NIR‐II‐Excited Intravital Two‐Photon Microscopy Distinguishes Deep Cerebral and Tumor Vasculatures with an Ultrabright NIR‐I AIE Luminogen

Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal‐to‐background ratio (SBR) is highly desirable for the study and theranostics of vascular‐related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long‐wavelength absorption and aggregation‐induced near‐infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two‐photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR‐II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR‐II excitation. This study highlights the importance of developing NIR‐II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.     

Bright Aggregation‐Induced‐Emission Dots for Targeted Synergetic NIR‐II Fluorescence and NIR‐I Photoacoustic Imaging of Orthotopic Brain Tumors

Precise diagnostics are of significant importance to the optimal treatment outcomes of patients bearing brain tumors. NIR‐II fluorescence imaging holds great promise for brain‐tumor diagnostics with deep penetration and high sensitivity. This requires the development of organic NIR‐II fluorescent agents with high quantum yield (QY), which is difficult to achieve. Herein, the design and synthesis of a new NIR‐II fluorescent molecule with aggregation‐induced‐emission (AIE) characteristics is reported for orthotopic brain‐tumor imaging. Encapsulation of the molecule in a polymer matrix yields AIE dots showing a very high QY of 6.2% with a large absorptivity of 10.2 L g^?1 cm^?1 at 740 nm and an emission maximum near 1000 nm. Further decoration of the AIE dots with c‐RGD yields targeted AIE dots, which afford specific and selective tumor uptake, with a high signal/background ratio of 4.4 and resolution up to 38 µm. The large NIR absorptivity of the AIE dots facilitates NIR‐I photoacoustic imaging with intrinsically deeper penetration than NIR‐II fluorescence imaging and, more importantly, precise tumor‐depth detection through intact scalp and skull. This research demonstrates the promise of NIR‐II AIE molecules and their dots in dual NIR‐II fluorescence and NIR‐I photoacoustic imaging for precise brain cancer diagnostics.     

Near‐Infrared Excitation/Emission and Multiphoton‐Induced Fluorescence of Carbon Dots

Carbon dots (CDs) have significant potential for use in various fields including biomedicine, bioimaging, and optoelectronics. However, inefficient excitation and emission of CDs in both near‐infrared (NIR‐I and NIR‐II) windows remains an issue. Solving this problem would yield significant improvement in the tissue‐penetration depth for in vivo bioimaging with CDs. Here, an NIR absorption band and enhanced NIR fluorescence are both realized through the surface engineering of CDs, exploiting electron‐acceptor groups, namely molecules or polymers rich in sulfoxide/carbonyl groups. These groups, which are bound to the outer layers and the edges of the CDs, influence the optical bandgap and promote electron transitions under NIR excitation. NIR‐imaging information encryption and in vivo NIR fluorescence imaging of the stomach of a living mouse using CDs modified with poly(vinylpyrrolidone) in aqueous solution are demonstrated. In addition, excitation by a 1400 nm femtosecond laser yields simultaneous two‐photon‐induced NIR emission and three‐photon‐induced red emission of CDs in dimethyl sulfoxide. This study represents the realization of both NIR‐I excitation and emission as well as two‐photon‐ and three‐photon‐induced fluorescence of CDs excited in an NIR‐II window, and provides a rational design approach for construction and clinical applications of CD‐based NIR imaging agents.     

Second‐Harmonic Generation from a Single Core/Shell Quantum Dot

Nanoparticles emitting two‐photon luminescence are broadly used as photostable emitters for nonlinear microscopy. Second‐harmonic generation (SHG) as another two‐photon mechanism offers complementary optical properties but the reported sizes of nanoparticles are still large, of a few tens of nanometers. Herein, coherent SHG from single core/shell CdTe/CdS nanocrystals with a diameter of 10 to 15 nm is reported. The nanocrystal excitation spectrum reveals resonances in the nonlinear efficiency with an overall maximum at about 970 nm. Polarization analysis of the second‐harmonic emission confirms the expected zinc blende symmetry, and allows extraction of the three‐dimensional nanocrystal orientation. The small size of these nonlinearly active quantum dots, together with the intrinsic coherence and orientation sensitivity of the SHG process, are well adapted for ultrafast probing of optical near‐fields with high resolution as well as for orientation tracking for bioimaging applications.     

Precise Two‐Photon Photodynamic Therapy using an Efficient Photosensitizer with Aggregation‐Induced Emission Characteristics

Two‐photon photodynamic therapy (PDT) is able to offer precise 3D manipulation of treatment volumes, providing a target level that is unattainable with current therapeutic techniques. The advancement of this technique is greatly hampered by the availability of photosensitizers with large two‐photon absorption (TPA) cross section, high reactive‐oxygen‐species (ROS) generation efficiency, and bright two‐photon fluorescence. Here, an effective photosensitizer with aggregation‐induced emission (AIE) characteristics is synthesized, characterized, and encapsulated into an amphiphilic block copolymer to form organic dots for two‐photon PDT applications. The AIE dots possess large TPA cross section, high ROS generation efficiency, and excellent photostability and biocompatibility, which overcomes the limitations of many conventional two‐photon photosensitizers. Outstanding therapeutic performance of the AIE dots in two‐photon PDT is demonstrated using in vitro cancer cell ablation and in vivo brain‐blood‐vessel closure as examples. This shows therapy precision up to 5 µm under two‐photon excitation.     

Corannulene‐Incorporated AIE Nanodots with Highly Suppressed Nonradiative Decay for Boosted Cancer Phototheranostics In Vivo

Fluorescent nanoparticles (NPs) based on luminogens with aggregation‐induced emission characteristic (AIEgens), namely AIE dots, have received wide attention because of their antiquenching attitude in emission and reactive oxygen species (ROS) generation when aggregated. However, few reports are available on how to control and optimize their fluorescence and ROS generation ability. Herein, it is reported that enhancing the intraparticle confined microenvironment is an effective approach to advanced AIE dots, permitting boosted cancer phototheranostics in vivo. Formulation of a “rotor‐rich” and inherently charged near‐infrared (NIR) AIEgen with 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy(polyethylene glycol)‐2000] and corannulene‐decorated PEG affords DSPE‐AIE dots and Cor‐AIE dots, respectively. Compared to DSPE‐AIE dots, Cor‐AIE dots show 4.0‐fold amplified fluorescence quantum yield and 5.4‐fold enhanced ROS production, because corannulene provides intraparticle rigidity and strong interactions with the AIEgen to restrict the intramolecular rotation of AIEgen to strongly suppress the nonradiative decay and significantly facilitate the fluorescence pathway and intersystem crossing. Thus, it tremendously promotes phototheranostic efficacies in terms of NIR image‐guided cancer surgery and photodynamic therapy using a peritoneal carcinomatosis‐bearing mouse model. Collectively, it not only provides a novel strategy to advanced AIE dots for cancer phototheranostics, but also brings new insights into the design of superior fluorescent NPs for biomedical applications.     

Molecular Engineering of an Organic NIR‐II Fluorophore with Aggregation‐Induced Emission Characteristics for In Vivo Imaging

Design and synthesis of new fluorophores with emission in the second near‐infrared window (NIR‐II, 1000–1700 nm) have fueled the advancement of in vivo fluorescence imaging. Organic NIR‐II probes particularly attract tremendous attention due to excellent stability and biocompatibility, which facilitate clinical translation. However, reported organic NIR‐II fluorescent agents often suffer from low quantum yield and complicated design. In this study, the acceptor unit of a known NIR‐I aggregation‐induced emission (AIE) luminogen (AIEgen) is molecularly engineered by varying a single atom from sulfur to selenium, leading to redshifted absorption and emission spectra. After formulation of the newly prepared AIEgen, the resultant AIE nanoparticles (referred as L897 NPs) have an emission tail extending to 1200 nm with a high quantum yield of 5.8%. Based on the L897 NPs, noninvasive vessel imaging and lymphatic imaging are achieved with high signal‐to‐background ratio and deep penetration. Furthermore, the L897 NPs can be used as good contrast agents for tumor imaging and image‐guided surgery due to the high tumor/normal tissue ratio, which peaks at 9.0 ± 0.6. This work suggests a simple strategy for designing and manufacturing NIR‐II AIEgens and demonstrates the potential of NIR‐II AIEgens in vessel, lymphatic, and tumor imaging.     

Real‐Time and High‐Resolution Bioimaging with Bright Aggregation‐Induced Emission Dots in Short‐Wave Infrared Region

Fluorescence imaging in the spectral region beyond the conventional near‐infrared biological window (700–900 nm) can theoretically afford high resolution and deep tissue penetration. Although some efforts have been devoted to developing a short‐wave infrared (SWIR; 900–1700 nm) imaging modality in the past decade, long‐wavelength biomedical imaging is still suboptimal owing to the unsatisfactory materials properties of SWIR fluorophores. Taking advantage of organic dots based on an aggregation‐induced emission luminogen (AIEgen), herein microscopic vasculature imaging of brain and tumor is reported in living mice in the SWIR spectral region. The long‐wavelength emission of AIE dots with certain brightness facilitates resolving brain capillaries with high spatial resolution (≈3 µm) and deep penetration (800 µm). Owning to the deep penetration depth and real‐time imaging capability, in vivo SWIR microscopic angiography exhibits superior resolution in monitoring blood–brain barrier damage in mouse brain, and visualizing enhanced permeability and retention effect in tumor sites. Furthermore, the AIE dots show good biocompatibility, and no noticeable abnormalities, inflammations or lesions are observed in the main organs of the mice. This work will inspire new insights on development of advanced SWIR techniques for biomedical imaging.     

AIE Nanoparticles with High Stimulated Emission Depletion Efficiency and Photobleaching Resistance for Long‐Term Super‐Resolution Bioimaging

Stimulated emission depletion (STED) nanoscopy is a typical super‐resolution imaging technique that has become a powerful tool for visualizing intracellular structures on the nanometer scale. Aggregation‐induced emission (AIE) luminogens are ideal fluorescent agents for bioimaging. Herein, long‐term super‐resolution fluorescence imaging of cancer cells, based on STED nanoscopy assisted by AIE nanoparticles (NPs) is realized. 2,3‐Bis(4‐(phenyl(4‐(1,2,2‐triphenylvinyl)phenyl)amino)phenyl) fumaronitrile (TTF), a typical AIE luminogen, is doped into colloidal mesoporous silica to form fluorescent NPs. TTF@SiO₂ NPs bear three significant features, which are all essential for STED nanoscopy. First, their STED efficiency can reach more than 60%. Second, they are highly resistant to photobleaching, even under long‐term and high‐power STED light irradiation. Third, they have a large Stokes' shift of ≈150 nm, which is beneficial for restraining the fluorescence background induced by the STED light irradiation. STED nanoscopy imaging of TTF@SiO₂‐NPs‐stained HeLa cells is performed, exhibiting a high lateral spatial resolution of 30 nm. More importantly, long‐term (more than half an hour) super‐resolution cell imaging is achieved with low fluorescence loss. Considering that AIE luminogens are widely used for organelle targeting, cellular mapping, and tracing, AIE‐NPs‐based STED nanoscopy holds great potential for many basic biomedical studies that require super‐resolution and long‐term imaging.     

Lipid‐PEG‐Folate Encapsulated Nanoparticles with Aggregation Induced Emission Characteristics: Cellular Uptake Mechanism and Two‐Photon Fluorescence Imaging

Folate functionalized nanoparticles (NPs) that contain fluorogens with aggregation‐induced emission (AIE) characteristics are fabricated to show bright far‐red/near‐infrared fluorescence, a large two‐photon absorption cross section and low cytotoxicity, which are internalized into MCF‐7 cancer cells mainly through caveolae‐mediated endocytosis. One‐photon excited in vivo fluorescence imaging illustrates that these AIE NPs can accumulate in a tumor and two‐photon excited ex vivo tumor tissue imaging reveals that they can be easily detected in the tumor mass at a depth of 400 μm. These studies indicate that AIE NPs are promising alternatives to conventional TPA probes for biological imaging.     

Multicolor Photo‐Crosslinkable AIEgens toward Compact Nanodots for Subcellular Imaging and STED Nanoscopy

Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane‐substituted AIEgens to generate compact and ultrastable AIE dots via photo‐crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation. Due to their small sizes, good stability, and surface functionalization, the cell‐surface markers and subcellular structures are specifically labeled by the AIE dot bioconjugates. Remarkably, stimulated emission depletion imaging with AIE dots is achieved for the first time, and the spatial resolution is significantly enhanced to ≈95 nm. This study provides a general approach for small functional molecules for preparing small sized and ultrastable nanodots.     

Super‐Resolution Mapping of Single Nanoparticles inside Tumor Spheroids

Cancer spheroids have structural, functional, and physiological similarities to the tumor, and have become a low‐cost in vitro model to study the physiological responses of single cells and therapeutic efficacy of drugs. However, the tiny spheroid, made of a cluster of high‐density cells, is highly scattering and absorptive, which prevents light microscopy techniques to reach the depth inside spheroids with high resolution. Here, a method is reported for super‐resolution mapping of single nanoparticles inside a spheroid. It first takes advantage of the self‐healing property of a “nondiffractive” doughnut‐shaped Bessel beam from a 980 nm diode laser as the excitation, and further employs the nonlinear response of the 800 nm emission from upconversion nanoparticles, so that both excitation and emission at the near‐infrared can experience minimal loss through the spheroid. These strategies lead to the development of a new nanoscopy modality with a resolution of 37 nm, 1/26th of the excitation wavelength. This method enables mapping of single nanoparticles located 55 µm inside a spheroid, with a resolution of 98 nm. It suggests a solution to track single nanoparticles and monitor their release of drugs in 3D multicellar environments.     

Silole‐Based Red Fluorescent Organic Dots for Bright Two‐Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging

Robust luminescent dyes with efficient two‐photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation‐caused quenching. In this work, a red fluorescent silole, 2,5‐bis[5‐(dimesitylboranyl)thiophen‐2‐yl]‐1‐methyl‐1,3,4‐triphenylsilole ((MesB)₂DTTPS), is synthesized and characterized. (MesB)₂DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation‐enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)₂DTTPS within lipid‐PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two‐photon absorption cross section of 3.43 × 10⁵ GM, which yields a two‐photon action cross section of 1.09 × 10⁵ GM. These (MesB)₂DTTPS dots show good biocompatibility and are successfully applied to one‐photon and two‐photon fluorescence imaging of MCF‐7 cells and two‐photon in vivo visualization of the blood vascular of mouse muscle in a high‐contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network.     

Luminescence Lifetime–Based In Vivo Detection with Responsive Rare Earth–Dye Nanocomposite

For years, luminescence lifetime imaging has served as a quantitative tool in indicating intracellular components and activities. However, very few studies involve the in vivo study of animals, especially in vivo stimuli‐responsive activities of animals, as both excitation and emission wavelengths should fall into the near‐infrared (NIR) optical transparent window (660–950 and 1000–1500 nm). Herein, this work reports a lifetime‐responsive nanocomposite with both excitation and emission in the NIR I window (800 nm) and lifetime in the microsecond region. The incorporation of Tm³⁺‐doped rare‐earth nanocrystals and NIR dye builds an efficient energy transfer pathway that enables a tunable luminescence lifetime range. The NaYF₄:Tm nanocrystal, which absorbs and emits photons at the same energy level, is found to be 33 times brighter than optimized core–shell upconversion nanocrystals, and proved to be an effective donor for NIR luminescence resonance energy transfer (LRET). The anti‐interference capability of luminescence lifetime signals is further confirmed by luminescence and lifetime imaging. In vivo studies also verify the lifetime response upon stimulation generated in an arthritis mouse model. This work introduces an intriguing tool for luminescence lifetime–based sensing in the microsecond region.     

Precise Deciphering of Brain Vasculatures and Microscopic Tumors with Dual NIR‐II Fluorescence and Photoacoustic Imaging

Diagnostics of cerebrovascular structures and microscopic tumors with intact blood–brain barrier (BBB) significantly contributes to timely treatment of patients bearing neurological diseases. Dual NIR‐II fluorescence and photoacoustic imaging (PAI) is expected to offer powerful strength, including good spatiotemporal resolution, deep penetration, and large signal‐to‐background ratio (SBR) for precise brain diagnostics. Herein, biocompatible and photostable conjugated polymer nanoparticles (CP NPs) are reported for dual‐modality brain imaging in the NIR‐II window. Uniform CP NPs with a size of 50 nm are fabricated from microfluidics devices, which show an emission peak at 1156 nm with a large absorptivity of 35.2 L g^?1 cm^?1 at 1000 nm. The NIR‐II fluorescence imaging resolves hemodynamics and cerebral vasculatures with a spatial resolution of 23 µm at a depth of 600 µm. The NIR‐II PAI enables successful noninvasive mapping of deep microscopic brain tumors (<2 mm at a depth of 2.4 mm beneath dense skull and scalp) with an SBR of 7.2 after focused ultrasound‐induced BBB opening. This study demonstrates that CP NPs are promising contrast agents for brain diagnostics.     

A Highly Efficient and Photostable Photosensitizer with Near‐Infrared Aggregation‐Induced Emission for Image‐Guided Photodynamic Anticancer Therapy

Photodynamic therapy (PDT), which relies on photosensitizers (PS) and light to generate reactive oxygen species to kill cancer cells or bacteria, has attracted much attention in recent years. PSs with both bright emission and efficient singlet oxygen generation have also been used for image‐guided PDT. However, simultaneously achieving effective ¹O₂ generation, long wavelength absorption, and stable near‐infrared (NIR) emission with low dark toxicity in a single PS remains challenging. In addition, it is well known that when traditional PSs are made into nanoparticles, they encounter quenched fluorescence and reduced ¹O₂ production. In this contribution, these challenging issues have been successfully addressed through designing the first photostable photosensitizer with aggregation‐induced NIR emission and very effective ¹O₂ generation in aggregate state. The yielded nanoparticles show very effective ¹O₂ generation, bright NIR fluorescence centered at 820 nm, excellent photostability, good biocompatibility, and negligible dark in vivo toxicity. Both in vitro and in vivo experiments prove that the nanoparticles are excellent candidates for image‐guided photodynamic anticancer therapy.     

Stereotactic Photodynamic Therapy Using a Two‐Photon AIE Photosensitizer

Two‐photon photodynamic therapy (TP‐PDT) is emerging as a powerful strategy for stereotactic targeting of diseased areas, but ideal photosensitizers (PSs) are currently lacking. This work reports a smart PS with aggregation‐induced emission (AIE) feature, namely DPASP, for TP‐PDT with excellent performances. DPASP exhibits high affinity to mitochondria, superior photostability, large two‐photon absorption cross section as well as efficient reactive oxygen species generation, enabling it to achieve photosensitization both in vitro and in vivo under two‐photon excitation. Moreover, its capability of stereotactic ablation of targeted cells with high‐precision is also successfully demonstrated. All these merits make DPASP a promising TP‐PDT candidate for accurate ablation of abnormal tissues with minimal damages to surrounding areas in the treatment of various diseases.     

Eccentric Loading of Fluorogen with Aggregation‐Induced Emission in PLGA Matrix Increases Nanoparticle Fluorescence Quantum Yield for Targeted Cellular Imaging

A simple strategy is developed to prepare eccentrically or homogeneously loaded nanoparticles (NPs) using poly (DL‐lactide‐co‐glycolide) (PLGA) as the encapsulation matrix in the presence of different amounts of polyvinyl alcohol (PVA) as the emulsifier. Using 2,3‐bis(4‐(phenyl(4‐(1,2,2‐triphenylvinyl)‐phenyl)amino)‐phenyl)‐fumaronitrile (TPETPAFN), a fluorogen with aggregation‐induced emission (AIE) characteristics, as an example, the eccentrically loaded PLGA NPs show increased fluorescence quantum yields (QYs) as compared to the homogeneously loaded ones. Field emission transmission electron microscopy and fluorescence lifetime measurements reveal that the higher QY of the eccentrically loaded NPs is due to the more compact aggregation of AIE fluorogens that restricts intramolecular rotations of phenyl rings, which is able to more effectively block the non‐radiative decay pathways. The eccentrically loaded NPs show far red/near infrared emission with a high fluorescence QY of 34% in aqueous media. In addition, by using poly([lactide‐co‐glycolide]‐b‐folate [ethylene glycol]) (PLGA‐PEG‐folate) as the co‐encapsulation matrix, the obtained NPs are born with surface folic acid groups, which are successfully applied for targeted cellular imaging with good photostability and low cytotoxicity. Moreover, the developed strategy is also demonstrated for inorganic‐component eccentrically or homogeneously loaded PLGA NPs, which facilitates the synthesis of polymer NPs with controlled internal architectures.     

Upconversion fluorescent nanoparticles as a potential tool for in-depth imaging

Upconversion nanoparticles (UCNs) are nanoparticles that are excited in the near infrared (NIR) region with emission in the visible or NIR regions. This makes these particles attractive for use in biological imaging as the NIR light can penetrate the tissue better with minimal absorption/scattering. This paper discusses the study of the depth to which cells can be imaged using these nanoparticles. UCNs with NaYF(4) nanocrystals doped with Yb(3+), Er(3+) (visible emission)/Yb(3+), Tm(3+) (NIR emission) were synthesized and modified with silica enabling their dispersion in water and conjugation of biomolecules to their surface. The size of the sample was characterized using transmission electron microscopy and the fluorescence measured using a fluorescence spectrometer at an excitation of 980 nm. Tissue phantoms were prepared by reported methods to mimic skin/muscle tissue and it was observed that the cells could be imaged up to a depth of 3 mm using the NIR emitting UCNs. Further, the depth of detection was evaluated for UCNs targeted to gap junctions formed between cardiac cells.     

Aggregation‐Induced Dual‐Phosphorescence from Organic Molecules for Nondoped Light‐Emitting Diodes

Aggregation‐induced emission (AIE) is a beneficial strategy for generating highly effective solid‐state molecular luminescence without suffering losses in quantum yield. However, the majority of reported AIE‐active molecules exhibit only strong fluorescence, which is not ideal for electrical excitation in organic light‐emitting diodes (OLEDs). By introducing various substituent groups onto the biscarbazole compound, a series of molecular materials with aggregation‐induced phosphorescence (AIP) is designed, which exhibits two distinctly different phosphorescence bands and an absolute solid‐state room‐temperature phosphorescence quantum yield up to 64%. Taking advantage of the AIE feature, the AIP molecules are fabricated into OLEDs as a homogeneous light‐emitting layer, which allows for relatively small efficiency roll‐off and shows an external electroluminescence quantum yield of up to 5.8%, more than the theoretical limit for purely fluorescent OLED devices. The design showcases a promising strategy for the production of cost‐effective and highly efficient OLED technology.