Ultralong Phosphorescence of Water‐Soluble Organic Nanoparticles for In Vivo Afterglow Imaging

Afterglow or persistent luminescence eliminates the need for light excitation and thus circumvents the issue of autofluorescence, holding promise for molecular imaging. However, current persistent luminescence agents are rare and limited to inorganic nanoparticles. This study reports the design principle, synthesis, and proof‐of‐concept application of organic semiconducting nanoparticles (OSNs) with ultralong phosphorescence for in vivo afterglow imaging. The design principle leverages the formation of aggregates through a top‐down nanoparticle formulation to greatly stabilize the triplet excited states of a phosphorescent molecule. This prolongs the particle luminesce to the timescale that can be detected by the commercial whole‐animal imaging system after removal of external light source. Such ultralong phosphorescent of OSNs is inert to oxygen and can be repeatedly activated, permitting imaging of lymph nodes in living mice with a high signal‐to‐noise ratio. This study not only introduces the first category of water‐soluble ultralong phosphorescence organic nanoparticles but also reveals a universal design principle to prolong the lifetime of phosphorescent molecules to the level that can be effective for molecular imaging.     

Room-Temperature Phosphorescence Resonance Energy Transfer for Construction of Near-Infrared Afterglow Imaging Agents

Afterglow imaging that detects photons after cessation of optical excitation avoids tissue autofluorescence and thus possesses higher sensitivity than traditional fluorescence imaging. Purely organic molecules with room-temperature phosphorescence (RTP) have emerged as a new library of benign afterglow agents. However, most RTP luminogens only emit visible light with shallow tissue penetration, constraining their in vivo applications. This study presents an organic RTP nanoprobe (mTPA-N) with emission in the NIR range for in vivo afterglow imaging. Such a probe is composed of RTP molecule (mTPA) as the phosphorescent generator and an NIR-fluorescent dye as the energy acceptor to enable room-temperature phosphorescence resonance energy transfer (RT-PRET), ultimately resulting in redshifted phosphorescent emission at 780 nm. Because of the elimination of background noise and redshifted afterglow luminescence in a biologically transparent window, mTPA-N permits imaging of lymph nodes in living mice with a high signal-to-noise ratio. This study thus opens up a universal approach to develop organic RTP luminogens into NIR afterglow imaging agents via construction of RT-PRET.     

Inorganic–Organic Hybrid Nanoprobe for NIR‐Excited Imaging of Hydrogen Sulfide in Cell Cultures and Inflammation in a Mouse Model

Hydrogen sulfide (H₂S) is an important gaseous signaling agent mediated by many physiological processes and diseases. In order to explore its role in biological signaling, much effort has been focused on developing organic fluorescent probes to image H₂S. However, these downconversion H₂S probes are impractical for bio‐imaging beyond a certain depth because of the short tissue penetration of UV/visible light (as an excitation source). In most circumstance, these probes are also not suitable for long‐term assay due to photo‐bleaching. Herein, a new design to detect H₂S based on the coumarin‐hemicyanine (CHC1)‐modified upconversion nanophosphors is reported. This inorganic–organic integrated nanoprobe is demonstrated to display a fast response time with a large ratiometric upconversion luminescence (UCL) enhancement, and extraordinary photo‐stability. CHC1‐UCNPs not only can be used for ratiometric UCL monitoring of pseudo‐enzymatic H₂S production in living cells, but can also be used to identify the risk of endotoxic shock through ratiometric UCL imaging of tissue and measurement of endogenous H₂S levels in plasma. The first ratiometric UCL H₂S nanoprobe reported here may be further developed as the next‐generation diagnostic tool for the detection of inflammatory‐related diseases.     

Self‐Assembly of a Monochromophore‐Based Polymer Enables Unprecedented Ratiometric Tracing of Hypoxia

The accuracy of traditional bischromophore‐based ratiometric probes is always compromised by undesirable energy/charge transferring interactions between the internal reference moiety and the sensing chromophore. In this regard, ratiometric sensing with a monochromophore system is highly desirable. Herein, an unprecedented monochromophore‐based ratiometric probe, which consists of a hydrophilic backbone poly(N‐vinylpyrrolidone) (PVP) and single chromophore of platinum(II) tetraphenylporphyrin (Pt‐TPP) is reported. Combination of the specific assembled clustering‐triggered fluorescent emission (oxygen‐insensitive) with the original Pt‐TPP phosphorescence (oxygen‐sensitive) enables successful construction of a monochromophore‐based ratiometric nanosensor for directly tracing hypoxia in vivo, along with the preferable facilitation of enhanced permeation and retention effect and long excitation wavelength. The unique ratiometric signals enable the direct observation from normoxic to hypoxic environment in both living A549 cells and a tumor‐bearing mice model, providing a significant paradigm of a monochromophore‐based dual‐emissive system with the specific assembled cluster emission. The work satisfactorily demonstrates a valuable strategy for designing monochromophore‐based dual‐emissive materials, and validates its utility for in vivo ratiometric biological sensing without the common energy/charge interference in bischromophore‐based system.     

Bright afterglow illuminator made of phosphorescent material and fluorescent fibers

Phosphorescent oxides and fluorescent dyes were used together to create a fiber-type illuminator that glows in the dark without the need for electric power. Dye-doped polymer fibers, which were bundled at one end, were linearly arrayed in a polysiloxane resin that contained phosphorescent oxide particles. The phosphorescent resin continued to glow for a long time even after the excitation light was removed. Organic dyes in a polymer fiber were excited by the phosphorescence and emitted fluorescence toward the fiber end. Fluorescence from a number of dyes was combined in the long fiber, and, consequently a bright light beam emerged from the fiber end. Useful performance, i.e., high power density, narrow beam divergence, and long afterglow, is demonstrated by the prototype fiber illuminator.     

Ratiometric coumarin-neutral red (CONER) nanoprobe for detection of hydroxyl radicals

Excessive production of reactive oxygen species can lead to alteration of cellular functions responsible for many diseases including cardiovascular diseases, neurodegenerative diseases, cancer, and aging. Hydroxyl radical is a short-lived radical which is considered very aggressive due to its high reactivity toward biological molecules. In this study, a COumarin-NEutral Red (CONER) nanoprobe was developed for detection of hydroxyl radical based on the ratiometric fluorescence signal between 7-hydroxy coumarin 3-carboxylic acid and neutral red dyes. Biocompatible poly lactide-co-glycolide (PLGA) nanoparticles containing encapsulated neutral red were produced using a coumarin 3-carboxylic acid conjugated poly(sodium N-undecylenyl-Nε-lysinate) (C3C-poly-Nε-SUK) as moiety reactive to hydroxyl radicals. The response of the CONER nanoprobe was dependent on various parameters such as reaction time and nanoparticle concentration. The probe was selective for hydroxyl radicals as compared with other reactive oxygen species including O(2)(?-), H(2)O(2), (1)O(2), and OCl(-). Furthermore, the CONER nanoprobe was used to detect hydroxyl radicals in vitro using viable breast cancer cells exposed to oxidative stress. The results suggest that this nanoprobe represents a promising approach for detection of hydroxyl radicals in biological systems.     

Activatable Semiconducting Theranostics: Simultaneous Generation and Ratiometric Photoacoustic Imaging of Reactive Oxygen Species In Vivo

Enhancing the generation of reactive oxygen species (ROS) is an effective anticancer strategy. However, it is a great challenge to control the production and to image ROS in vivo, both of which are vital for improving the efficacy and accuracy of cancer therapy. Herein, an activatable semiconducting theranostic nanoparticle (NP) platform is developed that can simultaneously enhance ROS generation while self‐monitoring its levels through ratiometric photoacoustic (PA) imaging. The NP platform can further guide in vivo therapeutic effect in tumors. The theranostic NP platform is composed of: (i) cisplatin prodrug and ferric ion catalyst for ROS generation, a part of combination cancer therapy; and (ii) a ratiometric PA imaging nanoprobe consisting of inert semiconducting perylene‐diimide (PDI) and ROS activatable near‐infrared dye (IR790s), used in ratiometric PA imaging of ROS during cancer treatment. Ratiometric PA signals are measured at two near‐infrared excitation wavelengths: 680 and 790 nm for PDI and IR790s, respectively. The measurements show highly accurate visualization of ^?OH generation in vivo. This novel ROS responsive organic theranostic NP allows not only synergistic cancer chemotherapy but also real‐time monitoring of the therapeutic effect through ratiometric PA imaging.     

Theranostic Upconversion Nanobeacons for Tumor mRNA Ratiometric Fluorescence Detection and Imaging‐Monitored Drug Delivery

Remote optical detection and imaging of specific tumor‐related biomarkers and simultaneous activation of therapy according to the expression level of the biomarkers in tumor site with theranostic probes should be an effective modality for treatment of cancers. Herein, an upconversion nanobeacon (UCNPs‐MB/Dox) is proposed as a new theranostic nanoprobe to ratiometrically detect and visualize the thymidine kinase 1 (TK1) mRNA that can simultaneously trigger the Dox release to activate the chemotherapy accordingly. UCNPs‐MB/Dox is constructed with the conjugation of a TK1 mRNA‐specific molecular beacon (MB) bearing a quencher (BHQ‐1) and an alkene handle modified upconversion nanoparticle (UCNP) through click reaction and subsequently loading with a chemotherapy drug (Dox). With this nanobeacon, quantitative ratiometric upconversion detection of the target with high sensitivity and selectivity as well as the target triggered Dox release in vitro is demonstrated. The sensitive and selective ratiometric detection and imaging of TK1 mRNA under the irradiation of near infrared light (980 nm) and the mRNA‐dependent release of Dox for chemotherapy in the tumor MCF‐7 cells and A549 cells are also shown. This work provides a smart and robust platform for gene‐related tumor theranostics.     

Visible‐Light‐Excited Ultralong Organic Phosphorescence by Manipulating Intermolecular Interactions

Visible light is much more available and less harmful than ultraviolet light, but ultralong organic phosphorescence (UOP) with visible‐light excitation remains a formidable challenge. Here, a concise chemical approach is provided to obtain bright UOP by tuning the molecular packing in the solid state under irradiation of available visible light, e.g., a cell phone flashlight under ambient conditions (room temperature and in air). The excitation spectra exhibit an obvious redshift via the incorporation of halogen atoms to tune intermolecular interactions. UOP is achieved through H‐aggregation to stabilize the excited triplet state, with a high phosphorescence efficiency of 8.3% and a considerably long lifetime of 0.84 s. Within a brightness of 0.32 mcd m^?2 that can be recognized by the naked eye, UOP can last for 104 s in total. Given these features, ultralong organic phosphorescent materials are used to successfully realize dual data encryption and decryption. Moreover, well‐dispersed UOP nanoparticles are prepared by polymer‐matrix encapsulation in an aqueous solution, and their applications in bioimaging are tentatively being studied. This result will pave the way toward expanding metal‐free organic phosphorescent materials and their applications.     

Recent Progress of Singlet‐Exciton‐Harvesting Fluorescent Organic Light‐Emitting Diodes by Energy Transfer Processes

The external quantum efficiency (EQE) of organic light‐emitting diodes (OLEDs) has been dramatically improved by developing highly efficient organic emitters such as phosphorescent emitters and thermally activated delayed fluorescent (TADF) emitters. However, high‐EQE OLED technologies suffer from relatively poor device lifetimes in spite of their high EQEs. In particular, the short lifetimes of blue phosphorescent and TADF OLEDs remain a big hurdle to overcome. Therefore, the high‐EQE approach harvesting singlet excitons of fluorescent emitters by energy transfer processes from the host or sensitizer has been explored as an alternative for high‐EQE OLED strategies. Recently, there has been a big jump in the EQE and device lifetime of singlet‐exciton‐harvesting fluorescent OLEDs. Recent progress on the materials and device structure is discussed herein.     

Nanodroplet‐Containing Polymers for Efficient Low‐Power Light Upconversion

Sensitized triplet–triplet‐annihilation‐based photon upconversion (TTA‐UC) permits the conversion of light into radiation of higher energy and involves a sequence of photophysical processes between two dyes. In contrast to other upconversion schemes, TTA‐UC allows the frequency shifting of low‐intensity light, which makes it particularly suitable for solar‐energy harvesting technologies. High upconversion yields can be observed for low viscosity solutions of dyes; but, in solid materials, which are better suited for integration in devices, the process is usually less efficient. Here, it is shown that this problem can be solved by using transparent nanodroplet‐containing polymers that consist of a continuous polymer matrix and a dispersed liquid phase containing the upconverting dyes. These materials can be accessed by a simple one‐step procedure that involves the free‐radical polymerization of a microemulsion of hydrophilic monomers, a lipophilic solvent, the upconverting dyes, and a surfactant. Several glassy and rubbery materials are explored and a range of dyes that enable TTA‐UC in different spectral regions are utilized. The materials display upconversion efficiencies of up to ≈15%, approaching the performance of optimized oxygen‐free reference solutions. The data suggest that the matrix not only serves as mechanically coherent carrier for the upconverting liquid phase, but also provides good protection from atmospheric oxygen.     

Bright and monodispersed phosphorescent particles and their applications for biological assays

Halogen-containing polymers and copolymers have been discovered to provide excellent encapsulation matrixes for making bright and monodispersed phosphorescent nanoparticles. The phosphorescent nanoparticles exhibit strong phosphorescence with long lifetime and large Stoke shift under ambient conditions. The cross-linked phosphorescent particles using halogen-containing copolymers have been found to be very stable and easily resuspendable in aqueous media. The surface functional groups have been demonstrated to allow covalent tagging of biological recognition molecules such as antibodies to make particle-antibody conjugates. The conjugates can be used to provide very sensitive detection of analytes through time-resolved phosphorescence measurements.     

A Single Composition Architecture‐Based Nanoprobe for Ratiometric Photoacoustic Imaging of Glutathione (GSH) in Living Mice

As one of the reduction species, glutathione (GSH) plays a tremendous role in regulating the homeostasis of redox state in living body. Accurate imaging of GSH in vivo is highly desired to provide a real‐time visualization of physiological and pathological conditions while it is still a big challenge. Recently developed photoacoustic imaging (PAI) with high resolution and deep penetration characteristics is more promising for in vivo GSH detection. However, its application is dramatically limited by the difficult designation of photoacoustic probes with changeable near‐infrared (NIR)‐absorption under reductive activation. A cyanine derivative‐based activatable probe is developed for in vivo ratiometric PAI of GSH for the first time. The probe is structurally designed to output ratiometric signals toward GSH in NIR‐absorption region based on the cleavage of disulfide bond followed by a subsequent exchange between the secondary amine and sulfydryl group formed. Such a ratiometric manner provides high signal‐to‐noise imaging of blood vessels and their surrounding areas in tumor. Concomitantly, it also exhibits good specificity toward GSH over other thiols. Furthermore, the single composition architecture of the probe effectively overcomes the leakage issue compared with traditional multicomposition architecture‐based nanoprobe, thus enhancing the imaging accuracy and fidelity in living body.     

Tomographic imaging of oxygen by phosphorescence lifetime

Imaging of oxygen in tissue in three dimensions can be accomplished by using the phosphorescence quenching method in combination with diffuse optical tomography. We experimentally demonstrate the feasibility of tomographic imaging of oxygen by phosphorescence lifetime. Hypoxic phantoms were immersed in a cylinder with scattering solution equilibrated with air. The phantoms and the medium inside the cylinder contained near-infrared phosphorescent probe(s). Phosphorescence at multiple boundary sites was registered in the time domain at different delays (t(d)) following the excitation pulse. The duration of the excitation pulse (t(p)) was regulated to optimize the contrast in the images. The reconstructed integral intensity images, corresponding to delays t(d), were fitted exponentially to give the phosphorescence lifetime image, which was converted into the three-dimensional image of oxygen concentrations in the volume. The time-independent diffusion equation and the finite element method were used to model the light transport in the medium. The inverse problem was solved by the recursive maximum entropy method. We provide what we believe to be the first example of oxygen imaging in three dimensions using long-lived phosphorescent probes and establish the potential of these probes for diffuse optical tomography.     

Ratiometric Photoacoustic Molecular Imaging for Methylmercury Detection in Living Subjects

Photoacoustic molecular imaging is an emerging and promising diagnostic tool for heavy metal ions detection. Methylmercury (MeHg⁺) is one of the most potent neurotoxins, which damages the brain and nervous system of human beings through fish consumption. The development of a selective and sensitive method for MeHg⁺ detection is highly desirable. In this Communication, we develope a chemoselective photoacoustic sensor (LP‐hCy7) composed of the liposome (LP) and MeHg⁺‐responsive near‐infrared (NIR) cyanine dye (hCy7) for MeHg⁺ detection within living subjects, such as zebrafish and mouse. The as‐prepared LP‐hCy7 nanoprobe displays unique dual‐shift NIR absorbance peaks and produces a normalized turn‐on response after the reaction of MeHg⁺ and hCy7 through a mercury‐promoted cyclization reaction. The absorbance intensities of LP‐hCy7 nanoprobe at 690 and 860 nm are decreased and increased, respectively. The ratiometric photoacoustic signal (PA860/PA690) is noticeably increased in the presence of MeHg⁺. These findings not only provide a ratiometric photoacoustic molecular imaging probe for the detection of metal ions in vivo, but also provides a tool for spectroscopic photoacoustic molecular imaging.     

Color‐Convertible,Unimolecular, Micelle‐Based,Activatable Fluorescent Probe for Tumor‐Specific Detection and Imaging In Vitro and In Vivo

Recent years have witnessed significant progress in molecular probes for cancer diagnosis. However, the conventional molecular probes are designed to be “always‐on” by attachment of tumor‐targeting ligands, which limits their abilities to diagnose tumors universally due to the variations of targeting efficiency and complex environment in different cancers. Here, it is proposed that a color‐convertible, activatable probe is responding to a universal tumor microenvironment for tumor‐specific diagnosis without targeting ligands. Based on the significant hallmark of up‐regulated hydrogen peroxide (H₂O₂) in various tumors, a novel unimolecular micelle constructed by boronate coupling of a hydrophobic hyperbranched poly(fluorene‐co‐2,1,3‐benzothiadiazole) core and many hydrophilic poly(ethylene glycol) arms is built as an H₂O₂‐activatable fluorescent nanoprobe to delineate tumors from normal tissues through an aggregation‐enhanced fluorescence resonance energy transfer strategy. This color‐convertible, activatable nanoprobe is obviously blue‐fluorescent in various normal cells, but becomes highly green‐emissive in various cancer cells. After intravenous injection to tumor‐bearing mice, green fluorescent signals are only detected in tumor tissue. These observations are further confirmed by direct in vivo and ex vivo tumor imaging and immunofluorescence analysis. Such a facile and simple methodology without targeting ligands for tumor‐specific detection and imaging is worthwhile to further development.     

Recent progresses on materials for electrophosphorescent organic light-emitting devices

Although organic light-emitting devices have been commercialized as flat panel displays since 1997, only singlet excitons were emitted. Full use of singlet and triplet excitons, electrophosphorescence, has attracted increasing attentions after the premier work made by Forrest, Thompson, and co-workers. In fact, red electrophosphorescent dye has already been used in sub-display of commercial mobile phones since 2003. Highly efficient green phosphorescent dye is now undergoing of commercialization. Very recently, blue phosphorescence approaching the theoretical efficiency has also been achieved, which may overcome the final obstacle against the commercialization of full color display and white light sources from phosphorescent materials. Combining light out-coupling structures with highly efficient phosphors (shown in the table-of-contents image), white emission with an efficiency matching that of fluorescent tubes (90 lm/W) has now been realized. It is possible to tune the color to the true white region by changing to a deep blue emitter and corresponding wide gap host and transporting material for the blue phosphor. In this article, recent progresses in red, green, blue, and white electrophosphorescent materials for OLEDs are reviewed, with special emphasis on blue electrophosphorescent materials.     

Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo

Fluorescent polymer nanoparticles for long‐term labeling and tracking of living cells with any desired color code are developed. They are built from biodegradable poly(lactic‐co‐glycolic acid) polymer loaded with cyanine dyes (DiO, DiI, and DiD) with the help of bulky fluorinated counterions, which minimize aggregation‐caused quenching. At the single particle level, these particles are ≈20‐fold brighter than quantum dots of similar color. Due to their identical 40 nm size and surface properties, these nanoparticles are endocytosed equally well by living cells. Mixing nanoparticles of three colors in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding is validated on 7 cell lines (HeLa, KB, embryonic kidney (293T), Chinese hamster ovary, rat basophilic leucemia, U97, and D2A1), 13 color codes, and it enables simultaneous tracking of co‐cultured barcoded cell populations for >2 weeks. It is also applied to studying competition among drug‐treated cell populations. This technology enabled six‐color imaging in vivo for (1) tracking xenografted cancer cells and (2) monitoring morphogenesis after microinjection in zebrafish embryos. In addition to a robust method of multicolor cell labeling and tracking, this work suggests that multiple functions can be co‐localized inside cells by combining structurally close nanoparticles carrying different functions.     

O(2)/pH multisensor based on one phosphorescent dye

A new sensor is described based on a phosphorescent metalloporphyrin dye incorporated in a polymeric membrane, which allows measurement of the two important analytes, oxygen and pH. In such a sensor, the bifunctional dye is quenched by O(2) altering its phosphorescence intensity and lifetime (0-21 kPa O(2)), whereas protonation of the dye causes a major change in the absorption spectrum and also reduces the phosphorescence intensity. As a result, quantification and continuous monitoring of the two analytes can be achieved by (i) simultaneous phosphorescence intensity (O(2), pH) and lifetime (O(2)) measurements and (ii) parallel phosphorescence lifetime (O(2)) and ratiometric absorbance (pH) measurements. Although the first method generally requires sensor calibration in intensity mode, the second provides internal referencing and calibration-free capabilities for both analytes. This approach can be extended to sensing of some other analytes.     

Near‐Infrared‐II Molecular Dyes for Cancer Imaging and Surgery

Fluorescence bioimaging affords a vital tool for both researchers and surgeons to molecularly target a variety of biological tissues and processes. This review focuses on summarizing organic dyes emitting at a biological transparency window termed the near‐infrared‐II (NIR‐II) window, where minimal light interaction with the surrounding tissues allows photons to travel nearly unperturbed throughout the body. NIR‐II fluorescence imaging overcomes the penetration/contrast bottleneck of imaging in the visible region, making it a remarkable modality for early diagnosis of cancer and highly sensitive tumor surgery. Due to their convenient bioconjugation with peptides/antibodies, NIR‐II molecular dyes are desirable candidates for targeted cancer imaging, significantly overcoming the autofluorescence/scattering issues for deep tissue molecular imaging. To promote the clinical translation of NIR‐II bioimaging, advancements in the high‐performance small molecule–derived probes are critically important. Here, molecules with clinical potential for NIR‐II imaging are discussed, summarizing the synthesis and chemical structures of NIR‐II dyes, chemical and optical properties of NIR‐II dyes, bioconjugation and biological behavior of NIR‐II dyes, whole body imaging with NIR‐II dyes for cancer detection and surgery, as well as NIR‐II fluorescence microscopy imaging. A key perspective on the direction of NIR‐II molecular dyes for cancer imaging and surgery is also discussed.