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.

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.

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.

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.

Revealing the signaling regulation of hydrogen peroxide to cell pyroptosis using a ratiometric fluorescent probe in living cells.

A ratiometric fluorescent probe with a large emission shift was developed for the accurate measurement of hydrogen peroxide (H2O2) in sophisticated pyroptosis signaling pathways. The results reported here demonstrate that H2O2, as a principal member of ROS, is a critical upstream signaling molecule in regulating pyroptosis.

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.

An azacyclo-localizing fluorescent probe for the specific labeling of lysosome and autolysosome.

Understanding lysosome-related physiology needs specific lysosome probes to track the biological processes of lysosome in living cells. Here, we report an azacyclo-modified fluorescent probe that has a large Stokes shift, good photostability and negligible cytotoxicity for highly specific labeling of lysosome and autolysosome in living cells. The probes with different kinds of azacyclo groups on parent dye dansyl are screened to show that dansyl-cycleanine (DNS-C) with four nitrogen atoms possesses the best lysosome-localized ability. And DNS-C as a universal tracker exhibits excellent ability for lysosome labeling in different cell lines with high overlap coefficients (≥0.90). Different from a commercially available LysoTracker, the Stokes shift of DNS-C up to 240 nm (λex/em = 330/570 nm), is much larger than that of LysoTracker ~20 nm (λex/em = 573/595 nm). More importantly, the fluorescence of DNS-C keeps still high brightness after a time-lapsed imaging for 40 min in living cells, implying its remarkable photostability for long-term tracking. In addition, DNS-C can also clearly image the autolysosome, a critical subcellular compartment, forming by the fusion of lysosome with autophagosome in autophagy. These results suggest the promising utility of our probe as a powerful tool to real-time trace physiological processes of lysosomes.

Graphene Oxide: From Tunable Structures to Diverse Luminescence Behaviors.

Since the first discovery of luminescent graphene oxide (GO), exponentially increasing investigations on the tunable structures and surfaces for modulating its optical properties have struggled to expand applications in imaging, sensing, biomedical diagnostics, and so on. Here, the latest works on reconstructing or modifying the structures and surfaces of GO to achieve diverse luminescence are systematically reviewed, including fluorescence, electroluminescence, and chemiluminescence. Moreover, the fundamental difficulties of the investigations and applications of luminescent GO nanomaterials are clarified to inspire more constructive thoughts for expanding their application boundaries.

Semiquantitative Visual Detection of Lead Ions with a Smartphone via a Colorimetric Paper-Based Analytical Device.

A simple, instrument-free, paper-based analytical device with dual-emission carbon dots (CDs) (blue CDs and red CDs) was developed for the semiquantitative, visual, and sensitive speciation analysis of lead ions in a real sample with a sensitive detection limit of 2.89 nM. When a paper strip was immersed into the sample solution, the blue fluorescence was quenched by Pb2+ in solution, while the red fluorescence served as a background reference without color change, and significant color evolutions from blue to red were observed under the ultraviolet lamp, resulting in a semiquantitative visual detection. Furthermore, a smartphone was used in the visual detection of lead ions by identifying the RGB value of the fluorescent probe solution and corresponding paper strip. The application of smartphones and fluorescent paper strips has greatly shortened the detection time and reduced the cost of detection, providing a new strategy for the on-site and semiquantitative detection of heavy-metal ions in water samples.

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.

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.

Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids.

Distinguishing tumor cells from normal cells holds the key to precision diagnosis and effective intervention of cancers. The fundamental difficulties, however, are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers. Here, we report a concept of tumor cell detection, bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going toward the hallmark of cancer, uncontrollable growth. Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms, ranging from solution-based assay, to single cell imaging and in vivo solid tumor imaging (unlike PCR that is restricted to cell lysates). We envision the SNAB technology will impact cancer diagnosis, therapeutic response assessment, and image-guided surgery.

Ratiometric Fluorescent Biosensor for Visual Discrimination of Cancer Cells with Different Telomerase Expression Levels.

Telomerase is inactive in normal somatic cells but highly activated in tumor cells to maintain their indefinite proliferation and immortal phenotype. As a specific marker for the generation and progress of almost all tumors, the detection of telomerase activity by classical PCR techniques has served in the biological research of tumors. However, the detection of in situ telomerase activity in cell extracts to evaluate the malignancy, progress, and metastasis of tumors remains a daunting challenge. Here, a precisely designed FRET-based ratiometric fluorescent oligonucleotide probe has achieved high-fidelity detection of telomerase activity for accurate discrimination of different cancer cells toward advanced diagnosis of tumors. Our method is superior to other methods in its capabilities to quantify telomerase activity in cell extracts and visualize various tumor cell extracts with different telomerase expression levels by the naked eye for clinical diagnosis. In particular, the ratiometric fluorescent probe used in the assay could exclude other experimental factors influence, and further avoid false positive signal generation. The method reported here could provide a reliable, accurate, and convenient way in medical diagnostics and therapeutic response assessment.

Efficient removal of hexavalent chromium from water by an adsorption-reduction mechanism with sandwiched nanocomposites.

Hexavalent chromium Cr(vi), one of the most toxic contaminants, is released in the environment due to various anthropogenic activities. This study presents a novel sandwiched nanocomposite synthesized using graphene oxide (GO), manganese dioxide (MnO2) nanowires, iron oxide (Fe3O4) nanoparticles and polypyrrole (PPy) to remove hexavalent chromium ion Cr(vi) from water by an adsorption-reduction mechanism. In the sandwiched nanocomposites, GO provided enough surface area, functional groups, and hydrophilic surface for efficient absorption. Fe3O4 nanoparticles with excellent magnetic properties make it easy to separate and recover from water. Under acidic conditions, MnO2 nanowires act as both template and oxidant to initiate the polymerization of pyrrole monomers on its freshly activated surface to obtain GO/MnO2/Fe3O4/PPy (designated as GMFP) nanocomposite. GMFP could effectively adsorb Cr(vi) through electrostatic attraction, and the adsorbed Cr(vi) ions were partly reduced to trivalent chromium Cr(iii) (62%), resulting in the efficient adsorption and high removal of Cr(vi) from water. Hexavalent chromium adsorption by GMFP is strongly pH dependent and the adsorption kinetics followed the pseudo-second-order model. The Langmuir isothermal model described the adsorption isotherm data well and the maximum adsorption capacity was up to 374.53 mg g-1 at pH 2.0. These experimental results suggested that GMFP had great potential as an economic and efficient adsorbent of hexavalent chromium from wastewater, which has huge application potential.

Click-Functionalized SERS Nanoprobes with Improved Labeling Efficiency and Capability for Cancer Cell Imaging.

Precise identification and detection of cancer cells using nanoparticle probes are critically important for early cancer diagnosis and subsequent therapy. We herein develop novel folate receptor (FR)-targeted surface-enhanced Raman scattering (SERS) nanoprobes for cancer cell imaging based on a click coupling strategy. A Raman-active derivative (5,5'-dithiobis(2-nitrobenzoic acid)-N3 (DNBA-N3)) is designed with a disulfide bond for covalently anchoring to the surface of hollow gold nanoparticles (HAuNPs) and a terminal azide group for facilitating highly efficient conjugation with the bioligand. Modification of HAuNPs with DNBA-N3 yields monolayer coverage of Raman labels absorbed on the nanoparticle surface (HAuNP-DNBA-N3) and strong SERS signals. HAuNP-DNBA-N3 can be simply and effectively conjugated with folate bicyclo[6.1.0]nonyne derivatives via a copper-free click reaction. The synthesized nanoprobes (HAuNP-DNBA-folic acid (FA)) exhibit excellent targeted capacities to FR-positive cancer cells relative to FR-negative cells through SERS mappings. The receptor-mediated delivery behaviors are confirmed by comparison with the uptake of HAuNP-DNBA-N3 and free FA competition experiments. In addition to its good stability and benign biocompatibility, the developed SERS nanoprobes have great potential for applications in targeted tumor imaging.

Dual-Colored Carbon Dot Ratiometric Fluorescent Test Paper Based on a Specific Spectral Energy Transfer for Semiquantitative Assay of Copper Ions.

Classical pH test papers are widely used to measure the acid-base degree of media in a qualitative or semiquantitative manner. However, the extension of portable and inexpensive methods to a wide range of analytes so as to eliminate the tediousness of instrumental assays remains unsuccessful. Here, we report a novel kind of dual-colored carbon dot (CD) ratiometric fluorescent test paper for the semiquantitative assay of copper ions (Cu2+) by a dose-sensitive color evolution. The preparation of the test paper is based on the following two interesting findings: on the one hand, residual p-phenylenediamine at the surface of as-synthesized red CDs (r-CDs) efficiently binds Cu2+ ions to produce a strong visible absorption that overlaps the emission of blue CDs (b-CDs); on the other hand, the Cu2+ ions render the adsorption of small b-CDs onto the surface of larger r-CDs through their dual-coordinating interactions with the surface ligands of both r-CDs and b-CDs. These two mechanisms lead to a specific spectral energy transfer to quench the fluorescence of b-CDs with a sensitive detection limit of 8.82 nM Cu2+, whereas the red fluorescence of r-CDs is unaffected as a stable internal standard. Ratiometric fluorescent test papers have been prepared using a mixture of r-CDs and b-CDs (1:7) as ink by jetprinting on a piece of paper. With the addition of Cu2+ ions, the blue test paper produces a consecutive wide-colored evolution from blue to orange-red, with a dose-discerning ability as low as 25 nM.

A silica-based SERS chip for rapid and ultrasensitive detection of fluoride ions triggered by a cyclic boronate ester cleavage reaction.

Chemical sensing for the convenient detection of trace aqueous fluoride ions (F-) has been widely explored with the use of various sensing materials and techniques. It still remains a challenge to achieve ultrasensitive but simple, rapid, and inexpensive detection of F- for environmental monitoring and protection. Here we reported a novel surface-enhanced Raman scattering (SERS) nanosensor, fluorescein phenylboronic acid covalently linked to 1,4-dimercapto-2,3-butanediol modified Au@Ag NPs by a cyclic boronate ester (Flu-PBA-Diol-Au@Ag NPs), for the rapid and ultrasensitive detection of F-. Once the Flu-PBA approached the surface of Au@Ag NPs, the Raman signals of Flu-PBA were remarkably enhanced due to the strong SERS effect. However, the presence of F- will induce the cleavage reaction of the cyclic boronate ester into the trifluoroborate anion (3F-Flu-PBA) and diol. The 3F-Flu-PBA molecules exfoliated from the surface of Au@Ag NPs, and the SERS signals of the nanosensor were quenched. Following the sensing mechanism, a silica-based SERS chip has been fabricated by the assembly of Flu-PBA-Diol-Au@Ag NPs on a piece of silicon wafer. The silica-based SERS chips showed high sensitivity for aqueous F-, and the limit of detection (LOD) could reach as low as 0.1 nM. Each test using the SERS chip only needs a droplet of 20 µL sample and is accomplished within ∼10 min. The silica-based SERS chip has also been applied to the quantification of F- in tap water and lake water.

A single dual-emissive nanofluorophore test paper for highly sensitive colorimetry-based quantification of blood glucose.

Fluorescent test papers are promising for the wide applications in the assays of diagnosis, environments and foods, but unlike classical dye-absorption-based pH test paper, they are usually limited in the qualitative yes/no type of detection by fluorescent brightness, and the colorimetry-based quantification remains a challenging task. Here, we report a single dual-emissive nanofluorophore probe to achieve the consecutive color variations from blue to red for the quantification of blood glucose on its as-prepared test papers. Red quantum dots were embedded into silica nanoparticles as a stable internal standard emission, and blue carbon dots (CDs) were further covalently linked onto the surface of silica, in which the ratiometric fluorescence intensity of blue to red is controlled at 5:1. While the oxidation of glucose induced the formation of Fe(3+) ions, the blue emission of CDs was thus quenched by the electron transfer from CDs to Fe(3+), displaying a serial of consecutive color variations from blue to red with the dosage of glucose. The high-quality test papers printed by the probe ink exhibited a dosage-sensitive allochromatic capability with the clear differentiations of ~5, 7, 9, 11mM glucose in human serum (normal: 3-8mM). The blood glucose determined by the test paper was almost in accordance with that measured by a standard glucometer. The method reported here opens a window to the wide applications of fluorescent test paper in biological assays.

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.