Unexpected omega-3 activities in intracellular lipolysis and macrophage foaming revealed by fluorescence lifetime imaging.

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo.

Synchronous Photoactivation-Imaging Fluorophores Break Limitations of Photobleaching and Phototoxicity in Live-cell Microscopy.

Fluorescence microscopy is one of the most important tools in the studies of cell biology and many other fields, but two fundamental issues, photobleaching and phototoxicity, associated with the fluorophores have still limited its use for long-term and strong-illumination imaging of live cells. Here, we report a new concept of fluorophore engineering chemistry, synchronous photoactivation-imaging (SPI) fluorophores, activating and exciting fluorophores by a single light source to thus avoid the repeated switches between activation and excitation lights. The chemically reconstructed, nonemissive fluorophores can be photolyzed to allow continuous replenishing of "bright-state" probes detectable by standard fluorescent microscopes in the imaging process so as to bypass the photobleaching barrier to greatly extend the imaging period. Equally importantly, SPI fluorophores substantially reduce photocytotoxicity due to the scavenging of reactive oxygen species (ROS) by a photoactivable group and the slow release of "bright-state" probes to minimize ROS generation. Using SPI fluorophores, the time-lapsed confocal (>16 h) and super-resolution (>3 h) imaging of subcellular organelles under intensive illumination (50 MW/cm2) were achieved in live cells.

Long-Lived Phosphorescent Carbon Dots as Photosensitizers for Total Antioxidant Capacity Assay.

Free radicals and their induced oxidative damage in living organisms are related to many diseases. Natural substances with antioxidant capacity are effective in scavenging free radicals, which could slow down aging and prevent diseases. However, the existing methods for the evaluation of antioxidant activity mostly required the use of complex instruments and operations. In this work, we proposed a unique method to determine the total antioxidant capacity (TAC) in real samples through a photosensitization-mediated oxidation system. N- and P-doped long-lived phosphorescent carbon dots (NPCDs) were developed, which exhibited the effective intersystem crossing from the singlet to the triplet state under UV light irradiation. Mechanism study confirmed that the energy of excited triplet state in NPCDs generated superoxide radicals and singlet oxygen through type I and type II photoreactions, respectively. On this basis, the quantitative determination of TAC in fresh fruits was achieved using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic bridge in the photosensitization-mediated oxidation system. This demonstration will not only provide a facile way to analyze antioxidant capacity in practical samples but also broaden the applications of phosphorescent carbon dots.

Recovery Mechanism of Endoplasmic Reticulum Revealed by Fluorescence Lifetime Imaging in Live Cells.

Endoplasmic reticulum (ER) is an important organelle of a membranous tubule network in cells for the synthesis, assembly, and modification of peptides, proteins, and enzymes. Autophagy and destruction of ER commonly occur during normal cellular activities. These processes have been studied extensively, but the spontaneous ER regeneration process is poorly understood because of the lack of molecular tools capable of distinguishing the intact, damaged, autophagic, and regenerative ER in live cells. Herein, we report a dual-localizing, environment-responsive, and lifetime-sensitive fluorescent probe for real-time monitoring ER autophagy and regeneration in live cells. Using this tool, the fluorescence lifetime imaging can quantitatively determine the degrees of ER destruction and spontaneous recovery. Significantly, we show that triglycerides supplied in lipid droplets can efficiently repair ER via the two critical pathways: (i) supplying materials for ER repair by converting triglycerides into fatty acids and diglycerides and (ii) partially inhibiting autophagy for stressed ER.

Fluorescence imaging of intracellular telomerase activity for tumor cell identification by oligonucleotide-functionalized gold nanoparticles.

As a specific biological marker for the occurrence and progression of tumor cells, detection of telomerase activity is of great importance for the physiological research of tumors. However, in situ measurement of telomerase activity in living cells still remains a challenge. Herein, we report a precisely designed oligonucleotide-functionalized gold nanoparticle probe that has realized high-efficiency detection of telomerase activity for cellular imaging toward the identification of tumors. Our method has achieved intracellular imaging of telomerase activity and shows good performance towards the distinction of tumor cells from normal ones. Moreover, the method reported here for tracking tumor cells in blood has wide applications in cancer diagnosis. This strategy offers an opportunity for cancer diagnosis, guiding therapy and evaluating prognosis.

Surface engineered bimetallic gold/silver nanoclusters for in situ imaging of mercury ions in living organisms.

Chemical sensing for the sensitive and reliable detection of mercury(II) ions (Hg2+) is of great importance in environmental protection, food safety, and biomedical applications. Due to the bio-enrichment property of Hg2+ in organisms, it is particularly meaningful to develop an effective tool that can in situ and rapidly monitor the level of Hg2+ in living organisms. In this work, we report ligand functionalized gold-silver bimetallic nanoclusters with bright red fluorescence as intracellular probes for imaging Hg2+ in living cells and zebrafish. The bimetallic nanoclusters of DTT-GSH@Au/AgNCs (DG-Au/AgNCs) with strong fluorescence that benefited from the synergistic effect of Au and Ag atoms were obtained through a one-pot synthesis method, incorporating glutathione (GSH) and dithiothreitol (DTT) as the reducers and functionalized ligands. Attractively, the bright red fluorescence of DG-Au/AgNCs could be rapidly and selectively quenched by Hg2+ within 1 min with a very low detection limit of 1.01 nM. Additionally, DG-Au/AgNCs had a great advantage in the detection of Hg2+ in living cells and zebrafish owing to its notably strong red fluorescence at 665 nm, which could avoid effectively auto-fluorescence interference from the organism. Such easily prepared bimetallic fluorescent nanoclusters would be expected to provide a noninvasive and sensitive approach in the detection of heavy metals in situ for environmental protection.

Revealing Sulfur Dioxide Regulation to Nucleophagy in Embryo Development by an Adaptive Coloration Probe.

Understanding signaling molecules in regulating organelles dynamics and programmed cell death is critical for embryo development but is also challenging because current imaging probes are incapable of simultaneously imaging the signaling molecules and the intracellular organelles they interact with. Here, we report a chemically and environmentally dual-responsive imaging probe that can react with gasotransmitters and label cell nuclei in distinctive fluorescent colors, similar to the adaptive coloration of chameleons. Using this intracellular chameleon-like probe in three-dimensional (3D) super-resolution dynamic imaging of live cells, we discovered SO2 as a critical upstream signaling molecule that activates nucleophagy in programmed cell death. An elevated level of SO2 prompts kiss fusion between the lysosomal and nuclear membranes and nucleus shrinkage and rupture. Significantly, we revealed that the gasotransmitter SO2 is majorly generated in the yolk, induces autophagy there at the initial stage of embryo development, and is highly related to the development of the auditory nervous system.

A Multi-responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super-resolution Imaging.

Understanding the biomolecular interactions in a specific organelle has been a long-standing challenge because it requires super-resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super-resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H2 O2 ), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H2 O2 level leads to disassociation of nucleic acid-protein complexes.

Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three-Channel Imaging Correlation.

Enzyme activity in live cells is dynamically regulated by small-molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long-standing challenge. Herein, we report a three-channel imaging correlation by a single dual-reactive fluorescent probe to measure the dependence of phosphatase activity on the H2 S level in cells. The two sites of the probe reactive to H2 S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three-channel imaging correlation shows that cells have an ideal level of H2 S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H2 S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H2 S in cellular signaling and in various human diseases.

Membrane-Penetrating Carbon Quantum Dots for Imaging Nucleic Acid Structures in Live Organisms.

The dynamics of DNA and RNA structures in live cells are important for understanding cell behaviors, such as transcription activity, protein expression, cell apoptosis, and hereditary disease, but are challenging to monitor in live organisms in real time. The difficulty is largely due to the lack of photostable imaging probes that can distinguish between DNA and RNA, and more importantly, are capable of crossing multiple membrane barriers ranging from the cell/organelle to the tissue/organ level. We report the discovery of a cationic carbon quantum dot (cQD) probe that emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA in live cells, thereby enabling real-time monitoring of DNA and RNA localization and motion. A surprising finding is that the probe can penetrate through various types of biological barriers in vitro and in vivo. Combined with standard and super-resolution microscopy, photostable cQDs allow time-lapse imaging of chromatin and nucleoli during cell division and Caenorhabditis elegans (C. elegans) growth.

Dynamic mapping of spontaneously produced H2S in the entire cell space and in live animals using a rationally designed molecular switch.

Hydrogen sulfide (H2S) is a key signaling molecule in the cytoprotection, vascular mediation and neurotransmission of living organisms. In-depth understanding of its production, trafficking, and transformation in cells is very important in the way H2S mediates cellular signal transductions and organism functions; it also motivates the development of H2S probes and imaging technologies. A fundamental challenge, however, is how to engineer probes with sensitivity and cellular penetrability that allow detection of spontaneous production of H2S in the entire cell space and live animals. Here, we report a rationally designed molecular switch capable of accessing all intracellular compartments, including the nucleus, lysosomes and mitochondria, for H2S detection. Our probe comprised three functional domains (H2S sensing, fluorescence, and biomembrane penetration), could enter almost all cell types readily, and exhibit a rapid and ultrasensitive response to H2S (≤120-fold fluorescence enhancement) for the dynamic mapping of spontaneously produced H2S as well as its distribution in the whole cell. In particular, the probe traversed blood/tissue/cell barriers to achieve mapping of endogenous H2S in metabolic organs of a live Danio rerio (zebrafish). These results open-up exciting opportunities to investigate H2S physiology and H2S-related diseases.

Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe.

Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.

Visualization of exhaled hydrogen sulphide on test paper with an ultrasensitive and time-gated luminescent probe.

Luminescent chemosensors for hydrogen sulphide (H2S) are of great interest because of the close association of H2S with our health. However, current probes for H2S detection have problems such as low sensitivity/selectivity, poor aqueous-solubility or interference from background fluorescence. This study reports an ultrasensitive and time-gated "switch on" probe for detection of H2S, and its application in test paper for visualization of exhaled H2S. The complex probe is synthesized with a luminescent Tb(3+) centre and three ligands of azido (-N3) substituted pyridine-2,6-dicarboxylic acid, giving the probe high hydrophilicity and relatively fast reaction dynamics with H2S because there are three -N3 groups in each molecule. The introduced -N3 group as a strong electron-withdrawing moiety effectively changes the energy level of ligand via intramolecular charge transfer (ICT), and thus breaks the energy transferring from ligand to lanthanide ion, resulting in quenching of Tb(3+) luminescence. On addition of H2S, the -N3 group can be reduced to an amine group to break the process of ICT, and the luminescence of Tb(3+) is recovered at a nanomolar sensitivity level. With a long lifetime of luminescence of Tb(3+) centre (1.9 ms), use of a time-gated technique effectively eliminates the background fluorescence by delaying fluorescence collection for 0.1 ms. The test paper imprinted by the complex probe ink can visualize clearly the trace H2S gas exhaled by mice.

Fluorescent Gold Nanoclusters with Interlocked Staples and a Fully Thiolate-Bound Kernel.

The structural features that render gold nanoclusters intrinsically fluorescent are currently not well understood. To address this issue, highly fluorescent gold nanoclusters have to be synthesized, and their structures must be determined. We herein report the synthesis of three fluorescent Au24 (SR)20 nanoclusters (R=C2 H4 Ph, CH2 Ph, or CH2 C6 H4 (t) Bu). According to UV/Vis/NIR, differential pulse voltammetry (DPV), and X-ray absorption fine structure (XAFS) analysis, these three nanoclusters adopt similar structures that feature a bi-tetrahedral Au8 kernel protected by four tetrameric Au4 (SR)5 motifs. At least two structural features are responsible for the unusual fluorescence of the Au24 (SR)20 nanoclusters: Two pairs of interlocked Au4 (SR)5 staples reduce the vibration loss, and the interactions between the kernel and the thiolate motifs enhance electron transfer from the ligand to the kernel moiety through the Au-S bonds, thereby enhancing the fluorescence. This work provides some clarification of the structure-fluorescence relationship of such clusters.

Label-free surface-enhanced Raman scattering imaging to monitor the metabolism of antitumor drug 6-mercaptopurine in living cells.

The molecular processes of drugs from cellular uptake to intracellular distribution as well as the intracellular interaction with the target molecule are critically important for the development of new antitumor drugs. In this work, we have successfully developed a label-free surface-enhanced Raman scattering (SERS) technique to monitor and visualize the metabolism of antitumor drug 6-mercaptopurine in living cells. It has been clearly demonstrated that Au@Ag NPs exhibit an excellent Raman enhancement effect to both 6-mercaptopurine and its metabolic product 6-mercaptopurine-ribose. Their different ways to absorb at the surface of Au@Ag NPs lead to the obvious spectral difference for distinguishing the antitumor drug and its metabolite by SERS spectra. The Au@Ag NPs can easily pass through cell membranes in a large amount and sensitively respond to the biological conversion of 6-mercaptopurine in tumor cells. The Raman imaging can visualize the real-time distribution of 6-mercaptopurine and its biotransformation with the concentrations in tumor cells. The SERS-based method reported here is simple and efficient for the assessments of drug efficacy and the understanding of the molecular therapeutic mechanism of antitumor drugs at the cellular level.

Inkjet-printed silver nanoparticle paper detects airborne species from crystalline explosives and their ultratrace residues in open environment.

An electronic nose can detect highly volatile chemicals in foods, drugs, and environments, but it is still very much a challenge to detect the odors from crystalline compounds (e.g., solid explosives) with a low vapor pressure using the present chemosensing techniques in such way as a dog's olfactory system can do. Here, we inkjet printed silver nanoparticles (AgNPs) on cellulose paper and established a Raman spectroscopic approach to detect the odors of explosive trinitrotoluene (TNT) crystals and residues in the open environment. The layer-by-layer printed AgNP paper was modified with p-aminobenzenethiol (PABT) for efficiently collecting airborne TNT via a charge-transfer reaction and for greatly enhancing the Raman scattering of PABT by multiple spectral resonances. Thus, a Raman switch concept by the Raman readout of PABT for the detection of TNT was proposed. The AgNPs paper at different sites exhibited a highly uniform sensitivity to TNT due to the layer-by-layer printing, and the sensitive limit could reach 1.6 × 10(-17) g/cm(2) TNT. Experimentally, upon applying a beam of near-infrared low-energy laser to slightly heat (but not destruct) TNT crystals, the resulting airborne TNT in the open environment was probed at the height of 5 cm, in which the concentration of airborne species was lower than 10 ppt by a theoretical analysis. Similarly, the odors from 1.4 ppm TNT in soil and 7.2, 2.9, and 5.7 ng/cm(2) TNT on clothing, leather, and envelope, respectively, were also quickly sensed for 2 s without destoying these inspected objects.

A viscosity-sensitivity probe for cross-platform multimodal imaging from mitochondria to animal.

Viscosity in biological systems is a critical factor for various physiological process, including signal transduction and metabolisms of substance and energy. Abnormal viscosity has been proven as a key feature of many diseases, thereby real-time monitoring of viscosities in cells and in vivo is of great significance for the diagnosis and therapy of related diseases. Up to date, it is still challenging to monitor viscosity cross-platform from organelles to cells to animals with a single probe. Here, we report a benzothiazolium-xanthene probe with rotatable bonds that switch on the optical signals in high viscosity environment. The enhancements of absorption, fluorescence intensity and lifetime signals allow to dynamically monitoring the viscosity change in mitochondria and cells, while near infrared absorption and emission facilitate imaging the viscosity with both fluorescence and photoacoustic imaging in animals. The cross-platform strategy is capable of monitoring the microenvironment with multifunctional imaging across various levels.

Shell thickness-dependent Raman enhancement for rapid identification and detection of pesticide residues at fruit peels.

Here, we report the shell thickness-dependent Raman enhancement of silver-coated gold nanoparticles (Au@Ag NPs) for the identification and detection of pesticide residues at various fruit peels. The Raman enhancement of Au@Ag NPs to a large family of sulfur-containing pesticides is ~2 orders of magnitude stronger than those of bare Au and Ag NPs, and there is a strong dependence of the Raman enhancement on the Ag shell thickness. It has been shown for the first time that the huge Raman enhancement is contributed by individual Au@Ag NPs rather than aggregated Au@Ag NPs with "hot spots" among the neighboring NPs. Therefore, the Au@Ag NPs with excellent individual-particle enhancement can be exploited as stand-alone-particle Raman amplifiers for the surface identification and detection of pesticide residues at various peels of fruits, such as apple, grape, mango, pear, and peach. By casting the particle sensors onto fruit peels, several types of pesticide residues (e.g., thiocarbamate and organophosphorous compounds) have been reliably/rapidly detected, for example, 1.5 nanograms of thiram per square centimeter at apple peel under the current unoptimized condition. The surface-lifting spectroscopic technique offers great practical potentials for the on-site assessment and identification of pesticide residues in agricultural products.

One-step synthesized amphiphilic carbon dots for the super-resolution imaging of endoplasmic reticulum in live cells.

Stimulated emission depletion (STED) microscopy provides a powerful tool for visualizing the ultrastructure and dynamics of subcellular organelles, however, the photobleaching of organelle trackers have limited the application of STED imaging in living cells. Here, we report photostable and amphiphilic carbon dots (Phe-CDs) with bright orange fluorescence via a simple one-pot hydrothermal treatment of o-phenylenediamine and phenylalanine. The obtained Phe-CDs not only had high brightness (quantum yield ∼18%) but also showed excellent photostability under ultraviolet irradiation. The CDs can quickly penetrate into cells within 2 min and are specific for intracellular ER. The further investigations by Phe-CDs revealed the reconstitution process of ER from loosely spaced tubes into a continuously dense network of tubules and sheets during cell division. Importantly, compared with the standard microscopy, STED super-resolution imaging allowed the tracking of the ER ultrastructure with a lateral resolution less than 100 nm and the pores within the ER network are clearly visible. Moreover, the three dimensional (3D) structure of ER was also successfully reconstructed from z-stack images due to the excellent photostability of Phe-CDs.

Real-time imaging of viscosity in the mitochondrial matrix by a red-emissive molecular rotor.

Mitochondrial matrix contains numerous metabolism-related proteins/enzymes and nucleic acids, which play key roles in the process of energy generation and signal transduction. The fluctuations in mitochondrial biomacromolecular levels lead to the changes in the mitochondrial matrix viscosity; therefore, real-time measuring the mitochondrial matrix viscosity is of great significance for the in-depth understanding of the mitochondrial physiology and pathobiology. However, investigations are limited due to the lack of a mitochondrial matrix-specific molecular rotor. Herein, we report a design of a molecular rotor that is specifically enriched in the mitochondrial matrix. The red fluorescence of the rotor switches on when the viscosity increases, enabling the real-time monitoring of the viscosity change therein. Interestingly, the rotor showed non-fluorescence behaviour in the liposome (mimicking membrane structure), avoiding fluorescence interference from the mitochondrial bilayer membrane. Super-resolution imaging reveals that the viscosity is uneven in an individual mitochondrion.