Strategies for Constructing Upconversion Luminescence Nanoprobes to Improve Signal Contrast

Lanthanide‐doped upconversion nanoparticles (UCNPs) can convert two or more lower‐energy near‐infrared photons to a single photon with higher energy, which makes them particularly suitable for constructing nanoprobes with large imaging depth and minimal interference of autofluorescence and light scattering from biosamples. Furthermore, they feature excellent photostability, sharp and narrow emissions, and large anti‐Stokes shift, which confer them the capability of long‐period bioimaging and real‐time tracking. In recent years, UCNPs‐based nanoprobes (UC‐nanoprobes) have been attracting increasing interest in biological and medical research. Signal contrast, the ratio of signal intensity after and before the reaction of the probe and target, is the determinant factor of the sensitivity of all reaction‐based probes. This progress report presents the methods of constructing UC‐nanoprobes, with a focus fixed on recent strategies to improve the signal contrast, which have kept on promoting the bioapplication of this type of probe.     

Gold and Hairpin DNA Functionalization of Upconversion Nanocrystals for Imaging and In Vivo Drug Delivery

Although multifunctional upconversion imaging probes have recently attracted considerable interest in biomedical research, there are currently few methods for stabilizing these luminescent nanoprobes with oligonucleotides in biological systems. Herein, a method to robustly disperse upconversion nanoprobes in physiological buffers based on rational design and synthesis of nanoconjugates comprising hairpin‐DNA‐modified gold nanoparticles is presented. This approach imparts the upconversion nanoprobes with excellent biocompatibility and circumvents the problem of particle agglomeration. By combining single‐band anti‐Stokes near‐infrared emission and the photothermal effect mediated by the coupling of gold to upconversion nanoparticles, a simple, versatile nanoparticulate system for simultaneous deep‐tissue imaging and drug molecule release in vivo is demonstrated.     

Responsive Upconversion Nanoprobe for Background‐Free Hypochlorous Acid Detection and Bioimaging

Responsive nanoprobes play an important role in bioassay and bioimaging, early diagnosis of diseases and treatment monitoring. Herein, a upconversional nanoparticle (UCNP)‐based nanoprobe, Ru@UCNPs, for specific sensing and imaging of hypochlorous acid (HOCl) is reported. This Ru@UCNP nanoprobe consists of two functional components,, i.e., NaYF₄:Yb, Tm UCNPs that can convert near infrared light‐to‐visible light as the energy donor, and a HOCl‐responsive ruthenium(II) complex [Ru(bpy)₂(DNCH‐bpy)](PF₆)₂ (Ru‐DNPH) as the energy acceptor and also the upconversion luminescence (UCL) quencher. Within this luminescence resonance energy transfer nanoprobe system, the UCL OFF–ON emission is triggered specifically by HOCl. This triggering reaction enables the detection of HOCl in aqueous solution and biological systems. As an example of applications, the Ru@UCNPs nanoprobe is loaded onto test papers for semiquantitative HOCl detection without any interference from the background fluorescence. The application of Ru@UCNPs for background‐free detection and visualization of HOCl in cells and mice is successfully demonstrated. This research has thus shown that Ru@UCNPs is a selective HOCl‐responsive nanoprobe, providing a new way to detect HOCl and a new strategy to develop novel nanoprobes for in situ detection of various biomarkers in cells and early disgnosis of animal diseases.     

Recent Advances in Nanotechnology for Autophagy Detection

Autophagy is closely related to various diseases, and is a diagnostic and therapeutic target for some diseases. In recent years, tremendous efforts have been made to develop excellent probes for detection of autophagy. Nanostructure‐based probes are interesting and promising approaches for in vivo biological imaging due to their unique structural and functional characteristics, e.g., modulating pharmacokinetics property by biocompatible coatings, multimodality capacity by delivering multiple imaging agents and highly specific targeting by antibody ligands. In this Review, we first introduce recent advancements in the development of nanostructure‐based probes for detection of autophagy, including inorganic hybrid nanomaterials and self‐assembled peptide polymeric nanoparticles. Meanwhile, a nanoprobe based on a “in vivo self‐assembly” strategy is highlighted. The “in vivo self‐assembly” endows nanoprobes with higher accumulation, and longer and better signal stability for in vivo detection of autophagy. Furthermore, this novel strategy could be widely used for biomedical imaging/diagnostics and therapeutics, which would attract more attention to this research area.     

Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.     

Fluorescence Quenching Nanoprobes Dedicated to In Vivo Photoacoustic Imaging and High‐Efficient Tumor Therapy in Deep‐Seated Tissue

Photoacoustic imaging (PAI) and photoacoustic (PA) therapy have promising applications for treating tumors. It is known that the utilization of high‐absorption‐coefficient probes can selectively enhance the PAI target contrast and PA tumor therapy efficiency in deep‐seated tissue. Here, the design of a probe with the highest availability of optical‐thermo conversion by using graphene oxide (GO) and dyes via π–π stacking interactions is reported. The GO serves as a base material for loading dyes and quenching dye fluorescence via fluorescence resonance energy transfer (FRET), with the one purpose of maximum of PA efficiency. Experiments verify that the designed fluorescence quenching nanoprobes can produce stronger PA signals than the sum of the separate signals generated in the dye and the GO. Potential applications of the fluorescence quenching nanoprobes are demonstrated, dedicating to enhance PA contrast of targets in deep‐seated tissues and tumors in living mice. PA therapy efficiency both in vitro and in vivo by using the fluorescence quenching nanoprobes is found to be higher than with the commonly used PA therapy agents. Taken together, quenching dye fluorescence via FRET will provide a valid means for developing high‐efficiency PA probes. Fluorescence quenching nanoprobes are likely to become a promising candidate for deep‐seated tumor imaging and therapy.     

Near‐Infrared Fluorescent Ag2Se–Cetuximab Nanoprobes for Targeted Imaging and Therapy of Cancer

Theranostic nanoprobes integrated with diagnostic imaging and therapy capabilities have shown great potential for highly effective tumor therapy by realizing imaging‐guided drug delivery and tumor treatment. Developing novel high‐performance nanoprobes is an important basis for tumor theranostic application. Here, near‐infrared (NIR) fluorescent and low‐biotoxicity Ag₂Se quantum dots (QDs) have been coupled with cetuximab, a clinical antiepidermal growth factor receptor antibody drug for tumor therapy, via a facile bioconjugation strategy to prepare multifunctional Ag₂Se–cetuximab nanoprobes. Compared with the Ag₂Se QDs alone, the Ag₂Se–cetuximab nanoprobes display faster and more enrichment at the site of orthotopic tongue cancer, and thus present better NIR fluorescence contrast between the tumor and the surrounding regions. At 24 h postinjection, the NIR fluorescence of Ag₂Se–cetuximab nanoprobes at the tumor site is still easily detectable, whereas no fluorescence is observed for the Ag₂Se QDs. Moreover, the Ag₂Se–cetuximab nanoprobes have also significantly inhibited the tumor growth and improved the survival rate of orthotopic tongue cancer‐bearing nude mice from 0% to 57.1%. Taken together, the constructed multifunctional Ag₂Se–cetuximab nanoprobes have achieved combined targeted imaging and therapy of orthotopic tongue cancer, which may greatly contribute to the development of nanotheranostics.     

Sensing of Vimentin mRNA in 2D and 3D Models of Wounded Skin Using DNA‐Coated Gold Nanoparticles

Wound healing is a highly complex biological process, which is accompanied by changes in cell phenotype, variations in protein expression, and the production of active biomolecules. Currently, the detection of proteins in cells is done by immunostaining where the proteins in fixed cells are detected by labeled antibodies. However, immunostaining cannot provide information about dynamic processes in living cells, within the whole tissue. Here, an easy method is presented to detect the transition of epithelial to mesenchymal cells during wound healing. The method employs DNA‐coated gold nanoparticle fluorescent nanoprobes to sense the production of Vimentin mRNA expressed in mesenchymal cells. Fluorescence microscopy is used to achieve temporal detection of Vimentin mRNA in wounds. 3D light‐sheet microscopy is utilized to observe the dynamic expression of Vimentin mRNA spatially around the wounded site in skin tissue. The use of DNA–gold nanoprobes to detect mRNA expression during wound healing opens up new possibilities for the study of real‐time mechanisms in complex biological processes.     

Layered Nanoprobe for Long‐Lasting Fluorescent Cell Label

A long‐lasting particle‐based fluorescent label is designed for extended cell imaging studies. This onion‐like nanoprobe is constructed through layer‐by‐layer fabrication technology. The nanoprobes are assembled with multiple layers of optically quenched polyelectrolytes, the fluorescence signal of which can be released later by intracellular proteolysis. Upon incubation with cells, the assembled nanoprobes are taken up efficiently. The tight packing and layered assembly of the quenched polyelectrolytes slow subsequent intracellular degradation, and then result in a prolonged intracellular fluorescence signal for up to 3 weeks with no noticeable toxicity.     

Plasmofluidics: Merging Light and Fluids at the Micro‐/Nanoscale

Plasmofluidics is the synergistic integration of plasmonics and micro/nanofluidics in devices and applications in order to enhance performance. There has been significant progress in the emerging field of plasmofluidics in recent years. By utilizing the capability of plasmonics to manipulate light at the nanoscale, combined with the unique optical properties of fluids and precise manipulation via micro/nanofluidics, plasmofluidic technologies enable innovations in lab‐on‐a‐chip systems, reconfigurable photonic devices, optical sensing, imaging, and spectroscopy. In this review article, the most recent advances in plasmofluidics are examined and categorized into plasmon‐enhanced functionalities in microfluidics and microfluidics‐enhanced plasmonic devices. The former focuses on plasmonic manipulations of fluids, bubbles, particles, biological cells, and molecules at the micro/nanoscale. The latter includes technological advances that apply microfluidic principles to enable reconfigurable plasmonic devices and performance‐enhanced plasmonic sensors. The article is concluded with perspectives on the upcoming challenges, opportunities, and possible future directions of the emerging field of plasmofluidics.     

Aptamer‐Directed Synthesis of Multifunctional Lanthanide‐Doped Porous Nanoprobes for Targeted Imaging and Drug Delivery

Multifunctional lanthanide‐doped porous nanoparticles are prepared via a facile one‐step solvothermal route by employing aptamers as the biotemplate. The nanoparticles feature excellent aqueous dispersibility and biospecific properties and could work as effective nanoprobes for targeted imaging and drug delivery. With aptamer being in principle available for any kind of target, this synthetic strategy may open the door to a new generation of nanoprobes for bioapplications such as time‐resolved biodetection, multimode bioimaging/biolabeling, and targeted cancer therapy.     

J‐Aggregates of Organic Dye Molecules Complexed with Iron Oxide Nanoparticles for Imaging‐Guided Photothermal Therapy Under 915‐nm Light

Recently, the development of nano‐theranostic agents aiming at imaging guided therapy has received great attention. In this work, a near‐infrared (NIR) heptamethine indocyanine dye, IR825, in the presence of cationic polymer, polyallylamine hydrochloride (PAH), forms J‐aggregates with red‐shifted and significantly enhanced absorbance. After further complexing with ultra‐small iron oxide nanoparticles (IONPs) and the followed functionalization with polyethylene glycol (PEG), the obtained IR825@PAH‐IONP‐PEG composite nanoparticles are highly stable in different physiological media. With a sharp absorbance peak, IR825@PAH‐IONP‐PEG can serve as an effective photothermal agent under laser irradiation at 915 nm, which appears to be optimal in photothermal therapy application considering its improved tissue penetration compared with 808‐nm light and much lower water heating in comparison to 980‐nm light. As revealed by magnetic resonance (MR) imaging, those nanoparticles after intravenous injection exhibit high tumor accumulation, which is then harnessed for in vivo photothermal ablation of tumors, achieving excellent therapeutic efficacy in a mouse tumor model. This study demonstrates for the first time that J‐aggregates of organic dye molecules are an interesting class of photothermal material, which when combined with other imageable nanoprobes could serve as a theranostic agent for imaging‐guided photothermal therapy of cancer.     

Photoacoustic Imaging: Contrast Agents and Their Biomedical Applications

Photoacoustic (PA) imaging as a fast‐developing imaging technique has great potential in biomedical and clinical applications. It is a noninvasive imaging modality that depends on the light‐absorption coefficient of the imaged tissue and the injected PA‐imaging contrast agents. Furthermore, PA imaging provides superb contrast, super spatial resolution, and high penetrability and sensitivity to tissue functional characteristics by detecting the acoustic wave to construct PA images. In recent years, a series of PA‐imaging contrast agents are developed to improve the PA‐imaging performance in biomedical applications. Here, recent progress of PA contrast agents and their biomedical applications are outlined. PA contrast agents are classified according to their components and function, and gold nanocrystals, gold‐nanocrystal assembly, transition‐metal chalcogenides/MXene‐based nanomaterials, carbon‐based nanomaterials, other inorganic imaging agents, small organic molecules, semiconducting polymer nanoparticles, and nonlinear PA‐imaging contrast agents are discussed. The applications of PA contrast agents as biosensors (in the sensing of metal ions, pH, enzymes, temperature, hypoxia, reactive oxygen species, and reactive nitrogen species) and in bioimaging (lymph nodes, vasculature, tumors, and brain tissue) are discussed in detail. Finally, an outlook on the future research and investigation of PA‐imaging contrast agents and their significance in biomedical research is presented.     

Nanotransducers for Near‐Infrared Photoregulation in Biomedicine

Photoregulation, which utilizes light to remotely control biological events, provides a precise way to decipher biology and innovate in medicine; however, its potential is limited by the shallow tissue penetration and/or phototoxicity of ultraviolet (UV)/visible light that are required to match the optical responses of endogenous photosensitive substances. Thereby, biologically friendly near‐infrared (NIR) light with improved tissue penetration is desired for photoregulation. Since there are a few endogenous biomolecules absorbing or emitting light in the NIR region, the development of molecular transducers is essential to convert NIR light into the cues for regulation of biological events. In this regard, optical nanomaterials able to convert NIR light into UV/visible light, heat, or free radicals are suitable for this task. Here, the recent developments of optical nanotransducers for NIR‐light‐mediated photoregulation in medicine are summarized. The emerging applications, including photoregulation of neural activity, gene expression, and visual systems, as well as photochemical tissue bonding, are highlighted, along with the design principles of nanotransducers. Moreover, the current challenges and perspectives in this field are discussed.     

Bio‐Hybrid Cell‐Based Actuators for Microsystems

As we move towards the miniaturization of devices to perform tasks at the nano and microscale, it has become increasingly important to develop new methods for actuation, sensing, and control. Over the past decade, bio‐hybrid methods have been investigated as a promising new approach to overcome the challenges of scaling down robotic and other functional devices. These methods integrate biological cells with artificial components and therefore, can take advantage of the intrinsic actuation and sensing functionalities of biological cells. Here, the recent advancements in bio‐hybrid actuation are reviewed, and the challenges associated with the design, fabrication, and control of bio‐hybrid microsystems are discussed. As a case study, focus is put on the development of bacteria‐driven microswimmers, which has been investigated as a targeted drug delivery carrier. Finally, a future outlook for the development of these systems is provided. The continued integration of biological and artificial components is envisioned to enable the performance of tasks at a smaller and smaller scale in the future, leading to the parallel and distributed operation of functional systems at the microscale.     

Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish

The zebrafish is an important vertebrate model for disease, drug discovery, toxicity, embryogenesis, and neuroscience. In vivo fluorescence microscopy can reveal cellular and subcellular details down to the molecular level with fluorescent proteins (FPs) currently the main tool for zebrafish imaging. However, long maturation times, low brightness, photobleaching, broad emission spectra, and sample autofluorescence are disadvantages that cannot be easily overcome by FPs. Here, a bright and photostable terbium-to-quantum dot (QD) Förster resonance energy transfer (FRET) nanoprobe with narrow and tunable emission bands for intracellular in vivo imaging is presented. The long photoluminescence (PL) lifetime enables time-gated (TG) detection without autofluorescence background. Intracellular four-color multiplexing with a single excitation wavelength and in situ assembly and FRET to mCherry demonstrate the versatility of the TG-FRET nanoprobes and the possibility of in vivo bioconjugation to FPs and combined nanoprobe-FP FRET sensing. Upon injection at the one-cell stage, FRET nanoprobes can be imaged in developing zebrafish embryos over seven days with toxicity similar to injected RNA and strongly improved signal-to-background ratios compared to non-TG imaging. This work provides a strategy for advancing in vivo fluorescence imaging applications beyond the capabilities of FPs.     

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes

In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near‐infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850–1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post‐acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes.     

Development of optical nanoprobes for molecular imaging of reactive oxygen and nitrogen species

Reactive oxygen and nitrogen species (RONS) play important roles in cell signal transduction. However, overproduction of RONS is associated with a series of pathological processes and may disrupt cellular homeostasis, causing oxidative and nitrosative stress. Accurate methods to selectively and specifically monitor RONS in living systems are required to further elucidate the biological functions of these species. Optical imaging possesses high sensitivity, high spatiotemporal resolution, and real-time imaging capability. These qualities are advantageous for the detection of RONS in living systems. This review summarizes the development of optical nanoprobes with near-infrared (NIR) fluorescent, upconversion luminescent, chemiluminescent, or photoacoustic signals for molecular imaging of RONS in living systems. In this review, we discuss the design principles and advantages of RONS-responsive activatable nanoprobes, as well as applications of these optical imaging modalities in different disease models.     

Targetable gold nanorods for epithelial cancer therapy guided by near-IR absorption imaging

Well-designed nanoparticle-mediated, image-guided cancer therapy has attracted interest for increasing the efficacy of cancer treatment. A new class of smart theragnostic nanoprobes employing cetuximab (CET)-conjugated polyethylene glycol (PEG)ylated gold nanorods (CET-PGNRs) is presented; these nanoprobes target epithelial cancer cells using near-infrared light. The cetyltrimethylammonium bromide bilayer on GNRs is replaced with heterobifunctional PEG (COOH-PEG-SH) to serve as a biocompatible stabilizer and to increase specificity. The carboxylated GNRs are further functionalized with CET using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry. To assess the potential of such GNRs, their optical properties, biocompatibility, colloidal stability, in vitro/in vivo binding affinities for cancer cells, absorption imaging, and photothermal therapy effects are investigated. CET-PGNRs exhibit excellent tumor targeting ability and strong potential for simultaneous absorption imaging and photothermal ablation of epithelial cancer cells.     

Optical Imaging Approaches to Monitor Static and Dynamic Cell‐on‐Chip Platforms: A Tutorial Review

Miniaturized laboratories on chip platforms play an important role in handling life sciences studies. The platforms may contain static or dynamic biological cells. Examples are a fixed medium of an organ‐on‐a‐chip and individual cells moving in a microfluidic channel, respectively. Due to feasibility of control or investigation and ethical implications of live targets, both static and dynamic cell‐on‐chip platforms promise various applications in biology. To extract necessary information from the experiments, the demand for direct monitoring is rapidly increasing. Among different microscopy methods, optical imaging is a straightforward choice. Considering light interaction with biological agents, imaging signals may be generated as a result of scattering or emission effects from a sample. Thus, optical imaging techniques could be categorized into scattering‐based and emission‐based techniques. In this review, various optical imaging approaches used in monitoring static and dynamic platforms are introduced along with their optical systems, advantages, challenges, and applications. This review may help biologists to find a suitable imaging technique for different cell‐on‐chip studies and might also be useful for the people who are going to develop optical imaging systems in life sciences studies.