used to distinguish between normal and cancerous tissues.

H2S is an important signaling molecule in living systems and participates in a series of physiological and pathological processes. In situ and rapid detection of H2S levels in living systems is of great importance to the in-depth study of its physiological and pathological roles. Herein, we present a new fluorescent probe (DFAN) for the detection of H2S in live cells and zebrafish. The probe DFAN utilized the auxiliary action of the ortho-aldehyde group to promote the thiolysis of 2,4-dinitrophenyl ether, and achieved a highly sensitive and selective response to H2S. The response mechanism of the probe DFAN to H2S is an integration of PET and ESIPT mechanisms. Moreover, the probe DFAN was successfully used for the fluorescence imaging of H2S in live cells and zebrafish.

Developing a reliable method to detect Na2S2O4 in real time is of great importance for the in-depth study of its toxicity to humans and to allow it to be safely handled. Currently, research on the fluorescence imaging of Na2S2O4 is very scarce. Herein, we present azo-based turn-on fluorescent probes with a high signal-to-noise ratio (emission enhancement >100-fold) for the visualization of Na2S2O4 in living cells.

As an important reactive oxygen species, hypochlorous acid (HClO) could irreversibly destroy Ca2+-ATPase to inhibit Ca2+ transport in the endoplasmic reticulum (ER), which may eventually lead to cell dysfunction and cell death. Herein, we present a novel ER-targeting fluorescent probe (NHS-ER) for the two-photon detection of HClO in living cells. NHS-ER was constructed using naphthalimide and showed strong green fluorescence. In response to HClO, the fluorescence intensity at 502 nm gradually decreased, and the probe showed excellent selectivity to HClO over other biologically relevant ROS. Moreover, the probe NHS-ER showed ER-targeting properties and could be successfully applied to the two-photon imaging of HClO in living cells.

The in-depth study of the interplay and cooperation between multiple organelles is an important biological task. Single fluorescent probes for separate visualization of multiple organelles is a desirable molecular tool, but the construction of such a probe is extremely difficult owing to the lack of valid strategies. In this work, utilizing the reversible cyclization reaction and intermolecular π stacking mechanism, a robust fluorescent probe is constructed to discriminatively illuminate lipid droplets (LDs), mitochondria, and lysosomes with blue, green, and red emission colors, respectively. Using the probe, the interplays and cooperation between LDs, mitochondria, and lysosomes are successfully studied, and the critical roles of lysosomes and LDs during mitochondrial fission are successfully revealed. Furthermore, this unique probe reveals the sequential damage of mitochondria and lysosomes during apoptosis through the successive fading of green and red emission. Thereby, the probe enables the discrimination of health state, early apoptosis, and late apoptosis of cells with three different sets of fluorescent signals. Overall, the robust probe is a desirable molecular tool to reveal the interactions between the three organelles, and investigate cell apoptosis and relative areas.

Colon cancer is a malignant neoplasm with high mortality that has seriously threatened human life. Accumulating evidence reveals that the β-glucuronidase (GLU), a lysosomal exoglycosidase enzyme, plays important roles in the pathological progression of colon cancer. Unfortunately, understanding the pathological roles of GLU remains a challenge due to the lack of effective detection methods for visualization the fluctuations of GLU in tissues. In this paper, based on hydrolysis function of GLU, an enzyme-activated ratiometric two-photon (TP) fluorescent probe (RN-GLU) was designed. RN-GLU was synthesized by introducing a glucopyranuronic acid methyl ester as the recognition group and 1, 8-naphthalimide as a TP fluorophore. In the presence of GLU, the trigger group was removed made an ICT process occurred induced enhancement of fluorescence ratio (I

Sulfur dioxide (SO

Mitochondrial membrane potential (ΔΨ

Ferroptosis is characterized by the massive lipid peroxidation, and recently has been demonstrated to be closely associated with lipid droplets (LDs). However, the changes of LDs viscosity during ferroptosis are still unrevealed. Herein, we present the changes of the LDs viscosity during ferroptosis by a novel viscosity-sensitive near-infrared (NIR) fluorescent probe (BDHT). Probe BDHT (2-(benzo[

Lipid droplets (LDs) are storage organelles at the centre of lipid and energy homeostasis, which act as vital hubs of cellular metabolism and the key to maintaining lipid and energy homeostasis. We synthesized a new two-photon fluorescent probe (CIV) that could detect the viscosity of lipid droplets. The probe is constructed via the typical ICT system of D–π–A using carbazole as the donor and imidazole as the acceptor. With the increase in viscosity from PBS to 99% glycerol, the fluorescence intensity of CIV increased by 13-fold, showing sensitivity and specificity towards viscosity. In addition, CIV showed low toxicity and excellent biocompatibility in cytotoxicity tests, and was successfully used for living cell LD imaging. Taken together, the results widen the way for the development of novel fluorescent probe-based the visualization LDs and detection in solutions, physiology and pathology.

ER stress has close relation with various metabolic diseases including obesity and insulin resistance, and could result in the abnormal production of ROS including O

Very recently, aggregation-induced emission (AIE) and two-photon (TP) emission materials have attracted great attention owing to their widespread applications. Herein, interestingly, we described a unique single fluorophore core with different substituents that can show either outstanding AIE or TP fluorescence properties. The introduction of an electron donating unit such as a tertiary amine group on the phenanthrenequinone imidazole core afforded a novel AIE-based fluorescent material PIN. The optical studies of PIN revealed that appropriate structural modifications on the phenanthrenequinone imidazole core could result in AIE character. On the other hand, modification of an electron-withdrawing moiety such as an indolium moiety on the same phenanthrenequinone imidazole-core provided a new material PID, which exhibited favorable TP emission, indicating that phenanthrenequinone imidazole derivatives could be exploited as TP materials. Furthermore, we have demonstrated that the novel AIE or TP materials constructed herein can be successfully applied for sensing targets of interest in aqueous and biological settings owing to their highly desirable emission profiles. The intriguing finding that careful modification of the phenanthrenequinone imidazole scaffold could afford excellent AIE or TP materials may open a new avenue to engineer robust materials with diverse properties based on a versatile core for various applications.

Herein, we report a unique D–π–A–π–D type fluorescent probe. The probe, with a large π system and two ICT processes, was sensitive to intracellular viscosity. Furthermore, the probe was successfully applied for the two-photon imaging of intracellular viscosity.

Developing a reliable method to detect Na

Propanedioyl dihydrazide (PDH), traditionally believed to be non-fluorescent, was first discovered to emit substantial fluorescence in both the solid state and solvents. Then, significantly, a novel strategy is provided: fluorescent non-aromatic small molecules with rigid molecular structures could be constructed by forming hydrogen bonds.

Currently, fluorescent sensors for the detection of H

Peroxynitrite (ONOO-) plays crucial roles in a variety of physiological and pathological processes in living systems, and therefore, real-time and in situ imaging of ONOO- is of great significance to in-depth study its biological roles. Herein, we have developed PET-ESIPT-based fluorescent probes (BCN and BCN-A) for the detection of ONOO- in living cells, zebrafishes and brain tissues. BCN was a highly sensitive ONOO- probe in which the fluorescence property of the fluorophore was simultaneously controlled by PET and ESIPT mechanisms, and transformed to BCN-A via acetylation. Especially, BCN showed large Stokes shift in response to ONOO-, and displayed high selectivity to ONOO-. BCN-A employed the acetate group to switch off ESIPT process of the fluorophore and improve the membrane permeability. In living cells, BCN-A released an ONOO--responsive probe (BCN) by the hydrolysis of esterase, and then detected ONOO-. Biological imaging demonstrated that although both metformin and rotenone are mitochondrial complex I inhibitors, metformin can increase the generation of ONOO- while rotenone had no significant influence on the generation of ONOO- in living cells and zebrafishes. Moreover, the amygdala and perirhinal/entorhinal cortex in the brain of depressive mouse both showed increasing fluorescence intensity relative to those of normal mouse, which suggested that the LPS-induced depressive disorder could result in the generation of ONOO- in the two brain areas of mouse. We expect that the probes (BCN and BCN-A) could extensively serve as the powerful molecular tools to investigate the biological roles of ONOO- for the in-depth study of drug mechanism and depressive disorder.

Sulfur dioxide (SO2) is an important signal molecule in living systems, and plays a wide range of physiological functions. Real-time and in situ detection of the dynamic balance of SO2 in mitochondria is of great significance to in-depth study its biological roles. Herein, we have developed a mitochondria-targeted fluorescent probe Nap-L based on the FRET mechanism to detect SO2 in living cells. The probe Nap-L employed naphthalimide and positively charged benzopyridine as the donor and acceptor in the FRET system, and emitted green and red fluorescence under excitation. In respond to SO2, the nucleophilic addition of bisulfite to benzopyridine and then interrupted the FRET process from naphthalimide to benzopyridine fluorophore, thereby triggering an obvious change in the fluorescence ratio. The probe Nap-L showed high selectivity to SO2 over the biothiols (Hcy, GSH, Cys) and other biologically related species. Biological experiments suggested that the probe Nap-L mainly distributed in mitochondria, and can be successfully used to detect mitochondrial endogenous SO2 in living cells.

1,4-Dithiothreitol (DTT) is an important small-molecular reducing agent and has extensive applications in biochemistry, peptide/protein chemistry and clinical medicine. The development of effective methods for monitoring DTT is of great importance for its safe use and studying its toxicity to human. In this work, we present a two-photon red-emissive probe for the imaging of DTT in living cells, tissues and animals. The probe employed a two-photon red-emissive xanthene dye as the fluorophore and selected 2,4-dinitrophenylate as the novel recognition site for DTT. In response to DTT, the probe displayed excellent sensitivity and selectivity. The probe was successfully applied to the two-photon imaging of DTT in living cells, and the imaging of DTT in living tissues and animals.

Mitochondria and nucleus play crucial roles during cell apoptosis process. In this work, a unique fluorescent probe capable of reversible migration between mitochondria and nucleus, as well as detection of cell viability in a dual-color mode is presented. The dual-color probe targets mitochondria in healthy cells, to form aggregates with deep-red emission. It migrates into nucleus and binds to DNA to form monomers with green fluorescence during apoptosis. Interestingly, the migration is reversible dependent on cell viability, which enables the dynamic visualization of apoptosis process. With the probe, mitochondria and nucleus can be visualized in dual colors during apoptosis, and the cell viability could be monitored by the emission color and localization of the probe.

The cytotoxicity of hydrazine (N2H4) is closely associated with mitochondria damages, and real-timely detecting mitochondrial N2H4 is of great importance for the in-depth study of the pathophysiological functions of N2H4 in living system. Although some of N2H4 fluorescent probes have been developed, the selective fluorescence imaging of mitochondrial N2H4 has rarely been reported. Herein, we present a novel mitochondria-targeted fluorescent probe (Rho-N2H4) for imaging N2H4 in living system. In the Rho-N2H4 system, a rhodamine derivative Rho with red emission was employed as the fluorophore, and acetyl group was utilized as response site for hydrazine. Upon treatment with N2H4, Rho-N2H4 displayed a drastically fluorescent signal at 645 nm. With the aid of Rho-N2H4, the fluorescence imaging of mitochondrial N2H4 in living cells was achieved. Assisted by high-definition 3D imaging, Rho-N2H4 can be applied for the imaging of N2H4 in living tissues with a penetration depth of about 50 μm with red emission manner. Furthermore, in vivo experiments demonstrated that Rho-N2H4 can be capable of imaging N2H4 in living animals. We expect that Rho-N2H4 could act as a promising tool for in-depthly unveiling the physiological and pathological roles of mitochondrial N2H4 in living system.