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.     

808‐nm‐Light‐Excited Lanthanide‐Doped Nanoparticles: Rational Design,Luminescence Control and Theranostic Applications

808 nm‐light‐excited lanthanide (Ln³⁺)‐doped nanoparticles (LnNPs) hold great promise for a wide range of applications, including bioimaging diagnosis and anticancer therapy. This is due to their unique properties, including their minimized overheating effect, improved penetration depth, relatively high quantum yields, and other common features of LnNPs. In this review, the progress of 808 nm‐excited LnNPs is reported, including their i) luminescence mechanism, ii) luminescence enhancement, iii) color tuning, iv) diagnostic and v) therapeutic applications. Finally, the future outlook and challenges of 808 nm‐excited LnNPs are presented.     

Engineering of Lanthanide‐Doped Upconversion Nanoparticles for Optical Encoding

Lanthanide‐doped upconversion nanoparticles (UCNPs) are an emerging class of luminescent materials that emit UV or visible light under near infra‐red (NIR) excitations, thereby possessing a large anti‐Stokes shift property. Due to their sharp excitation and emission bands, excellent photo‐ and chemical stability, low autofluorescence, and high tissue penetration depth of the NIR light used for excitation, UCNPs have surpassed conventional fluorophores in many bioapplications. A better understanding of the mechanism of upconversion, as well as the development of better approaches to preparing UCNPs, have provided more opportunities to explore their use for optical encoding, which has the potential for applications in multiplex detection and imaging. With the current ability to precisely control the microstructure and properties of UCNPs to produce particles of tunable emission, excitation, luminescence lifetime, and size, various strategies for optical encoding based on UCNPs can now be developed. These optical properties of UCNPs (such as emission and excitation wavelengths, ratiometric intensity, luminescence lifetime, and multicolor patterns), and the strategies employed to engineer these properties for optical encoding of UCNPs through homogeneous ion doping, heterogeneous structure fabrication and microbead encapsulation are reviewed. The challenges and potential solutions faced by UCNP optical encoding are also discussed.     

Albumin Nanoshell Encapsulation of Near‐Infrared‐Excitable Rare‐Earth Nanoparticles Enhances Biocompatibility and Enables Targeted Cell Imaging

The use of traditional fluorophores for in vivo imaging applications is limited by poor quantum yield, poor tissue penetration of the excitation light, and excessive tissue autofluorescence, while the use of inorganic fluorescent particles that offer a high quantum yield is frequently limited due to particle toxicity. Rare‐earth‐doped nanoparticles that utilize near‐infrared upconversion overcome the optical limitations of traditional fluorophores, but are not typically suitable for biological application due to their insolubility in aqueous solution, lack of functional surface groups for conjugation of biomolecules, and potential cytotoxicity. A new approach to establish highly biocompatible and biologically targetable nanoshell complexes of luminescent rare‐earth‐doped NaYF₄ nanoparticles (REs) excitable with 920–980 nm near‐infrared light for biomedical imaging applications is reported. The approach involves the encapsulation of NaYF₄ nanoparticles doped with Yb and Er within human serum albumin nanoshells to create water‐dispersible, biologically functionalizable composite particles. These particles exhibit narrow size distributions around 200 nm and are stable in aqueous solution for over 4 weeks. The albumin shell confers cytoprotection and significantly enhances the biocompatibility of REs even at concentrations above 200 µg REs mL^?1. Composite particles conjugated with cyclic arginine‐glycine‐aspartic acid (cRGD) specifically target both human glioblastoma cell lines and melanoma cells expressing α_vβ₃ integrin receptors. These findings highlight the promise of albumin‐encapsulated rare‐earth nanoparticles for imaging cancer cells in vitro and the potential for targeted imaging of disease sites in vivo.     

Integration of IR‐808 Sensitized Upconversion Nanostructure and MoS2 Nanosheet for 808 nm NIR Light Triggered Phototherapy and Bioimaging

Near infrared (NIR) light triggered phototherapy including photothermal therapy (PTT) and photodynamic therapy (PDT) affords superior outcome in cancer treatment. However, the reactive oxygen species (ROS) generated by NIR‐excited upconversion nanostructure is limited by the feeble upconverted light which cannot activate PDT agents efficiently. Here, an IR‐808 dye sensitized upconversion nanoparticle (UCNP) with a chlorin e6 (Ce6)‐functionalized silica layer is developed for PDT agent. The two booster effectors (dye‐sensitization and core–shell enhancement) synergistically amplify the upconversion efficiency, therefore achieving superbright visible emission under low 808 nm light excitation. The markedly amplified red light subsequently triggers the photosensitizer (Ce6) to produce large amount of ROS for efficient PDT. After the silica is endowed with positive surface, these PDT nanoparticles can be easily grafted on MoS₂ nanosheet. As the optimal laser wavelength of UCNPs is consistent with that of MoS₂ nanosheet for PTT, the invented nanoplatform generates both abundant ROS and local hyperthermia upon a single 808 nm laser irradiation. Both the in vitro and in vivo assays validate that the innovated nanostructure presents excellent cancer cell inhibition effectiveness by taking advantages of the synergistic PTT and PDT, simultaneously, posing trimodal (upconversion luminescence/computed tomography (CT)/magnetic resonance imaging (MRI) imaging capability.     

Near-infrared light activated persistent luminescence nanoparticles via upconversion

Persistent luminescence nanoparticles (PLNPs) and upconversion nanoparticles (UCNPs) are two special optical imaging nanoprobes.In this study,efficient upconverted persistent luminescence (UCPL) is realized by combining their unique features into polymethyl methacrylate,forming a film composed of both PLNPs and UCNPs.The red persistent luminescence (～640 nm) of the PLNPs (CaS∶Eu,Tm,Ce) can be activated by upconverted green emission of UCNPs (β-NaYF4∶Yb,Er@NaYF4) excited by near-infrared light (NIR).Using this strategy,both the unique optical properties of PLNPs and UCNPs can be optimally synergized,thus generating efficient upconversion,photoluminescence,and UCPL simultaneously.The UCPL system has potential applications in in vivo bioimaging by simply monitoring the biocompatible low power density of NIR-light-excited persistent luminescence.Due to its simplicity,we anticipate that this method for the preparation of UCPL composite can be easily adjusted using other available upconversion and persistent phosphor pairs for a number of biophotonic and photonic applications.     

New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third‐Harmonic Generation Bioimaging

Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth‐inspired synthesis is developed combined with primary mineralization and a ligand‐assisted secondary growth strategy to prepare mesostructured α‐FeOOH nanorods (NRs). The sub‐wavelength heterogeneity of the refractive index leads to enhanced third‐harmonic generation (THG) signals under near‐infrared excited wavelengths at 1230 nm. The as‐prepared NRs exhibit an 11‐fold stronger THG intensity compared to bare α‐FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α‐FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long‐term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe‐based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.     

Tunable Resonator‐Upconverted Emission (TRUE) Color Printing and Applications in Optical Security

Lanthanide‐doped nanophosphors are promising in anti‐counterfeiting and security printing applications. These nanophosphors can be incorporated as transparent inks that fluoresce by upconverting near‐infrared illumination into visible light to allow easy verification of documents. However, these inks typically exhibit a single luminescent color, low emission efficiency, and low print resolutions. Tunable resonator‐upconverted emission (TRUE) is achieved by placing upconversion nanoparticles (UCNPs) within plasmonic nanoresonators. A range of TRUE colors are obtained from a single‐UCNP species self‐assembled within size‐tuned gap‐plasmon resonances in Al nanodisk arrays. The luminescence intensities are enhanced by two orders of magnitude through emission and absorption enhancements. The enhanced emissive and plasmonic colors are simultaneously employed to generate TRUE color prints that exhibit one appearance under ambient white light, and a multicolored luminescence appearance that is revealed under near‐infrared excitation. The printed color and luminescent images are of ultrahigh resolutions (≈50 000 dpi), and enable multiple colors from a single excitation source for increased level of security.     

Mesostructured Composite Materials with Electrically Tunable Upconverting Properties

A promising approach of designing mesostructured materials with novel physical behavior is to combine unique optical and electronic properties of solid nanoparticles with long‐range ordering and facile response of soft matter to weak external stimuli. Here, orientationally ordered nematic liquid crystalline dispersions of rod‐like upconversion nanoparticles are designed, practically realized, and characterized. Boundary conditions on particle surfaces, defined through surface functionalization, promote spontaneous unidirectional self‐alignment of the dispersed rod‐like nanoparticles, mechanically coupled to the molecular ordering direction of the thermotropic nematic liquid crystal host. As host is electrically switched at low voltages ≈1 V, nanorods rotate, yielding tunable upconversion and polarized luminescence properties of the composite. Spectral and polarization dependencies are characterized and explained through invoking models of electrical switching of liquid crystals and upconversion dependence on crystalline matrices of nanorods, and their potential practical uses are discussed.     

Nanocomposite‐Based Photodynamic Therapy Strategies for Deep Tumor Treatment

Photodynamic therapy (PDT), as an emerging clinically approved modality, has been used for treatment of various cancer diseases. Conventional PDT strategies are mainly focused on superficial lesions because the wavelength of illumination light of most clinically approved photosensitizers (PSs) is located in the UV/VIS range that possesses limited tissue penetration ability, leading to ineffective therapeutic response for deep‐seated tumors. The combination of PDT and nanotechnology is becoming a promising approach to fight against deep tumors. Here, the rapid development of new PDT modalities based on various smartly designed nanocomposites integrating with conventionally used PSs for deep tumor treatments is introduced. Until now many types of multifunctional nanoparticles have been studied, and according to the source of excitation energy they can be classified into three major groups: near infrared (NIR) light excited nanomaterials, X‐ray excited scintillating/afterglow nanoparticles, and internal light emission excited nanocarriers. The in vitro and in vivo applications of these newly developed PDT modalities are further summarized here, which highlights their potential use as promising nano‐agents for deep tumor therapy.     

Thermally Activated Upconversion Near‐Infrared Photoluminescence from Carbon Dots Synthesized via Microwave Assisted Exfoliation

Upconversion near‐infrared (NIR) fluorescent carbon dots (CDs) are important for imaging applications. Herein, thermally activated upconversion photoluminescence (UCPL) in the NIR region, with an emission peak at 784 nm, which appears under 808 nm continuous‐wave laser excitation, are realized in the NIR absorbing/emissive CDs (NIR‐CDs). The NIR‐CDs are synthesized by microwave‐assisted exfoliation of red emissive CDs in dimethylformamide, and feature single or few‐layered graphene‐like cores. This structure provides an enhanced contact area of the graphene‐like plates in the core with the electron‐acceptor carbonyl groups in dimethylformamide, which contributes to the main NIR absorption band peaked at 724 nm and a tail band in 800–850 nm. Temperature‐dependent photoluminescence spectra and transient absorption spectra confirm that the UCPL of NIR‐CDs is due to the thermally activated electron transitions in the excited state, rather than the multiphoton absorption process. Temperature dependent upconversion NIR luminescence imaging is demonstrated for NIR‐CDs embedded in a polyvinyl pyrrolidone film, and the NIR upconversion luminescence imaging in vivo using NIR‐CDs in a mouse model is accomplished.     

Aggregation‐Induced Nonlinear Optical Effects of AIEgen Nanocrystals for Ultradeep In Vivo Bioimaging

Nonlinear optical microscopy has become a powerful tool in bioimaging research due to its unique capabilities of deep optical sectioning, high‐spatial‐resolution imaging, and 3D reconstruction of biological specimens. Developing organic fluorescent probes with strong nonlinear optical effects, in particular third‐harmonic generation (THG), is promising for exploiting nonlinear microscopic imaging for biomedical applications. Herein, a simple method for preparing organic nanocrystals based on an aggregation‐induced emission (AIE) luminogen (DCCN) with bright near‐infrared emission is successfully demonstrated. Aggregation‐induced nonlinear optical effects, including two‐photon fluorescence (2PF), three‐photon fluorescence (3PF), and THG, of DCCN are observed in nanoparticles, especially for crystalline nanoparticles. The nanocrystals of DCCN are successfully applied for 2PF microscopy at 1040 nm NIR‐II excitation and THG microscopy at 1560 nm NIR‐II excitation, respectively, to reconstruct the 3D vasculature of the mouse cerebral vasculature. Impressively, the THG microscopy provides much higher spatial resolution and brightness than the 2PF microscopy and can visualize small vessels with diameters of ≈2.7 µm at the deepest depth of 800 µm in a mouse brain. Thus, this is expected to inspire new insights into the development of advanced AIE materials with multiple nonlinearity, in particular THG, for multimodal nonlinear optical microscopy.     

Recent Progress in Time‐Resolved Biosensing and Bioimaging Based on Lanthanide‐Doped Nanoparticles

Luminescent nanomaterials have attracted great attention in luminescence‐based bioanalysis due to their abundant optical and tunable surface physicochemical properties. However, luminescent nanomaterials often suffer from serious autofluorescence and light scattering interference when applied to complex biological samples. Time‐resolved luminescence methodology can efficiently eliminate autofluorescence and light scattering interference by collecting the luminescence signal of a long‐lived probe after the background signals decays completely. Lanthanides have a unique [Xe]4f^N electronic configuration and ladder‐like energy states, which endow lanthanide‐doped nanoparticles with many desirable optical properties, such as long luminescence lifetimes, large Stokes/anti‐Stokes shifts, and sharp emission bands. Due to their long luminescence lifetimes, lanthanide‐doped nanoparticles are widely used for high‐sensitive biosensing and high‐contrast bioimaging via time‐resolved luminescence methodology. In this review, recent progress in the development of lanthanide‐doped nanoparticles and their application in time‐resolved biosensing and bioimaging are summarized. At the end of this review, the current challenges and perspectives of lanthanide‐doped nanoparticles for time‐resolved bioapplications are discussed.     

Lanthanide‐Doped Core@Multishell Nanoarchitectures: Multimodal Excitable Upconverting/Downshifting Luminescence and High‐Level Anti‐Counterfeiting

The development of luminescent materials with concurrent multimodal emissions is a great challenge to improve security and data storage density. Lanthanide‐doped nanocrystals are particularly appropriate for such applications for their abundant intermediate energy states and distinguishable spectroscopic profiles. However, traditional lanthanide luminescent nanoparticles have a limited capacity for information storage or complexity to shield against counterfeiting. Herein, it is demonstrated that the combination of upconverting and downshifting emissions in a particulate designed lanthanide‐doped core@multishell nanoarchitecture allows the generation of multicolor dual‐modal luminescence over a wide spectral range for complex information storage. Precise control of lanthanide dopants distribution in the core and distinct shells enables simultaneous excitation of 980/808 nm focusing/defocusing laser and 254 nm light and produces complex upconverting emissions from Er, Tm, Eu, and Tb via multiphoton energy transfer processes and downshifting emissions from Eu and Tb via efficient energy transfer from Ce to Eu/Tb in Gd‐assisted lattices. It is experimentally proven that multiple visualized anti‐counterfeit and information encryption with facile decryption and authentication using screen‐printing inks containing the present core@multishell nanocrystals are practically applicable by selecting different excitation modes.     

Photoreversible Fluorescent Modulation of Nanoparticles via One‐Step Miniemulsion Polymerization

A nitrobenzoxadiazolyl(NBD)‐based fluorescent dye and a photochromic spiropyran derivative are incorporated into polymeric nanoparticles via a one‐step miniemulsion polymerization. The diameter of the nanoparticles can be varied from approximately 40 nm to 80 nm by adjusting the polymerization conditions. The prepared nanoparticles exhibit the spectral properties of both NBD dye and spiropyran, indicating that the two chromophores are incorporated into the nanoparticles. The determined amount of NBD and spiropyran in the nanoparticles are about ≈85–90% of the feed amount, while the determined weight ratios of spiropyran to NBD in nanoparticles are very close to that of feed ratios, suggesting the miniemulsion polymerization is a suitable approach for incorporating multiple chromophores into individual nanoparticles with controlled amounts (content) and ratio. UV and visible light can be applied to modulate the fluorescence emission of NBD dye in nanoparticles. Upon UV irradiation, the spiropyran moieties in nanoparticles are converted to the open‐ring (McH form) structure and upon visible‐light irradiation they return to the closed‐ring (SP form) structure; as a result, the fluorescence of NBD can be reversibly “switched off” and “switched on”. Fluorescence resonance energy transfer from the excited NBD dye molecules to the McH form of the spiropyran moieties is the drives the fluorescence modulation. The nanoparticles display fairly good photoreversibility, photostability, and relatively fast photoresponsivity upon alternate UV/Vis irradiation. This class of photoresponsive nanoparticles may find applications in biological fields, such as labeling and imaging, as well as in optical fields, for example, individually light‐addressable nanoscale devices.     

An Excitation Navigating Energy Migration of Lanthanide Ions in Upconversion Nanoparticles

Upconversion nanoparticles (UCNPs) doped with lanthanide ions that possess ladder-like energy levels can give out multiple emissions at specific ultra-violet or visible wavelengths irrespective of excitation light. However, precisely controlling energy migration processes between different energy levels of the same lanthanide ion to generate switchable emissions remains elusive. Herein, a novel dumbbell-shaped UCNP is reported with upconverted red emission switched to green emission when excitation wavelength changed from 980 to 808 nm. The sensitizer Yb ions are doped with activator Er ions and energy modulator Mn ions in NaYF₄ core nanocrystal coated with an inner NaYF₄:Yb shell to generate red emission after harvesting 980 nm excitation light, while an outer NaNdF₄:Yb shell is coated to form a dumbbell shape to generate green emission upon 808 nm excitation. Such specially designed UCNPs with switchable green and red emissions are further explored for imaging of latent fingerprint and detection of explosive residues in the fingerprint simultaneously. This work suggests a novel research interest in fine-tuning of upconversion emissions through precisely controlling energy migration processes of the same lanthanide activator ion. Furthermore, use of these nanoparticles in other applications such as simultaneous dual-color imaging or orthogonal bidirectional photoactivation can be explored.     

Engineering Porous Emitting Framework Nanoparticles with Integrated Sensitizers for Low‐Power Photon Upconversion by Triplet Fusion

The conversion of low‐energy light into photons of higher energy based on sensitized triplet–triplet annihilation (sTTA) upconversion is emerging as the most promising wavelength‐shifting methodology because it operates efficiently at excitation powers as low as the solar irradiance. However, the production of solid‐state upconverters suited for direct integration in devices is still an ongoing challenge owing to the difficulties concerning the organization of two complementary moieties, the triplet sensitizer, and the annihilator, which must interact efficiently. This problem is solved by fabricating porous fluorescent nanoparticles wherein the emitters are integrated into robust covalent architectures. These emitting porous aromatic framework (ePAF) nanoparticles allow intimate interaction with the included metallo‐porphyrin as triplet sensitizers. Remarkably, the high concentration of framed chromophores ensures hopping‐mediated triplet diffusion required for TTA, yet the low density of the framework promotes their high optical features without quenching effects, typical of the solid state. A green‐to‐blue photon upconversion yield as high as 15% is achieved: a record performance among annihilators in a condensed phase. Furthermore, the engineered ePAF architecture containing covalently linked sensitizers produces full‐fledge solid‐state bicomponent particles that behave as autonomous nanodevices.     

Tuning Excitonic Properties of Monolayer MoS2 with Microsphere Cavity by High‐Throughput Chemical Vapor Deposition Method

Tuning the optical properties of 2D direct bandgap semiconductors is crucial for applications in photonic light source, optical communication, and sensing. In this work, the excitonic properties of molybdenum disulphide (MoS₂) are successfully tuned by directly depositing it onto silica microsphere resonators using chemical vapor deposition method. Multiple whispering gallery mode (WGM) peaks in the emission wavelength range of ≈650–750 nm are observed under continuous wave excitation at room temperature. Time‐resolved photoluminescence (TRPL) and femtosecond transient absorption (TA) spectroscopy are conducted to study light‐matter interaction dynamics of the MoS₂ microcavities. TRPL study suggests radiative recombination rate of carrier‐phonon scattering and interband transition processes in MoS₂ is enhanced by a factor of ≈1.65 due to Purcell effect in microcavities. TA spectroscopy study shows modulation of the interband transition process mainly occurs at PB‐A band with an estimated F ≈ 1.60. Furthermore, refractive index sensing utilizing WGM peaks of MoS₂ is established with sensitivity up to ≈150 nm per refractive index unit. The present work provides a large‐scale and straightforward method for coupling atomically thin 2D gain media with cavities for high‐performance optoelectronic devices and sensors.     

Visible upconversion emission of Pr3+ doped gadolinium gallium garnet nanocrystals

The luminescence properties of a Pr3+-doped gadolinium gallium garnet (GGG, Gd3Ga5O12) nanocrystalline host were investigated. Dominant blue/green emission was observed emanating from the 3P0 --> 3H4 transition after excitation using a wavelength of 457.9 nm. Continuous wave excitation into the 1D2 level of the Pr3+ ion at 606.9 nm transition produced blue upconversion luminescence spectra, ascribed to emission from the 3P1 --> 3H4 and 3P0 --> 3H4 transitions. The increase in the decay times of the observed transitions following excitation with 606.9 nm is indicative of the dominance of an energy transfer upconversion (ETU) mechanism relative to excited state absorption (ESA). Furthermore, blue, green and red upconversion emission was observed from the 3P0, 3P1 and 1D2 states following excitation into the 1G4 energy level with 980 nm. No change in the decay times of the emitting states was observed following excitation with a wavelength of 980 or 457.9 nm; hence, upconversion was determined to primarily occur through ESA. The luminescence properties of the nanocrystals are compared to a single crystal of GGG:Pr3+ (bulk) with an identical Pr3+ concentration (1%).     

A Modular System for the Design of Stimuli‐Responsive Multifunctional Nanoparticle Aggregates by Use of Host–Guest Chemistry

A self‐assembly approach for the design of multifunctional nanomaterials consisting of different nanoparticles (gold, iron oxide, and lanthanide‐doped LiYF₄) is developed. This modular system takes advantage of the light‐responsive supramolecular host–guest chemistry of β‐cyclodextrin and arylazopyrazole, which enables the dynamic and reversible self‐assembly of particles to spherical nanoparticle aggregates in aqueous solution. Due to the magnetic iron oxide nanoparticles, the aggregates can be manipulated by an external magnetic field leading to the formation of linear structures. As a result of the integration of upconversion nanoparticles, the aggregates are additionally responsive to near‐infrared light and can be redispersed by use of the upconversion effect. By varying the nanoparticle and linker concentrations the composition, size, shape, and properties of the multifunctional nanoparticle aggregates can be fine‐tuned.