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.

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.

Online Quantitative Analysis of Chlorine Contents in Chlorinated Paraffins by Facile Raman Spectroscopy.

Online analysis of industrial chemicals is extremely important for managing product quality and performance. The chlorine (Cl) content is one of the most important technical metrics for chlorinated paraffins (CPs), and the conventional approaches to estimate Cl contents require transforming the Cl element to chloride followed by quantitative analysis with either titration or instrumentation, which are normally tedious and time-consuming and cannot simultaneously guide the industrial production. Here, we developed a rapid, real-time, and online approach to determine the Cl content of CPs with facile Raman spectroscopy. The chlorination of paraffins generated two new Raman peaks at 610-618 and 668-690 cm-1, which are associated with the vibrational modes of the SHH and SHC conformations of the C-Cl bond in CPs, respectively. More importantly, the corresponding peak of the SHH conformation decreased and that of the SHC conformation increased with the enhancement of the chlorination degree of CPs. The ratiometric calculation of the two respective Raman peak areas leads to a quantitative analysis of the Cl content of CPs. The developed approach can online provide the Cl contents of CPs within seconds accurately but without the tedious sample treatment required by conventional approaches. The strategy of integrating Raman analysis with the industrial pipeline will help in managing the production and quality control of industrial chemicals.

Integration of quantum dots with Zn2GeO4 nanoellipsoids to expand the dynamic detection range of uranyl ions in fluorescent test strips.

Fluorescent colorimetric test strips normally have a narrow dynamic detection-range due to the limited responsive range from single responsive materials, which cannot meet the wide detection requirement in practical applications. Herein, we developed an approach to detect uranyl ions (UO22+) with a broad detection range using the synthesized ZnS:Mn quantum dots (QDs) modified Zn2GeO4 nanoellipsoids (Zn2GeO4 @ZnS:Mn NEs), containing two responsive materials with the opposite signal responses at different UO22+ concentrations. Specifically, a red to chocolate color change was observed at low analyte concentrations (0.01-100 µM) resulting from the photoinduced electron transfer effect from ZnS:Mn QDs to UO22+. A sequentially olive drab to green color change has been observed when further increasing the UO22+ concentration (100-1000 µM) as a result of the antenna effect between Zn2GeO4 nanoellipsoids and UO22+. In addition, a low-cost and portable fluorescent test strip has been further fabricated through embedding Zn2GeO4 @ZnS:Mn NEs on a microporous structure membrane, demonstrating a facile yet effective colorimetric response to UO22+ in lab water, lake water, and seawater with a wide dynamic range. Therefore, it is potentially attractive for real-time and on-site detection of UO22+ in sudden-onset situations.

3D microscopy and deep learning reveal the heterogeneity of crown-like structure microenvironments in intact adipose tissue.

Crown-like structures (CLSs) are adipose microenvironments of macrophages engulfing adipocytes. Their histological density in visceral adipose tissue (VAT) predicts metabolic disorder progression in obesity and is believed to initiate obesity comorbidities. Here, we use three-dimensional (3D) light sheet microscopy and deep learning to quantify 3D features of VAT CLSs in lean and obese states. Obese CLS densities are significantly higher, composing 3.9% of tissue volume compared with 0.46% in lean tissue. Across the states, individual CLS structural characteristics span similar ranges; however, subpopulations are distinguishable. Obese VAT contains large CLSs absent from lean tissues, located near the tissue center, while lean CLSs have higher volumetric cell densities and prolate shapes. These features are consistent with inefficient adipocyte elimination in obesity that contributes to chronic inflammation, representing histological biomarkers to assess adipose pathogenesis. This tissue processing, imaging, and analysis pipeline can be applied to quantitatively classify 3D microenvironments across diverse tissues.

Antibody Self-Assembly Maximizes Cytoplasmic Immunostaining Accuracy of Compact Quantum Dots.

Antibody conjugates of quantum dots (QDs) are expected to transform immunofluorescence staining by expanding multiplexed analysis and improving target quantification. Recently, a new generation of small QDs coated with multidentate polymers has improved QD labeling density in diverse biospecimens, but new challenges prevent their routine use. In particular, these QDs exhibit nonspecific binding to fixed cell nuclei and their antibody conjugates have random attachment orientations. This report describes four high-efficiency chemical approaches to conjugate antibodies to compact QDs. Methods include click chemistry and self-assembly through polyhistidine coordination, both with and without adaptor proteins that directionally orient antibodies. Specific and nonspecific labeling are independently analyzed after application of diverse blocking agent classes, and a new assay is developed to quantitatively measure intracellular labeling density based on microtubule stain connectivity. Results show that protein conjugation to the QD surface is required to simultaneously eliminate nonspecific binding and maintain antigen specificity. Of the four conjugation schemes, polyhistidine-based coordination of adaptor proteins with antibody self-assembly yields the highest intracellular staining density and the simplest conjugation procedure. Therefore, antibody and adaptor protein orientation, in addition to blocking optimization, are important determinants of labeling outcomes, insights that can inform translational development of these more compact nanomaterials.

Short-Wave Infrared Quantum Dots with Compact Sizes as Molecular Probes for Fluorescence Microscopy.

Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on HgxCd1-xSe alloy cores red shifted to the SWIR by epitaxial deposition of thin HgxCd1-xS shells with a small band gap. By tuning alloy composition alone, the emission can be shifted across the visible-to-SWIR (VIR) spectra while maintaining a small and equal size, allowing direct comparisons of molecular labeling performance across a broad range of wavelength. After coating with click-functional multidentate polymers, the VIR-QD spectral series has high quantum yield in the SWIR (14-33%), compact size (13 nm hydrodynamic diameter), and long-term stability in aqueous media during continuous excitation. We show that these properties enable diverse applications of SWIR molecular probes for fluorescence microscopy using conjugates of antibodies, growth factors, and nucleic acids. A broadly useful outcome is a 10-55-fold enhancement of the signal-to-background ratio at both the single-molecule level and the ensemble level in the SWIR relative to visible wavelengths, primarily due to drastically reduced autofluorescence. We anticipate that VIR-QDs with SWIR emission will enable ultrasensitive molecular imaging of low-copy number analytes in biospecimens with high autofluorescence.

Multifunctional Conjugated Polymer Nanoparticles for Image-Guided Photodynamic and Photothermal Therapy.

A multifunctional theranostic platform based on conjugated polymer nanoparticles (CPNs) with tumor targeting, fluorescence detection, photodynamic therapy (PDT), and photothermal therapy (PTT) is developed for effective cancer imaging and therapy. Two conjugated polymers, poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)-ethyl)fluorenyldivinylene]-alt-4,7-(2,1,3-benzothiadiazole) with bright red emission and photosensitizing ability and poly[(4,4,9,9-tetrakis(4-(octyloxy)phenyl)-4,9-dihydro-s-indacenol-dithiophene-2,7-diyl)-alt-co-4,9-bis(thiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-thiadiazolo-quinoxaline] with strong near-infrared absorption and excellent photothermal conversion ability are co-loaded into one single CPN via encapsulation approach using lipid-polyethylene glycol as the matrix. The obtained co-loaded CPNs show sizes of around 30 nm with a high singlet oxygen quantum yield of 60.4% and an effective photothermal conversion efficiency of 47.6%. The CPN surface is further decorated with anti-HER2 affibody, which bestows the resultant anti-HER2-CPNs superior selectivity toward tumor cells with HER2 overexpression both in vitro and in vivo. Under light irradiation, the PDT and PTT show synergistic therapeutic efficacy, which provides new opportunities for the development of multifunctional biocompatible organic materials in cancer therapy.

Bovine Serum Albulmin Protein-Templated Silver Nanocluster (BSA-Ag13 ): An Effective Singlet Oxygen Generator for Photodynamic Cancer Therapy.

This paper reports a novel synthesis approach of bovine serum albumin (BSA) protein-templated ultrasmall (<2 nm) Ag nanocluster (NC) with strong singlet oxygen generation capacity for photodynamic therapy (PDT). An atomically precise BSA-Ag13 NC (i.e., 13 Ag atoms per cluster) is successfully synthesized for the first time by using NaOH-dissolved NaBH4 solution as the controlling reducing agent. The ubiquitous size of BSA-Ag13 NC results in unique behaviors of its photoexcited states as characterized by the ultrafast laser spectroscopy using time-correlated single photon counting and transient absorption techniques. In particular, triply excited states can be largely present in the excited BSA-Ag13 NC and readily sensitized molecular oxygen to produce singlet oxygen (1 O2 ) with a high quantum efficiency (≈1.26 using Rose Bengal as a standard). This value is much higher than its Au analogue (i.e., ≈0.07 for BSA-Au25 NC) and the commonly available photosensitizers. Due to the good cellular uptake and inherent biocompatibility imparted by the surface protein, BSA-Ag13 NC can be applied as an effective PDT agent in generating 1 O2 to kill cancer cell as demonstrated in this study.

Fluorogens with Aggregation Induced Emission: Ideal Photoacoustic Contrast Reagents Due to Intramolecular Rotation.

Exogenous contrast agents with high sensitivity are highly desirable for photoacoustic (PA) imaging. In this work, we show that fluorogens with aggregation induced emission (AIE) characteristics are born with strong PA signals. In addition, we find that the PA signal of conventional fluorophores could be significantly enhanced through conjugation with tetraphenylethene (TPE), an iconic AIE fluorogen. Taking 2,3-bis[4-(diphenylamino)phenyl]fumaronitrile (TPAFN) as an example, conjugation between TPAFN and TPE affords 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl) fumaroni-trile (TPETPAFN), a molecule with significant AIE characteristics, which shows 170% higher PA signals as compared to that of TPAFN. The higher PA signal of TPETPAFN is mainly ascribed to the enhanced molecular rotation, which is beneficial to its thermal expansion upon light absorption. Moreover, the significantly reduced PA signals for TPETPAFN in solvents with high viscosity or as nanoparticles further highlight the contribution of molecular rotation on PA signals.

Silica shelled and block copolymer encapsulated red-emissive AIE nanoparticles with 50% quantum yield for two-photon excited vascular imaging.

A polymer and silica co-protection strategy has been developed to encapsulate organic fluorogens with aggregation-induced emission and charge transfer characteristics into small nanoparticles (NPs). The co-pretected NPs show bright red fluorescence (50% quantum yield) with a large two-photon action cross-section (450 GM at 840 nm), which have been sucessfully used for two-photon fluorescence imaging of vasculature of the mouse tibial muscle.

Biocompatible conjugated polymer nanoparticles for efficient photothermal tumor therapy.

Conjugated polymers (CPs) with strong near-infrared (NIR) absorption and high heat conversion efficiency have emerged as a new generation of photothermal therapy (PTT) agents for cancer therapy. An efficient strategy to design NIR absorbing CPs with good water dispersibility is essential to achieve excellent therapeutic effect. In this work, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) is synthesized through the combination of donor-acceptor moieties by Suzuki polymerization. PFTTQ nanoparticles (NPs) are fabricated through a precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000 ) as the encapsulation matrix. Due to the large NIR absorption coefficient (3.6 L g(-1) cm(-1) ), the temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased to more than 50 °C upon continuous 808 nm laser irradiation (0.75 W/cm(2) ) for 5 min. The PFTTQ NPs show good biocompatibility to both MDA-MB-231 cells and Hela cells at 400 µg/mL of NPs, while upon laser irradiation, effective cancer cell killing is observed at a NP concentration of 50 µg/mL. Moreover, PFTTQ NPs could efficiently ablate tumor in in vivo study using a Hela tumor mouse model. Considering the large amount of NIR absorbing CPs available, the general encapsulation strategy will enable the development of more efficient PTT agents for cancer or tumor therapy.

Conjugated polymer microparticles for selective cancer cell image-guided photothermal therapy.

Nanotechnology has recently attracted great attention in biomedical research. Current nanoparticle approaches generally require further surface decoration with targeting ligands, peptides or proteins to achieve selective cancer imaging and therapy. This surface functionalization often complicates nanoparticles and leads to protein corona or varied nanoparticle uptake. In this work, we report a facile approach for selective cancer cell image-guided photothermal therapy by fabricating theranostic microparticles (MPs) using conjugated polymers (CPs) as the imaging and therapeutic agents. Through fine tuning of the backbone structures, we synthesized two CPs, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) with high near infrared (NIR) molar absorptivity and poly(9,9-dihexylfluorene-alt-2,1,3-benzothiadiazole) (PFBT) with bright green emission. The two CPs were physically blended into single particles with ∼3 µm size, which was confirmed by scanning electron microscopy (SEM) and confocal fluorescence imaging. Although without any surface functionalization, the obtained CP MPs showed selective internalization into MCF-7 cancer cells over NIH-3T3 normal cells, while CP nanoparticles showed similar uptake into both cell lines. Moreover, the CP MPs could selectively kill MCF-7 cells upon NIR irradiation, which showed a half-maximal inhibitory concentration (IC50) of 30 µg mL-1 based on PFTTQ concentration.

A highly sensitive fluorescent light-up probe for real-time detection of the endogenous protein target and its antagonism in live cells.

Real-time detection and monitoring of cancer-related biomolecular interactions in live cells are of paramount importance for disease diagnostics and drug screening. Herein, we developed a target-specific fluorescent light-up probe for cellular detection of Mdm2, the key negative regulator of the p53 tumour suppressor protein. Conjugation of a uniquely designed fluorogen (TPECM) with aggregation induced-emission properties, to a specific p53-derived peptide (12.1Pep) targeting Mdm2, yielded a cell-permeable probe (TPECM-12.1Pep) with turn-on fluorescence properties for real-time live cell imaging of Mdm2. This specific light-up probe is almost non-fluorescent in its isolated state but is highly emissive upon binding to Mdm2, enabling quantitative detection of both Mdm2 and its antagonism. Using a model compound (Nutlin-3a), we demonstrate that the as-developed probes can be used to screen p53-Mdm2 inhibiting drug candidates, both in vitro and in cells. Furthermore, the probe activity can be accurately monitored in cells using a fluorescently activated cell sorting machine. These features will expedite research in the areas of drug discovery, clinical diagnostics and fundamental cell biology.

Fluorogen-peptide conjugates with tunable aggregation-induced emission characteristics for bioprobe design.

Fluorogens with aggregation-induced emission (AIE) characteristics are attracting intense research interest, and an AIE-peptide conjugate strategy has been reported for developing turn-on probes based on hydrophilic peptide ligands. To build a model also suitable for hydrophobic ligands, we propose to fine-tune the AIE characteristics for probe design. In this work, an iconic AIE fluorogen tetraphenylethene (TPE) was designed to conjugate with peptide fragments containing different numbers of aspartic acid (D) units. Relationships between the numbers of D and the hydrophilicity, optical properties, and aggregate sizes and the AIE characteristics of TPE-peptide conjugates were investigated carefully. Five carboxyl groups were found to be the threshold to "turn off" the fluorescence of TPE. As a proof-of-concept, TPE-SS-D5 containing a cleavable disulfide bond was synthesized for thiol turn-on detection. The validated tunable AIE characteristic offers new opportunities to design fluorescence turn-on probes based on hydrophobic recognition elements and AIE fluorogens.

Near-infrared fluorescence amplified organic nanoparticles with aggregation-induced emission characteristics for in vivo imaging.

Near-infrared (NIR) fluorescence signals are highly desirable to achieve high resolution in biological imaging. To obtain NIR emission with high brightness, fluorescent nanoparticles (NPs) are synthesized by co-encapsulation of 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenylamino)phenyl)fumaronitrile (TPETPAFN), a luminogen with aggregation-induced emission (AIE) characteristics, and a NIR fluorogen of silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775) using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] as the encapsulation matrix. The good spectral overlap between the emission of TPETPAFN and the absorption of NIR775 leads to efficient energy transfer, resulting in a 47-fold enhancement of the NIR775 emission intensity upon excitation of TPETPAFN at 510 nm as compared to that upon direct excitation of NIR775 at 760 nm. The obtained fluorescent NPs show sharp NIR emission with a band width of 20 nm, a large Stokes shift of 275 nm, good photostability and low cytotoxicity. In vivo imaging study reveals that the synthesized NPs are able to provide high fluorescence contrast in live animals. The Förster resonance energy transfer strategy overcomes the intrinsic limitation of broad emission spectra for AIE NPs, which opens new opportunities to synthesize organic NPs with high brightness and narrow emission for potential applications in multiplex sensing and imaging.

Water-soluble bioprobes with aggregation-induced emission characteristics for light-up sensing of heparin.

Two water-soluble cationic fluorene-based fluorescent probes for heparin detection are designed and synthesized. A slight change in the molecular design results in two probes with opposite optical properties in their solution and aggregation states as well as a response to heparin in buffer solution. The probe with a propeller-like conformation exhibits aggregation-induced emission (AIE) characteristics and shows a green fluorescence enhancement upon interaction with heparin; in contrast, the probe with a more planar conformation has a fluorescence quenching response. A comprehensive study on heparin detection using the two probes was conducted, which revealed that the AIE probe shows a better performance than the aggregation-caused quenching (ACQ) probe in terms of sensitivity. The AIE probe integrated with graphene oxide (GO) further improves the heparin detection sensitivity and selectivity. The solution of AIE probe/GO emits strong green fluorescence only in the presence of heparin, which allows for light-up visual discrimination of heparin from its analogues such as chondroitin-4-sulfate and hyaluronic acid. Moreover, the linear light-up response of AIE probe/GO enables heparin quantification in the range of 0-13.2 µM with a detection limit of 10 nM, which is of practical importance for heparin monitoring during surgery or therapy.

Rational design of fluorescent light-up probes based on an AIE luminogen for targeted intracellular thiol imaging.

A water-soluble fluorescent light-up bioprobe based on a luminogen with aggregation-induced emission characteristics was developed for targeted intracellular thiol imaging.

A ratiometric probe composed of an anionic conjugated polyelectrolyte and a cationic phosphorescent iridium(III) complex for time-resolved detection of Hg(II) in aqueous media.

A hybrid complex composed of an anionic conjugated polyelectrolyte (PFB-SO3Na) and a cationic phosphorescent Ir(III) oligomer is formed through electrostatic interaction by simple physical mixing in aqueous media. Due to their opposite charges and their effective spectral overlap, fluorescence resonance energy transfer occurs from the blue-emissive PFB-SO3Na to the red-emissive phosphorescent Ir(III) complex, which allows ratiometric and colorimetric Hg(2+) sensing in aqueous solution with good selectivity, sensitivity, as well as visible detection. Time-resolved photoluminescence is applied for Hg(2+) detection, which can effectively eliminate the background interference and improve the sensing sensitivity and signal-to-noise ratio in complicated media.