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.     

Retrosynthesis of Tunable Fluorescent Carbon Dots for Precise Long‐Term Mitochondrial Tracking

Mitochondria play a significant role in many cellular processes. Precise long‐term tracking of mitochondrial status and behavior is very important for regulating cell fate and treating mitochondrial diseases. However, developing probes with photostability, long‐term tracking capability, and tunable long‐wavelength fluorescence has been a challenge in mitochondrial targeting. Carbon dots (CDs) as new fluorescent nanomaterials with low toxicity and high stability show increasing advantages in bioimaging. Herein, the mitochondria tracking CDs (MitoTCD) with intrinsic mitochondrial imaging capability and tunable long‐wavelength fluorescence from green to red are synthesized where the lipophilic cation of rhodamine is served as the luminescent center of CDs. Due to the excellent photostability, superior fluorescence properties and favorable biocompatibility, these MitoTCD are successfully used for mitochondrial targeting imaging of HeLa cells in vitro and can be tracked as long as six passages, which is suitable for long‐term cell imaging. Moreover, these MitoTCD can also be used for zebrafish imaging in vivo.     

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.     

In Vivo NIR Fluorescence Imaging,Biodistribution, and Toxicology of Photoluminescent Carbon Dots Produced from Carbon Nanotubes and Graphite

Oxidization of carbon nanotubes by a mixed acid has been utilized as a standard method to functionalize carbon nanomaterials for years. Here, the products obtained from carbon nanotubes and graphite after a mixed‐acid treatment are carefully studied. Nearly identical carbon dot (Cdot) products with diameters of 3–4 nm are produced using this approach from a variety of carbon starting materials, including single‐walled carbon nanotubes, multiwalled carbon nanotubes, and graphite. These Cdots exhibit strong yellow fluorescence under UV irradiation and shifted emission peaks as the excitation wavelength is changed. In vivo fluorescence imaging with Cdots is then demonstrated in mouse experiments, by using varied excitation wavelengths including some in the near‐infrared (NIR) region. Furthermore, in vivo biodistribution and toxicology of those Cdots in mice over different periods of time are studied; no noticeable signs of toxicity for Cdots to the treated animals are discovered. This work provides a facile method to synthesize Cdots as safe non‐heavy‐metal‐containing fluorescent nanoprobes, promising for applications in biomedical imaging.     

Endoplasmic Reticulum–Targeted Fluorescent Nanodot with Large Stokes Shift for Vesicular Transport Monitoring and Long‐Term Bioimaging

Herein, a highly stable aggregation‐induced emission (AIE) fluorescent nanodot assembled by an amphiphilic quinoxalinone derivative‐peptide conjugate, namely Quino‐1‐Fmoc‐RACR (also termed as Q1‐PEP), which exhibits large Stokes shift and an endoplasmic reticulum (ER)‐targeting capacity for bioimaging is reported. It is found that the resulting nanodot can effectively enter the ER with high fluorescent emission. As the ER is mainly involved in the transport of synthesized proteins in vesicles to the Golgi or lysosomes, the Q1‐PEP nanodot with ER‐targeting capacity can be used to monitor vesicular transport inside the cells. Compared to conventional fluorescent dyes with small Stokes shifts, the self‐assembled fluorescent nanodot shows superior resistance to photobleaching and aggregation‐induced fluorescence quenching, and elimination of the spectra overlap with autofluorescence of biosubstrate owning to their AIE‐active and red fluorescence emission characteristics. All these optical properties make the fluorescent nanodot suitable for noninvasive and long‐term imaging both in vitro and in vivo.     

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.     

Förster resonance energy transfer-based biosensors for multiparameter ratiometric imaging of Ca2+ dynamics and caspase-3 activity in single cells

As one of the principal cytoplasmic second messengers, the calcium ion (Ca(2+)) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca(2+) in live cells using noninvasive methods such as genetically encoded biosensors based on F?rster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca(2+) biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca(2+) affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca(2+) in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.     

Light‐Activated Nanoprobes for Biosensing and Imaging

Fluorescent nanoprobes are indispensable tools to monitor and analyze biological species and dynamic biochemical processes in cells and living bodies. Conventional nanoprobes have limitations in obtaining imaging signals with high precision and resolution because of the interference with biological autofluorescence, off‐target effects, and lack of spatiotemporal control. As a newly developed paradigm, light‐activated nanoprobes, whose imaging and sensing activity can be remotely regulated with light irradiation, show good potential to overcome these limitations. Herein, recent research progress on the design and construction of light‐activated nanoprobes to improve bioimaging and sensing performance in complex biological systems is introduced. First, recent innovative strategies and their underlying mechanisms for light‐controlled imaging are reviewed, including photoswitchable nanoprobes and phototargeted nanosystems. Subsequently, a short highlight is provided on the development of light‐activatable nanoprobes for biosensing, which offer possibilities for the remote control of biorecognition and sensing activity in a precise manner both temporally and spatially. Finally, perspectives and challenges in light‐activated nanoprobes are commented.     

Nonbleaching fluorescence of gold nanoparticles and its applications in cancer cell imaging

In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays.     

Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull

Visualization of the brain in its native environment is important for understanding common brain diseases. Herein, bright luminogens with remarkable aggregation-induced emission (AIE) characteristics and high quantum yields of up to 42.6% in the solid state are synthesized through facile reaction routes. The synthesized molecule, namely BTF, shows ultrabright far-red/near-infrared emission and can be fabricated into AIE dots by a simple nanoprecipitation procedure. Due to their high brightness, large Stokes shift, good biocompatibility, satisfactory photostability, and large three-photon absorption cross section, the AIE dots can be utilized as efficient fluorescent nanoprobes for in vivo brain vascular imaging through the intact skull by a three-photon fluorescence microscopy imaging technique. This is the first example of using AIE dots for the visualization of the cerebral stroke process through the intact skull of a mouse with high penetration depth and good image contrast. Such good results are anticipated to open up a new venue in the development of efficient emitters with strong nonlinear optical effects for noninvasive bioimaging of living brain.     

Nanoscale Ultrasound‐Switchable FRET‐Based Liposomes for Near‐Infrared Fluorescence Imaging in Optically Turbid Media

A new approach for fluorescence imaging in optically turbid media centered on the use of nanoscale ultrasound‐switchable FRET‐based liposome contrast agents is reported. Liposomes containing lipophilic carbocyanine dyes as FRET pairs with emission wavelengths located in the near‐infrared window are prepared. The efficacy of FRET and self‐quenching for liposomes with a range of fluorophore concentrations is first calculated from measurement of the liposome emission spectra. Exposure of the liposomes to ultrasound results in changes in the detected fluorescent signal, the nature of which depends on the fluorophores used, detection wavelength, and the fluorophore concentration. Line scanning of a tube containing the contrast agents with 1 mm inner diameter buried at a depth of 1 cm in a heavily scattering tissue phantom demonstrates an improvement in image spatial resolution by a factor of 6.3 as compared with images obtained in the absence of ultrasound. Improvements are also seen in image contrast with the highest obtained being 9% for a liposome system containing FRET pairs. Overall the results obtained provide evidence of the potential the nanoscale ultrasound‐switchable FRET‐based liposomes studied here have for in vivo fluorescence imaging.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

See the Unseen: Red-Emissive Carbon Dots for Visualizing the Nucleolar Structures in Two Model Animals and In Vivo Drug Toxicity

Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cell or an organism. Here, bright red-emissive carbon dots (termed CPCDs) with excitation-independent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine. The CPCDs can achieve wash-free, real-time, long-term, and high-quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals—zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish-related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag⁺), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.     

Multicolor FRET silica nanoparticles by single wavelength excitation

Fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified St?ber synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size, and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift, which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines, and assemblies within living systems.     

A Non-Invasive Nanoprobe for In Vivo Photoacoustic Imaging of Vulnerable Atherosclerotic Plaque

Vulnerable atherosclerotic (AS) plaque is the major cause of cardiovascular death. However, clinical methods cannot directly identify the vulnerable AS plaque at molecule level. Herein, osteopontin antibody (OPN Ab) and NIR fluorescence molecules of ICG co-assembled Ti₃C₂ nanosheets are reported as an advanced nanoprobe (OPN Ab/Ti₃C₂/ICG) with enhanced photoacoustic (PA) performance for direct and non-invasive in vivo visual imaging of vulnerable AS plaque. The designed OPN Ab/Ti₃C₂/ICG nanoprobes successfully realize obvious NIR fluorescence imaging toward foam cells as well as the vulnerable AS plaque slices. After intravenous injection of OPN Ab/Ti₃C₂/ICG nanoprobes into AS model mice, in vivo imaging results show a significantly enhanced PA signal in the aortic arch accumulated with vulnerable plaque, well indicating the remarkable feasibility of OPN Ab/Ti₃C₂/ICG nanoprobes to distinguish the vulnerable AS plaque. The proposed OPN Ab/Ti₃C₂/ICG nanoprobes not only overcome the clinical difficulty to differentiate vulnerable plaque, but also achieve the non-invasively specific in vivo imaging of vulnerable AS plaque at molecule level, greatly promoting the innovation of cardiovascular diagnosis technology.     

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.     

Evaluation of red CdTe and near infrared CdHgTe quantum dots by fluorescent imaging

Non-invasive fluorescent imaging of preclinical animal models in vivo is a rapidly developing field with new emerging technologies and techniques. Quantum dot (QD) fluorescent probes with longer emission wavelengths in red and near infrared (NIR) emission ranges are more amenable to deep-tissue imaging, because both scattering and autofluorescence are reduced as wavelengths are increased. We have designed and synthesized red CdTe and NIR CdHgTe QDs for fluorescent imaging. We demonstrated fluorescent imaging by using CdTe and CdHgTe QDs as fluorescent probes both in vitro and in vivo. Both CdTe and CdHgTe QDs provided sensitive detection over background autofluorescence in tissue biopsies and live mice, making them attractive probes for in vivo imaging extending into deep tissues or whole animals. The studies suggest a basis of using QD-antibody conjugates to detect membrane antigens.     

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.     

Atomic‐Precision Gold Clusters for NIR‐II Imaging

Near‐infrared II (NIR‐II) imaging at 1100–1700 nm shows great promise for medical diagnosis related to blood vessels because it possesses deep penetration and high resolution in biological tissue. Unfortunately, currently available NIR‐II fluorophores exhibit slow excretion and low brightness, which prevents their potential medical applications. An atomic‐precision gold (Au) cluster with 25 gold atoms and 18 peptide ligands is presented. The Au₂₅ clusters show emission at 1100–1350 nm and the fluorescence quantum yield is significantly increased by metal‐atom doping. Bright gold clusters can penetrate deep tissue and can be applied in in vivo brain vessel imaging and tumor metastasis. Time‐resolved brain blood‐flow imaging shows significant differences between healthy and injured mice with different brain diseases in vivo. High‐resolution imaging of cancer metastasis allows for the identification of the primary tumor, blood vessel, and lymphatic metastasis. In addition, gold clusters with NIR‐II fluorescence are used to monitor high‐resolution imaging of kidney at a depth of 0.61 cm, and the quantitative measurement shows 86% of the gold clusters are cleared from body without any acute or long‐term toxicity at a dose of 100 mg kg^?1.