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Fluorescence imaging in the second infrared windows (1000–1700 nm) has emerged as a promising approach to tumor diagnostic. However, the currently available NIR-II imaging agents are based on “alwa...

Fluorescence imaging is a noninvasive and dynamic real-time imaging technique; however, it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation. The recently developed ultrasound-controlled fluorescence (UCF) imaging is a novel imaging technique that can overcome this bottleneck. Previous studies suggested that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo. Here, this review highlights the recent advances (2015–2020) in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues. The imaging performances of various UCF systems, including the signal-to-noise ratio, imaging resolution, and imaging depth, are specifically discussed. In addition, the challenges and prospects are highlighted. With continuously increasing research interest in this field and emerging multidisciplinary applications, UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly, which is expected to have a far-reaching impact on disease surveillance and/or therapy.

OBJECTIVE Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is nowadays widely applied for improved resection of glioblastomas (GBMs). Initially, pretreatment with dexamethasone was considered to be essential for optimal fluorescence effect. However, recent studies reported comparably high rates of visible fluorescence in GBMs despite absence of dexamethasone pretreatment. Recently, the authors proposed fluorescence lifetime imaging (FLIM) for the quantitative analysis of 5-ALA–induced protoporphyrin IX (PpIX) accumulation. The aim of this study was thus to investigate the influence of dexamethasone on visible fluorescence and quantitative PpIX accumulation. METHODS The authors prospectively analyzed the presence of visible fluorescence during surgery in a cohort of patients with GBMs. In this study, patients received dexamethasone preoperatively only if clinically indicated. One representative tumor sample was collected from each GBM, and PpIX accumulation was analyzed ex vivo by FLIM. The visible fluorescence status and mean FLIM values were correlated with preoperative intake of dexamethasone. RESULTS In total, two subgroups with (n = 27) and without (n = 20) pretreatment with dexamethasone were analyzed. All patients showed visible fluorescence independent from preoperative dexamethasone intake. Furthermore, the authors did not find a statistically significant difference in the mean FLIM values between patients with and without dexamethasone pretreatment (p = 0.097). CONCLUSIONS In this first study to date, the authors found no significant influence of dexamethasone pretreatment on either visible 5-ALA fluorescence during GBM surgery or PpIX accumulation based on FLIM. According to these preliminary data, the authors recommend administering dexamethasone prior to fluorescence-guided surgery of GBMs only when clinically indicated.

Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.

To test the hypothesis that the microviscosity changes of Endoplasmic Reticulum (ER) can be a useful indicator of ferroptosis promoted by ER Stresses (ERS), a new ER targeting viscosity rotor, L-Vis-1 was developed and applied in the quantitation of viscosity by FLIM imaging in live cells. The FLIM imaging exhibited an excellent resolution almost as good as the corresponding confocal imaging, more significantly, during ferroptosis processes promoted by different types of ERS, the viscosity increases were clearly monitored by FLIM of L-Vis-1 within ER, which has not been demonstrated before.

The small molecular second near-infrared (NIR-II, 1000-1700 nm) dye-based nanotheranostics can concurrently combine deep-tissue photodiagnosis with in situ phototherapy, which occupies a vital position in the early detection and precise treatment of tumors. However, the development of small molecular NIR-II dyes is still challenging due to the limited electron acceptors and cumbersome synthetic routes. Herein, we report a novel molecular electron acceptor, boron difluoride formazanate (BDF). Based on BDF, a new small molecular NIR-II dye BDF1005 is designed and synthesized with strong NIR-I absorption at 768 nm and bright NIR-II peak emission at 1034 nm. In vitro and in vivo experiments demonstrate that BDF1005-based nanotheranostics can be applied for NIR-II fluorescence imaging-guided photothermal therapy of 4T1 tumor-bearing mice. Under 808 nm laser irradiation, tumor growth can be effectively inhibited. This work opens up a new road for the exploitation of NIR-II small molecular dyes for cancer phototheranostics.

Cancer is one of the leading causes of human death around the world. Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is an emerging light-triggered cancer treatment and shows the advantages of non-invasiveness and low side effects. The design and preparation of efficient phototherapeutic agents are of great significance for phototherapy. Diketopyrrolopyrrole (DPP) is a small molecular organic dye featuring outstanding photophysical properties, facile tuning of structures and properties, and excellent photostability; thus, phototherapeutic agents based on organic small molecular DPP derivatives have attracted significant research attention for not only phototherapy but also photodiagnosis of fluorescence imaging (FLI) and photoacoustic imaging (PAI). This review summarizes the recent progress of various DPP-based organic small molecules on phototheranostics during the last five years. The molecular structure design and their phototheranostics performances are discussed in detail, as will be of great help for further creation of DPP-based phototheranostics.

Controllable self-assembly of photonic molecules for precise biomedicine is highly desirable but challenging to prepare multifunctional nano-phototheranostics. Herein, we developed a generic self-assembly approach to design nano-phototheranostics that provides NIR-II fluorescence imaging and phototherapy. We first designed and synthesized two amphiphilic photonic molecules, PEG2000-IR806 and BODIPY. Then, we prepared the co-self-assembled phototheranostic agents, PEG2000-IR806/BODIPY nanoparticles (PIBY NPs). The morphology of the PIBY NPs is controllable by adjusting the ratio of PEG2000-IR806 and BODIPY during self-assembly. The NIR-II fluorescence properties and phototherapy capability of the PIBY NPs were demonstrated in vitro and in vivo. By tuning the ratio of PEG2000-IR806 and BODIPY, the PIBY NPs showed various morphologies (e.g. spherical nanoparticles, nanovesicles and rod-like nanoparticles). The PEG2000-IR806 plays two roles in the co-self-assemblies, one is second near-infrared (NIR-II, 1000-1700 nm) agent, the other is the surfactant for BODIPY encapsulation. The phototherapeutic PIBY NPs all show bright NIR-II fluorescence and effective phototherapeutic (photothermal and photodynamic) properties, which are attributed to IR806 and BODIPY, respectively. The driving force of the self-assembly can be attributed to the electrostatic interaction between NIR806 and BODIPY and their hydrophobicity. The rod-like PIBY NPs (rPIBY NPs) demonstrated a low half inhibitory concentration (IC50) of 3.96 μg/mL on U87MG cells. The NIR-II imaging showed the accumulation of rPIBY NPs in the tumor region. After systemic injection of rPIBY NPs at low dose (0.5 mg/kg), the tumor growth was greatly inhibited upon laser irradiation without noticeable side effects. This study provides a generic self-assembly approach to fabricate NIR-II imaging and phototherapeutic platform for cancer phototheranostics. STATEMENT OF SIGNIFICANCE: Nanophototheranostics providing NIR-II fluorescence imaging and phototherapy are expected to play a critical role in modern precision medicine. Controllable self-assembly of optical molecules for the fabrication of efficient nanophototheranostics is highly desirable but challenging. This work reports for the first time the co-assembly of a NIR-II imaging contrast agent and a phototherapeutic agent to yield nanophototheranostics with various morphologies. The design of molecular co-assembly with complementary optical functions can be a generic method for future the development of phototheranostics.

The development of nanomedicine has recently achieved several breakthroughs in the field of cancer treatment; however, biocompatibility and targeted penetration of these nanomaterials remain as limitations, which lead to serious side effects and significantly narrow the scope of their application. The self-assembly of intermediate filaments with arginine–glycine–aspartate (RGD) peptide (RGD-IFP) was triggered by the hydrophobic cationic molecule 7-amino actinomycin D (7-AAD) to synthesize a bifunctional nanoparticle that could serve as a fluorescent imaging probe to visualize tumor treatment. The designed RGD-IFP peptide possessed the ability to encapsulate 7-AAD molecules through the formation of hydrogen bonds and hydrophobic interactions by a one-step method. This fluorescent nanoprobe with RGD peptide could be targeted for delivery into tumor cells and released in acidic environments such as endosomes/lysosomes, ultimately inducing cytotoxicity by arresting tumor cell cycling with inserted DNA. It is noteworthy that the RGD-IFP/7-AAD nanoprobe tail-vein injection approach demonstrated not only high tumor-targeted imaging potential, but also potent antitumor therapeutic effects in vivo. The proposed strategy may be used in peptide-driven bifunctional nanoparticles for precise imaging and cancer therapy.

STUDY QUESTION Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts? SUMMARY ANSWER FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts. WHAT IS KNOWN ALREADY Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts. STUDY DESIGN, SIZE, DURATION This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini–Hochberg’s false discovery rate, at a q-value of 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04). LIMITATIONS, REASONS FOR CAUTION This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis. WIDER IMPLICATIONS OF THE FINDINGS These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. There are no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A

STUDY QUESTION Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? SUMMARY ANSWER FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts. WHAT IS KNOWN ALREADY Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. STUDY DESIGN, SIZE, DURATION We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis. PARTICIPANTS/MATERIALS, SETTING, METHODS Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P MAIN RESULTS AND THE ROLE OF CHANCE We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P LIMITATIONS, REASONS FOR CAUTION Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy. WIDER IMPLICATIONS OF THE FINDINGS This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality. STUDY FUNDING/COMPETING INTEREST(S) Supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. TRIAL REGISTRATION NUMBER N/A.

The demand for in-situ detection of latent fingerprints (LFPs) in ways of high sensitivity, high selectivity, high contrast, low cost and user-friendly is still urgent. To overcome this challenge, a moisture-stable, red-emitting fluoride phosphor K3AlF6:Mn4+ (KAF:Mn4+) with an organic hydrophobic skin was prepared. The phosphor has a uniform and superfine morphology with excellent luminescence properties. More importantly, this non-ultraviolet (UV) or non-near infrared (NIR) induced phosphor was proved to be an ideal fluorescent label for LFP imaging, which is both friendly for touch DNA analysis and compatible to forensic light sources. The well-defined ridge details with little background interference on various surfaces were presented by the oleic acid (OA) modified KAF:Mn4+ (KAF:Mn4+-OA) phosphor in few seconds using the powder dusting method. To confirm the high selectivity of KAF:Mn4+-OA for LFP imaging, an efficient quantitative evaluation method is proposed with the aid of ImageJ & Origin software. Due to the superiority of the Mn4+-doped fluoride for the rapid imaging of LFPs in terms of low-cost, high compatibility and good availability, it is expected to be a promising candidate for forensic science as well as fluorescence imaging in other fields instead of rare earth luminescent materials.

As an essential part in the toolbox of super-resolution microscopy, stimulated emission depletion (STED) nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging. Among the applied STED fluorophores, silicon-substituted rhodamines (SiRs) belong to one of the most extensively employed fluorophores. The carboxy-SiR was favored in STED bioimaging with many advantages, including reliable photostability, cell permeability, tunable fluorogenicity, feasible structural decoration and so on. We reviewed the research of carboxy-SiR in the STED nanoscopy and hopefully this can inspire more efforts in the design and application of STED fluorophores.

Incorporation of multiple functions into one nanoplatform can improve cancer diagnostic efficacy and enhance anti-cancer outcomes. Here, we constructed doxorubicin (DOX)-loaded silk fibroin-based nanoparticles (NPs) with surface functionalization by photosensitizer (N770). The obtained nanotheranostics (N770-DOX@NPs) had desirable particle size (157 nm) and negative surface charge (−25 mV). These NPs presented excellent oxygen-generating capacity and responded to a quadruple of stimuli (acidic solution, reactive oxygen species, glutathione, and hyperthermia). Surface functionalization of DOX@NPs with N770 could endow them with active internalization by cancerous cell lines, but not by normal cells. Furthermore, the intracellular NPs were found to be preferentially retained in mitochondria, which were also efficient for near-infrared (NIR) fluorescence imaging, photothermal imaging, and photoacoustic imaging. Meanwhile, DOX could spontaneously accumulate in the nucleus. Importantly, a mouse test group treated with N770-DOX@NPs plus NIR irradiation achieved the best tumor retardation effect among all treatment groups based on tumor-bearing mouse models and a patient-derived xenograft model, demonstrating the unprecedented therapeutic effects of trimodal imaging-guided mitochondrial phototherapy (photothermal therapy and photodynamic therapy) and chemotherapy. Therefore, the present study brings new insight into the exploitation of an easy-to-use, versatile, and robust nanoplatform for programmable targeting, imaging, and applying synergistic therapy to tumors., Graphical abstract Surface-functionalized silk fibroin-based nanotherapeutics could self-produce oxygen, achieve multimodal image-guided mitochondrial phototherapy, and further exert synergistic therapeutic effects with chemotherapy.Image 1

To minimize unwanted reactions with high concentrations of reduced glutathione (GSH) in the tumor microenvironment (TME) during chemodynamic therapy (CDT), a simple and effective strategy was developed to fabricate a TME stimuli-responsive theranostic nanomedicine (Fe-CD) for fluorescence imaging-guided GSH depletion and cancer therapy by combining fluorescent imaging carbon dots (CD) and Fe(III). Introducing Fe(III) into Fe-CD not only quenched the fluorescence of CD while reacting with and consuming intracellular GSH for fluorescence imaging of the depletion of GSH but also provided a source of metal ions to generate more abundant hydroxyl radicals (•OH) with hydrogen peroxide (H2O2) through the Fenton reaction to improve CDT. Fe-CD showed promising •OH generation under H2O2 to effectively degrade methylene blue in vitro and obviously activate the green fluorescence of the reactive oxygen species (ROS) probe in cells. Benefiting from the fluorescence enhancement in response to TME stimulation, Fe-CD greatly enhanced CDT cytotoxicity while monitoring successful GSH depletion by fluorescence imaging. Fe-CD has the potential to act as a theranostic nanomedicine for fluorescence imaging-guided GSH depletion to amplify CDT.

Despite tremendous advances in gas therapy, there are major concerns about the inevitable concentration of toxicity and the ability to perform real-time tracking of drug delivery. Second near-infrared (NIR-II) window absorbing nanoplatforms hold great promise for precision medicine because of their excellent tissue penetration of light and non-invasive nature. In this study, we engineered an NIR-II laser-activated theranostic agent (named CP-bF@PEG) that was composed of amphiphilic polymers (Pluronic F127, with polyethylene glycol, PEG, moieties) coated with an NIR-II-absorbing conjugated polymer (PTTBBT, CP) and nitric oxide (NO) donor (benzofuroxan, bF), which served as an NIR-II photothermal inducer and NO nanogenerator. Under deep tissue penetration of NIR-II laser irradiation, CP-bF@PEG was found to possess fluorescence imaging ability to accurately identify tumor and excellent photothermal effect. Moreover, CP-bF@PEG could generate NO via glutathione activation in the tumor microenvironment in a controllable manner. This NIR-II-absorbing polymer for high-contrast NIR-II fluorescence imaging-guided precision photothermal therapy achieved synergistic effects with NO therapy, as evidenced by pronounced tumor therapeutic efficacy and few side effects. This nanotheranostic agent is a highly promising candidate for high-contrast NIR-II imaging-guided precision photothermal therapy combined with gas therapy against cancer.

Graphene quantum dots (GQD) have attracted much attention due to their unique properties in biomedical application, such as biosensing, imaging, and drug delivering. However, scale preparing red luminescing GQD is still challenging now. Herein, with the help of electron beam irradiation, a simple, rapid, and efficient up-to-down strategy was developed to synthesize GQD with size of 2.75 nm emitting 610 nm luminescence. GQD were further functionalized with polyethylene glycol (PEG) and exhibited good solubility and biocompatibility. The potential in vivo toxicity of PEGylated GQD could completely be eliminated by the clinic cholesterol-lowering drug simvastatin. PEGylated GQD could selectively accumulate in tumor after intravenous injection as a security, reliable and sensitive tumor fluorescence imaging agent. Therefore, this work presented a new method preparing red luminescing GQD for biomedical application.

Investigation of the intracellular generation of H2O2, one of the most important reactive oxygen species (ROS), is crucial for preventing various diseases since it is closely linked with different physiological and complex cell signaling pathways. Despite the development of various fluorescent probes, the majority of the fluorescent probes cannot move across the nuclear membrane. However, detection of the nuclear level of H2O2 is very important since it can directly cause oxidative DNA damage which ultimately leads to various diseases. Therefore, in this study, p-phenylenediamine based carbon quantum dots (B-PPD CDs) have been synthesized and integrated with 4-formylbenzeneboronic acid as a doping agent for the detection of H2O2. The detection mechanism showed that, upon exposure to H2O2, the fluorescence of the B-PPD CDs was immediately quenched. Further investigation has been done in the in vitro RAW 264.7 cell line by both exogenous and endogenous exposure of H2O2 to demonstrate the feasibility of the method. It is shown successfully that the exogenous presence and endogenous generation of H2O2 in RAW 264.7 cells can be detected using B-PPD CDs. The limit of detection (LOD) was determined to be 0.242 μM. The development of such imaging probes using carbon quantum dots will lead to live-cell imaging as well as ROS detection.

Liver cancer, of which hepatocellular carcinoma (HCC) is the most common form, is one of the most lethal cancers worldwide. The five-year survival rate for HCC is below 9%, which can be attributed to late diagnosis and limited treatment options at the late stage. Therefore, safe and efficient imaging strategies are urgently needed to facilitate HCC diagnosis and stage evaluation. The development of the second near infrared window (NIR-II, 1000–1700 nm) fluorescence imaging offers the advantages of enhanced resolutions, deeper penetration depth, and less autofluorescence compared to traditional NIR-I window (700–900 nm) imaging. Herein, an HCC targeted NIR-II fluorescent probe, GPC-ICG, was developed by labelling a humanized anti-GPC3 monoclonal antibody with indocyanine green (ICG). Compared to the negative control IgG-ICG probe, the GPC3-ICG probe demonstrated specific GPC3 targeting capability in vitro. And for GPC3 positive Huh-7 tumor bearing mice, the GPC3-ICG probe specifically accumulated in subcutaneous xenografts, with a tumor–background ratio (TBR) of up to 3. The NIR-II imaging of mice organs ex vivo also indicated that GPC3-ICG specifically targeted Huh-7 tumor tissue. Overall, GPC3-ICG is a promising NIR-II probe for GPC3 targeted imaging of HCC.

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular interactions within a sample can therefore be investigated independently of the poorly defined and usually unknown concentration of the fluorophores. Starting from a general description of the molecular effects involved in fluorescence, the first part of this article describes typical applications of FLIM in Biology. The second part concentrates on the technical details of FLIM. It summarizes the requirements to a FLIM technique for application in biology, and gives an overview on commonly used FLIM techniques.

A microfluidic device based on the polymerase chain reaction (PCR) technique for genetic diagnostics was designed and fabricated. The continuous-flow microfluidic PCR system was integrated with two heating blocks to increase the temperature in each zone (denaturation, annealing, and extension). To maintain a constant temperature, heat transfer was controlled by a temperature controller system while receiving real-time feedback of a thermocouple attached to the microchannel. The DNA amplification was evaluated by fluorescence imaging through agarose gel electrophoresis. The developed continuous-flow microfluidic PCR chip provides reliable amplification of the target DNA with a real-time system controller while maintaining the temperature.

Indocyanine green (ICG) is a fluorescent dye that is widely used in hepatobiliary surgery for fluorescence contrast. ICG is selectively absorbed by the liver after intravenous injection and then secreted into the bile. ICG's unique catabolism and fluorescence characteristics allow for multiple applications in liver transplantation. We have divided the applications of ICG fluorescence imaging in liver transplantation into: (i) cholangiography, (ii) evaluation of liver transplantation vessels, (iii) liver mapping, and (iv) evaluation of donor liver quality. In this review, we summarize the current status of applications of ICG fluorescence imaging in liver transplantation.

Super‐resolution fluorescence imaging provides critically improved information about the composition, organization, and dynamics of subcellular structures. Quantum dot triexciton imaging (QDTI) has been introduced as an easy‐to‐use sub‐diffraction imaging method that achieves an almost 2‐fold improvement in resolution when used with conventional confocal microscopes. Here, we report an overall 3‐fold increase in lateral and axial resolution compared to conventional confocal microscopes by combining QDTI with state‐of‐the‐art commercial laser scanning microscope systems., We report an overall 3‐fold increase in lateral and axial image resolution for confocal fluorescence microscopy by combining quantum dot triexciton imaging with state‐of‐the‐art commercial laser scanning microscope systems. Based on quantum dots as fluorescent labels, we present an easy‐to‐use super‐resolution method. A single confocal scan with minimal image post‐processing is sufficient to produce a sub‐diffraction image with an image resolution of approx. 80 nm lateral and 200 nm axial.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues. A combination of multiview imaging, structured illumination, reconstruction algorithms and deep-learning predictions realizes spatial- and temporal-resolution improvements in fluorescence microscopy to produce super-resolution images from diffraction-limited input images.

Fluorescence imaging, as one of the important means of biological lesion analysis, is widely used in medical analysis. To improve detection specificity, near-infrared emission fluorescent probes have been developed. Sensitive and selective near-infrared (NIR) fluorescent probes for Hg2+ , which is a heavy metal ion harmful to human health, are urgently needed to investigate the physiological toxicity of Hg2+ . The NIR fluorophore based on the traditional structure of rhodamine was prepared by introducing anthocyanin functional groups, and a rhodamine spiro ring structure was constructed to recognize Hg2+ (CCS-Hg). The probe CCS-Hg demonstrated good selectivity and high detection sensitivity for Hg2+ and the most likely mechanism was verified through theoretical calculations. We applied the probe CCS-Hg in the examination of Hg2+ distribution in living cells by NIR fluorescence imaging. This work provides a promising molecular tool for studying the toxicological effects of mercury ions in cell.

In this study, we synthesized a novel fluorescein isothiocyanate (FITC)-labeled prostate-specific membrane antigen (PSMA) ligand (PSMA-FITC) via the Fmoc solid-phase synthesis method, and the application value of PSMA-FITC in targeted fluorescence imaging of PSMA-positive prostate cancer was evaluated. The PSMA ligand developed based on the Glu-urea-Lys structure was linked to FITC by aminocaproic acid (Ahx) to obtain PSMA-FITC. The new probe was evaluated in vitro and in vivo. Fluorescence microscopy examination of PSMA-FITC in PSMA(+) LNCaP cells, PSMA(−) PC3 cells, and blocked LNCaP cells showed that the binding of PSMA-FITC with PSMA was target-specific. For in vivo optical imaging, PSMA-FITC exhibited rapid 22Rv1 tumor targeting within 30 min of injection, and the highest tumor-background ratio (TBR) was observed 60 min after injection. The TBR was 3.45 ± 0.31 in the nonblocking group and 0.44 ± 0.13 in the blocking group, which was consistent with the in vitro results. PSMA-FITC is a promising probe and has important reference value for the development of PSMA fluorescent probes. In the future, it can be applied to obtain accurate tumor images for radical prostatectomy.

Tumor microenvironment (TME) is the survival environment for tumor cells to proliferate and metastasize in deep tissue. TME contains tumor cells, immune cells, stromal cells and a variety of active molecules including reactive oxygen species (ROS). Inside the TME, ROS regulate the oxidation-reduction (redox) homeostasis and promote oxidative stress. Due to the rapid proliferation ability and specific metabolic patterns of the TME, ROS pervade virtually all complex physiological processes and play irreplaceable roles in protein modification, signal transduction, metabolism, and energy production in various tumors. Therefore, measurements of the dynamically, multicomponent simultaneous changes of ROS in the TME are of great significance to reveal the detailed proliferation and metastasis mechanisms of the tumor. Near-infrared (NIR) and two-photon (TP) fluorescence imaging techniques possess real-time, dynamic, highly sensitive, and highly signal-to-noise ratios with deep tissue penetration abilities. With the rationally designed probes, the NIR and TP fluorescence imaging techniques have been widely used to reveal the mechanisms of how ROS regulates and constructs complex signals and metabolic networks in TME. Therefore, we summarize the design principles and performances of NIR and TP fluorescence imaging of ROS in the TME in the last four years, as well as discuss the advantages and potentials of these works. This Review can provide guidance and prospects for future research work on TME and facilitate the development of antitumor drugs.

Graphene quantum dots (GQDs) have been of high interest due to their size and optical characteristics, which improves when functional groups are added to their borders and defects. In this work, th...

Chemodynamic therapy (CDT) is widely explored for tumor-specific therapy by converting endogenous H2O2 to lethal ·OH to destroy cancer cells. However, ·OH scavenging by glutathione (GSH) and insufficient intratumoral H2O2 levels seriously hinder the application of CDT. Herein, we reported the fabrication of copper ion-doped ZIF-8 loaded with gold nanozymes and doxorubicin hydrochloride (DOX) for the chemotherapy and CDT synergistic treatment of tumors with the assistance of tumor microenvironment (TME)-activated fluorescence imaging. The Cu2+-doped ZIF-8 shell was gradually degraded to release DOX and gold nanoclusters responding to the acidic TME. The fluorescence signal of the tumor region was acquired after the quenched fluorescence of the gold nanoclusters by Cu2+ and DOX by aggregation-induced quenching was turned on because of the interaction of GSH with Cu2+ and the release of free DOX. The Cu2+ ions could deplete the GSH via redox reactions and the generated Cu+ could convert internal H2O2 to ·OH for tumor CDT. The chemotherapeutic effect of DOX was strengthened through drug efflux inhibition and drug sensitivity increase due to the consumption of GSH and ·OH burst. Moreover, DOX could raise the level of H2O2 and augment the effect of CDT. In addition, the fluorescent gold nanoclusters not only served as a peroxidase to convert H2O2 to ·OH but also employed as an oxidase to consume GSH, resulting in the amplification of chemotherapy and CDT. This work presents an approach to construct tumor microenvironment-activated theranostic probes without external stimuli and to achieve the tumor elimination through cascade reactions and synergistic treatment.

Designing intelligent stimuli-responsive nanoplatforms that are integrated with a biological membrane system and nanomaterials to realize efficient imaging and therapy of tumors still remains to be challenging. Herein, we report a unique strategy to prepare redox-responsive yellow fluorescent carbon dot nanoclusters (y-CDCs) loaded with anticancer drug doxorubicin (DOX) and coated with the cancer cell membrane (CCM) for precision fluorescence imaging and homologous targeting chemotherapy of tumors. The y-CDs with a size of 7.2 nm were first synthesized via a hydrothermal method and crosslinked to obtain redox-responsive y-CDCs with a size of 150.0 nm. The formulated y-CDCs were physically loaded with DOX with an efficiency of up to 81.0% and coated with CCM to endow them with antifouling properties, immune escape ability to escape from macrophage uptake, and homologous targeting capability to cancer cells. Within the reductive tumor microenvironment, the y-CDCs with quenched fluorescence can dissociate to form single y-CDs with recovered fluorescence and improved tumor penetration ability and simultaneously release DOX from the "cluster bomb", thus realizing efficient targeted tumor fluorescence imaging and chemotherapy. The designed y-CDCs/DOX@CCM may represent an updated nanomedicine formulation based on CDs for improved theranostics of different types of tumors.

Well-designed second near-infrared (NIR-II) fluorophores are promising in optical diagnosis and therapy of tumors. In this work, we synthesized a donor-acceptor-donor (D-A-D) NIR-II fluorophore named BBTD-BET with dithienylethene as an electron donor and benzobisthiadiazole as an electron acceptor. To the best of our knowledge, this is the first report of using dithienylethene, a typical photochromic molecule, as a building block for NIR-II fluorophores. We studied the geometrical configuration, electronic state, and optical properties of BBTD-BET by both theoretical and experimental means. BBTD-BET had absorption and emission in the NIR-I and NIR-II spectral ranges, respectively. Using PEGylated BBTD-BET as a theranostic agent, we achieved NIR-II fluorescence/photoacoustic (PA) dual-modal imaging and attained high imaging resolution, desired signal-to-noise ratio, and excellent photothermal therapy (PTT) efficacy. After one PTT treatment, the tumors established in mice were eradicated. This work provides a novel organic conjugated molecule integrating NIR-II/PA dual-modal imaging and PTT functionalities that is very promising in the theranostic of tumors.

Aging-induced alterations to the blood-brain barrier (BBB) are increasingly being seen as a primary event in chronic progressive neurological disorders that lead to cognitive decline. With the goal of increasing delivery into the brain in hopes of effectively treating these diseases, a large focus has been placed on developing BBB permeable materials. However, these strategies have suffered from lack of specificity towards regions of disease progression. Here we report on the development of a nanoparticle (C1C2-NP) that targets regions of increased claudin-1 expression that reduces BBB integrity. Using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) we find that C1C2-NP accumulation and retention is significantly increased in brains from 12-month-old mice as compared to non-targeted NPs and brains from 2-month-old mice. Furthermore, we find C1C2-NP accumulation in brain endothelial cells with high claudin-1 expression, suggesting target-specific binding of the NPs, which was validated through fluorescence imaging, in vitro testing, and biophysical analyses. Our results further suggest a role of claudin-1 in reducing BBB integrity during aging and show altered expression of claudin-1 can be actively targeted with NPs. These findings could help develop strategies for longitudinal monitoring of tight junction protein expression changes during aging as well as be used as a delivery strategy for site-specific delivery of therapeutics at these early stages of disease development.

The progression of Alzheimer’s disease (AD) is characterized with the deposition and aggregation of β-amyloid (Aβ). Visualizing Aβ aggregates at high spatial resolution is beneficial for AD diagnosis and treatment. Herein, we designed a new molecule by conjugating corannulene (Cor) with rhodamine B isothiocyanate (Rhb), namely Cor-Rhb, for the nanoscopic imaging and modulating Aβ peptide fibrillation. The low duty cycle, high photon output and sufficient switching cycles enable Cor-Rhb suitable for localization-based nanoscopic fluorescence imaging. We find that Cor-Rhb can inhibit Aβ peptides fibrillization and interact directly with mature fibrils, triggering their disaggregation under light illumination. Noticeably reduced Aβ-mediated cytotoxicity after the addition of Cor-Rhb is also confirmed. These explorations suggest that Cor-Rhb displays great potential as a multifunctional therapeutic agent against amyloid-related diseases, and may largely facilitate a variety of super-resolution based biological applications.

Photothermal therapy (PTT) has inherent advantages in the treatment of hypoxic tumors due to its optically controlled selectivity on tumor ablation and oxygen-independent nature. The subcellular organelle-targeting capability and photothermal conversion efficiency (PCE) at near-infrared (NIR) wavelength are the key parameters in the assessment of the photothermal agent (PTA). Here, we report that carbon dots (CDs) prepared by the hydrothermal treatment of coronene derivatives show a high PCE of 54.7% at 808 nm, which can be attributed to the narrow band gap and the presence of amounts of continuous energy bands on CDs. Moreover, the vibrations in the layered graphite structures of the CDs also increase the rate of nonradiative transition and thus enhance the PCE. Furthermore, the CDs also possess excellent photostability, biocompatibility, and cell penetration capability and could mainly accumulate in the lysosomes. These experiment results have proved that the CDs are suitable as an efficient NIR light-triggered PTA for efficient PTT against cancer.

The nucleolus, the locus of ribosome biogenesis, was found to be the predominant intracellular target of a new fluorescent probe, V-P1. In solution, the probe demonstrated both a selectivity to RNA...

Nonalcoholic fatty liver disease (NAFLD) can cause serious liver damage. Early diagnosis and effective treatment of NAFLD can greatly improve treatment rates. The initiation and development of NAFLD has been closely linked to endoplasmic reticulum (ER) stress, which might cause ER viscosity variations. Therefore, if the internal relationship between ER viscosity and NAFLD is clarified, an effective approach for early diagnosis may result. Herein, we fabricated a novel near-infrared (NIR) fluorescence imaging probe, Er-V, for monitoring ER viscosity through a molecular rotor strategy. Er-V exhibited a strong NIR fluorescence signal (at 626 nm) when the environmental viscosity hindered the rotation of the malononitrile group. Using Er-V, we successfully observed a significant enhancement in viscosity in the liver of mice with NAFLD. Therefore, this imaging method based on Er-V is expected to provide a new approach for early detection and diagnosis of NAFLD.

The development of multifunctional theranostic nano-agents is an important resolution for personalized treatment of cancer. In this work, we synthesized a new kind of gadolinium boride nanoparticles (GBN) by a microwave-assisted chemical etching method, and discovered their optical characteristics including fluorescence imaging and near-infrared (NIR) photothermal conversion capability. Bright greenishyellow fluorescence enabled for intracellular localization, while effective NIR-photothermal conversion supported photothermal therapy (PTT). In vitro and in vivo results indicated that GBN exhibited a superior antitumor performance and high biocompatibility. This study demonstrated a promising multifunctional theranostic nanoplatform for cancer treatment.

Developing high-quality NIR-II fluorophores (emission in 1000–1700 nm) for in vivo imaging is of great significance. Benzothiadiazole-core fluorophores are an important class of NIR-II dyes, yet ongoing limitations such as aggregation-caused quenching in aqueous milieu and non-activatable response are still major obstacles for their biological applications. Here, we devise an activatable nanoprobe to address these limitations. A molecular probe named BTPE-NO2 is synthesized by linking a benzothiadiazole core with two tetraphenylene groups serving as hydrophobic molecular rotors, followed by incorporating two nitrophenyloxoacetamide units at both ends of the core as recognition moieties and fluorescence quenchers. An FDA-approved amphiphilic polymer Pluronic F127 is then employed to encapsulate the molecular BTPE-NO2 to render the nanoprobe BTPE-NO2@F127. The pathological levels of H2O2 in the disease sites cleave the nitrophenyloxoacetamide groups and activate the probe, thereby generating strong fluorescent emission (950~1200 nm) and ultrasound signal for multi-mode imaging of inflammatory diseases. The nanoprobe can therefore function as a robust tool for detecting and imaging the disease sites with NIR-II fluorescent and multispectral optoacoustic tomography (MSOT) imaging. Moreover, the three-dimensional MSOT images can be obtained for visualizing and locating the disease foci. Fluorescent imaging in the second biological window has advantages for in vivo applications. Here, the authors synthesise a molecular nanoprobe which activates upon binding H2O2, generating both strong fluorescent NIR-II emission and ultrasound signal for multi-mode imaging of inflammatory diseases.

A new rod-shaped MoO3-x@YF3:Yb3+,Er3+ integrated carrier for the NIR-II fluorescence imaging and chemotherapy has been successfully prepared by introducing the local surface plasmon resonance (LSPR) effect and regulating the morphology and surface state. The analysis methods such as XRD, SEM, EDX, TEM, UV–Vis-NIR, PL and IR are used to study the structure, morphology, composition and properties. XRD, SEM and TEM analysis shows that the surface of MoO3-x is successfully coated with YF3:Yb3+, Er3+ nanocrystals of well-crystallized orthogonality, and the carrier obtained has a unique rod-like morphology. XPS analysis shows that Mo element exists in two valence states: Mo (VI) and Mo (V). Furthermore, PL analysis shows that the carrier has stronger luminescence intensity of up-conversion and the near-infrared second region (NIR II) under the excitation of 980 nm and 1540 nm, respectively. The drug delivery capacity and release effect of carrier are studied by using doxorubicin (DOX) as an anti-tumor template drug. The results shows that the carrier with unmodified polyethylene glycol (PEG) has strong drug loading capacity and release effect. The cytotoxicity of the carrier is performed using Hela cells. The result show that the cytotoxicity was obviously related to the concentration of MoO3-x@YF3:Yb3+,Er3+ nanoparticles. This work will solve the bottleneck of fluorescence imaging of visual drug delivery carriers, tumor penetration rate and efficient drug delivery.

Shuai Ren, Lina Song, Ying Tian, Li Zhu, Kai Guo, Huifeng Zhang, Zhongqiu Wang Department of Radiology, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, 210029, People’s Republic of ChinaCorrespondence: Shuai Ren; Zhongqiu WangDepartment of Radiology, Jiangsu Province Hospital of Chinese Medicine, Affiliated Hospital of Nanjing University of Chinese Medicine, No. 155 Hanzhong Road, Nanjing, Jiangsu Province, 210029, People’s Republic of ChinaEmail sren001@163.com; zhongqiuwang0815@163.comBackground: Pancreatic cancer (PC) remains a difficult tumor to diagnose and treat. It is often diagnosed as advanced by reason of the anatomical structure of the deep retroperitoneal layer of the pancreas, lack of typical symptoms and effective screening methods to detect this malignancy, resulting in a low survival rate. Emodin (EMO) is an economical natural product with effective treatment and few side effects of cancer treatment. Magnetic nanoparticles (MNPs) can achieve multiplexed imaging and targeted therapy by loading a wide range of functional materials such as fluorescent dyes and therapeutic agents.Purpose: The purpose of this study was to design and evaluate a multifunctional theranostic nanoplatform for PC diagnosis and treatment.Methods: In this study, we successfully developed EMO-loaded, Cy7-functionalized, PEG-coated Fe3O4 (Fe3O4-PEG-Cy7-EMO). Characteristics including morphology, hydrodynamic size, zeta potentials, stability, and magnetic properties of Fe3O4-PEG-Cy7-EMO were evaluated. Fluorescence imaging (FI)/magnetic resonance imaging (MRI) and therapeutic treatment were examined in vitro and in vivo.Results: Fe3O4-PEG-Cy7-EMO nanoparticles had a core size of 9.9 ± 1.2 nm, which showed long-time stability and FI/MRI properties. Bio-transmission electron microscopy (bio-TEM) results showed that Fe3O4-PEG-Cy7-EMO nanoparticles were endocytosed into BxPC-3 cells, while few were observed in hTERT-HPNE cells. Prussian blue staining also confirmed that BxPC-3 cells have a stronger phagocytic ability as compared to hTERT-HPNE cells. Additionally, Fe3O4-PEG-Cy7-EMO had a stronger inhibition effect on BxPC-3 cells than Fe3O4-PEG and EMO. The hemolysis experiment proved that Fe3O4-PEG-Cy7-EMO can be used in vivo experiments. In vivo analysis demonstrated that Fe3O4-PEG-Cy7-EMO enabled FI/MRI dual-modal imaging and targeted therapy in pancreatic tumor xenografted mice.Conclusion: Fe3O4-PEG-Cy7-EMO may serve as a potential theranostic nanoplatform for PC.Keywords: pancreatic cancer, emodin, magnetic nanoparticles, passive targeting