MINIMIZING EXCITATION LIGHT LEAKAGE AND MAXIMIZING MEASUREMENT SENSITIVITY FOR MOLECULAR IMAGING WITH NEAR-INFRARED FLUORESCENCE

Near-infraredfluorescence(NIRF)imaging involves the separation of weakfluorescence signals from backscattered excitation light.The measurement sensitivity of current NIRF imaging systems is limited by the excitation light leakage through rejectionfilters.In this contribution,the authors demonstrate that the excitation light leakage can be suppressed upon using appropriatefilter combination and permutations.The excitation light leakage and measurement sensitivity were assessed and compared in this study by computing the transmission ratios of excitation to emission light collected and the signal-to-noise ratios in well-controlled phantom studies with differentfilter combinations and permutations.Using appropriatefilter combinations and permutations,we observe as much as two orders of magnitude reduction in the transmission ratio and higher signal-to-noise ratio.

Evaluation of COC183B2 antibody targeting ovarian cancer by near-infrared fluorescence imaging

Objective: To evaluate the imaging potential of a novel near-infrared(NIR) probe conjugated to COC183 B2 monoclonal antibodies(MAb) in ovarian cancer(OC).Methods: The expression of OC183 B2 antigen in OC was determined by immunohistochemical(IHC) staining using tissue microarrays with the H-score system and immunofluorescence(IF) staining of tumor cell lines.Imaging probes with the NIR fluorescent dye cyanine 7(Cy7) conjugated to COC183 B2 Mab were chemically engineered. OC183 B2-positive human OC cells(SKOV3-Luc) were injected subcutaneously into BALB/c nude mice. Bioluminescent imaging(BLI) was performed to detect tumor location and growth. COC183 B2-Cy7 at 1.1,3.3, 10, or 30 μg were used for in vivo fluorescence imaging, and phosphate-buffered saline(PBS), free Cy7 dye and mouse isotype immunoglobulin G(IgG)-Cy7(delivered at the same doses as COC183 B2-Cy7) were used as controls.Results: The expression of OC183 B2 with a high H-score was more prevalent in OC tissue than fallopian tube(FT) tissue. Among 417 OC patients, the expression of OC183 B2 was significantly correlated with the histological subtype, histological grade, residual tumor size, relapse state and survival status. IF staining demonstrated that COC183 B2 specifically expressed in SKOV3 cells but not HeLa cells. In vivo NIR fluorescence imaging indicated that COC183 B2-Cy7 was mainly distributed in the xenograft and liver with optimal tumor-to-background(T/B)ratios in the xenograft at 30 μg dose. The highest fluorescent signals in the tumor were observed at 96 h postinjection(hpi). Ex vivo fluorescence imaging revealed the fluorescent signals mainly from the tumor and liver. IHC analysis confirmed that xenografts were OC183 B2 positive.Conclusions: COC183 B2 is a good candidate for NIR fluorescence imaging and imaging-guided surgery in OC.

Advances in surgical techniques for gastric cancer:Indocyanine green and near-infrared fluorescence imaging.Is it ready for prime time?

Surgery is still the primary curative treatment for gastric cancer,which includes resection of the tumor with adequate margins and extended lymphadenectomy.In order to improve the operative results and the quality of life of patients,several endeavors have been made toward precision medicine through image-guided surgery,allowing access to real-time intraoperative anatomy and accurate tumor staging.The goal of the surgeon is to achieve a more precise,individualized,and less invasive surgery without compromising oncological efficiency and safety.In this perspective,we have demonstrated the role of indocyanine green(ICG)and near-infrared(NIR)fluorescence imaging method in gastric cancer surgery.This technique may be used to improve localization of the tumor,detection of sentinel lymph nodes(SLN),real-time lymphatic mapping,and blood flow assessment(anastomosis perfusion).

Novel imaging system for positioning of the indocyanine green (ICG) target;visible projection of the near-infrared fluorescence image

Background: Even though NIR fluorescence imaging has many advantages in SLN mapping and cancer detection, NIR fluorescence imaging shows a serious drawback that NIR cannot be detected by the naked eye without any detectors. This limitation further disturbs accurate SLN detection and adequate tumor resection resulting in the presence of cancerous cells near the boundaries of surgically removed tissues. Materials and methods: To overcome the drawback of the conventional NIR imaging method, we suggest a novel NIR imaging system which can make the NIR fluorescence image visible to the naked eye as NIR fluorescence image detected by a video camera is processed by a computer and then projected back onto the NIR fluorescence excitation position with a projector using conspicuous color light. Image processing techniques were used for projection onto the exact position of the NIR fluorescence image. Also, we implemented a phantom experiment to evaluate the performance of the developed NIR fluorescence projection system by use of the ICG. Results: The developed NIR fluorescence projection system was applied in normal mouse model to confirm the usefulness of the system in the clinical field. A BALB/c nude mouse was prepared to be applied in normal mouse model and 0.25 mg/ml stock solution of the ICG was injected through a tail vein of the mouse. From the application in normal mouse model, we could confirm that the injected ICG stayed in the liver of the mouse and verify that the projection system projected the ICG fluorescence image at the exact location of the ICG by performing laparotomy of the mouse. Conclusions: From the application in normal mouse model, we could verify that the ICG fluorescence image was precisely projected back on the site where ICG fluorescence generated. It can be demonstrated that the NIR fluorescence projection system can make it possible to visualize the invisible NIR fluorescence image and to realize that SLN mapping and cancer detection in clinical surgery.

Recent developments of fluorescence imaging technology in the second near-infrared window

Background:Fluorescence bio-imaging in the second near-infrared window(NIR-II FL,1000-1700nm)has great potential in clinical theranostics,which is of great importance providing precise locations of lesions and molecular dynamic actions simultaneously in a single nanoprobe.Methods:T here has been an upsurge of multidisciplinary research focusing on developing functional types of inorganic and organic nanoprobes that can be used for NIR-II FL with the high spatiotemporal resolution,deep tissue penetration,and negligible auto-fluorescence.Results:In this mini-review,we summarize recent progress in inorganic/organic NIR-II FL nanoprobes.We introduce the design and properties of inorganic and organic nanoprobes,in the order of single-walled carbon nanotubes,quantum dots,rare-earth-doped nanoparticles,metal nanoclusters and organic fluorophores,expect to realize precise diagnosis and efficient image-guided therapy.Conclusion:Meanwhile,to elucidate the problems and perspectives,we aim to offer diverse biological applications of inorganic/organic NIR-II FL nanoprobes and accelerate the clinical transformation progress.

Deep learning enhanced NIR-Ⅱ volumetric imaging of whole mice vasculature

Fluorescence imaging through the second near-infrared window(NIR-Ⅱ,1000–1700 nm) allows in-depth imaging.However, current imaging systems use wide-field illumination and can only provide low-contrast 2D information, without depth resolution. Here, we systematically apply a light-sheet illumination, a time-gated detection, and a deep-learning algorithm to yield high-contrast high-resolution volumetric images. To achieve a large Fo V(field of view) and minimize the scattering effect, we generate a light sheet as thin as 100.5 μm with a Rayleigh length of 8 mm to yield an axial resolution of 220 μm. To further suppress the background, we time-gate to only detect long lifetime luminescence achieving a high contrast of up to 0.45 Icontrast. To enhance the resolution, we develop an algorithm based on profile protrusions detection and a deep neural network and distinguish vasculature from a low-contrast area of 0.07 Icontrast to resolve the 100μm small vessels. The system can rapidly scan a volume of view of 75 × 55 × 20 mm3and collect 750 images within 6mins. By adding a scattering-based modality to acquire the 3D surface profile of the mice skin, we reveal the whole volumetric vasculature network with clear depth resolution within more than 1 mm from the skin. High-contrast large-scale 3D animal imaging helps us expand a new dimension in NIR-Ⅱ imaging.

Near-infrared fluorescence sentinel lymph node detection in gastric cancer: A pilot study

AIM: To investigate feasibility and accuracy of near-infrared fluorescence imaging using indocyanine green: nanocolloid for sentinel lymph node (SLN) detection in gastric cancer.METHODS: A prospective, single-institution, phase I feasibility trial was conducted. Patients suffering from gastric cancer and planned for gastrectomy were included. During surgery, a subserosal injection of 1.6 mL ICG:Nanocoll was administered around the tumor. NIR fluorescence imaging of the abdominal cavity was performed using the Mini-FLARE™ NIR fluorescence imaging system. Lymphatic pathways and SLNs were visualized. Of every detected SLN, the corresponding lymph node station, signal-to-background ratio and histopathological diagnosis was determined. Patients underwent standard-of-care gastrectomy. Detected SLNs outside the standard dissection planes were also resected and evaluated.RESULTS: Twenty-six patients were enrolled. Four patients were excluded because distant metastases were found during surgery or due to technical failure of the injection. In 21 of the remaining 22 patients, at least 1 SLN was detected by NIR Fluorescence imaging (mean 3.1 SLNs; range 1-6). In 8 of the 21 patients, tumor-positive LNs were found. Overall accuracy of the technique was 90% (70%-99%; 95%CI), which decreased by higher pT-stage (100%, 100%, 100%, 90%, 0% for respectively Tx, T1, T2, T3, T4 tumors). All NIR-negative SLNs were completely effaced by tumor. Mean fluorescence signal-to-background ratio of SLNs was 4.4 (range 1.4-19.8). In 8 of the 21 patients, SLNs outside the standard resection plane were identified, that contained malignant cells in 2 patients.CONCLUSION: This study shows successful use of ICG:Nanocoll as lymphatic tracer for SLN detection in gastric cancer. Moreover, tumor-containing LNs outside the standard dissection planes were identified.

A near-infrared fluorescent probe for fast and precise imaging of senescent cells and ovarian cancer cells via trackingβ-galactosidase

Aging-related diseases are gradually becoming a major problem with the rapid development of aged population in human society.Although many fluorescent probes have been employed to diagnosis senescence via imaging senescence-associatedβ-galactosidase(SA-β-Gal),which is proved to be closely associated with senescent cells,the similar catalytic effectiveness of enzymatic reaction of ovarian cancer-associatedβ-Gal(OA-β-Gal)will interfere with imaging accuracy.Herein,a near-infrared(NIR)hemicyanine based fluorescent probe HCyXA-βGal was designed for light-up imaging of live cells containingβ-Gal.With the organelle-targeting morpholinyl and positive charge moieties,HCyxA-βGal was successfully applicated to image the difference of enzymatic location in senescent cells and ovarian cancer cells.Furthermore,inspired by the fast response performance,fast and precise imaging of the two cell lines was realized via covering another dimension of fluorescence signal:time-dependent intensity.

Research on the detection of early caries based on hyperspectral imaging

Objective:We applied hyperspectral imaging(HSI)system to distinguish early caries from soundand pigmented areas.It will provide a theoretical basis and technical support,for research anddevelopment of an instrument that could be used for screening and detection of early dentalcaries.Methods:Eighteen extracted human teeth(molars and premolars),with varying degrees ofnatural pathology and no degree of decay involving dentin were obtained.HSI system with awavelength range from 400 to 1000nm was used to obtain images of all 18 teeth containingsound,carious and pigmented areas.We compared the spectra of the wavebands at both 500 nmand 780 nm from the different tooth states,and the reflectance diference bet ween sound versuscarious lesions and sound versus pigmented areas,respectively.Results:There was a slight diference in refectance bet ween carious areas and pigmented areas at500 nm.A substantial difference was additionally noted in refectance bet ween carious areas andpigmented areas at 780 nm.Conclusion:The results have shown that the interference of tooth surface pigment can be elim-inated in the near-infrared(NIR)waveband,and the caries can be effectively identifed from the pigmented areas.Thus,it could be used to detect carious areas of teeth in place of the traditionalvisual inspection method or white light endoscopy.Clinical significance:The NIR difused light signal enables the identification of early caries frompigment and other interference,providing a reasonable detection tool for early detection andearly treatment of teeth diseases.

Fabrication of PdSe2/GaAs Heterojunction for Sensitive Near-Infrared Photovoltaic Detector and Image Sensor Application

In this study,we have developed a high-sensitivity,near-infrared photodetector based on PdSe2/GaAs heterojunction,which was made by transferring a multilayered PdSe2 film onto a planar GaAs.The as-fabricated PdSe2/GaAs heterojunction device exhibited obvious photovoltaic behavior to 808 nm illumination,indicating that the near-infrared photodetector can be used as a self-driven device without external power supply.Further device analysis showed that the hybrid heterojunction exhibited a high on/off ratio of 1.16×10^5 measured at 808 nm under zero bias voltage.The responsivity and specific detectivity of photodetector were estimated to be 171.34 mA/W and 2.36×10^11 Jones,respectively.Moreover,the device showed excellent stability and reliable repeatability.After 2 months,the photoelectric characteristics of the near-infrared photodetector hardly degrade in air,attributable to the good stability of the PdSe2.Finally,the PdSe2/GaAs-based heterojunction device can also function as a near-infrared light sensor.

Mechanism of Dynamic Near-infrared Fluorescence Cholangiography of Extrahepatic Bile Ducts and Applications in Detecting Bile Duct Injuries Using Indocyanine Green in Animal Models

Fluorescence intraoperative cholangiography（IOC） is a potential alternative for identifying anatomical variation and preventing iatrogenic bile duct injuries by using the near-infrared probe indocyanine green（ICG）. However, the dynamic process and mechanism of fluorescence IOC have not been elucidated in previous publications. Herein, the optical properties of the complex of ICG and bile, dynamic fluorescence cholangiography and iatrogenic bile duct injuries were investigated. The emission spectrum of ICG in bile peaked at 844 nm and ICG had higher tissue penetration. Extrahepatic bile ducts could fluoresce 2 min after intravenous injection, and the fluorescence intensity reached a peak at 8 min. In addition, biliary dynamics were observed owing to ICG excretion from the bile ducts into the duodenum. Quantitative analysis indicated that ICG-guided fluorescence IOC possessed a high signal to noise ratio compared to the surrounding peripheral tissue and the portal vein. Fluorescence IOC was based on rapid uptake of circulating ICG in plasma by hepatic cells, excretion of ICG into the bile and then its interaction with protein molecules in the bile. Moreover, fluorescence IOC was sensitive to detect bile duct ligation and acute bile duct perforation using ICG in rat models. All of the results indicated that fluorescence IOC using ICG is a valid alternative for the cholangiography of extrahepatic bile ducts and has potential for measurement of biliary dynamics.

A transferrin receptor targeting dual-modal MR/NIR fluorescent imaging probe for glioblastoma diagnosis

The prognosis of glioblastoma(GBM)remains challenging,primarily due to the lack of a precise,effective imaging technique for comprehensively characterization.Addressing GBM diagnostic challenges,our study introduces an innovative dual-modal imaging that merges near-infrared(NIR)fluorescent imaging with magnetic resonance imaging(MR).This method employs superparamagnetic iron oxide nanoparticies coated with NIR fluorescent dyes,specifically Cyanine 7,and targeted peptides.This synthetic probe facilitates MRI functionality through superparamagnetic iron oxide nanoparticles,provides NIR imaging capability via Cyanine 7 and enhances tumor targeting trough peptide interactions,offering a comprehensive diagnostic tool for GBM.Notably,the probe traverses the blood-brain barrier,targeting GBM in vivo via peptides,producing clear and discermible images in both modalities.Cytotoxicity and histopathology assessments confirm the probe's favorable safet profile.These findings suggest that the dual-modal MRINIR fluorescent imaging probe could revolutionize GBM prognosis and survival rate which can also be extended to other tumors type.

Stable Near-infrared-emitting Radical Nanoparticles for Fluorescence Imaging

Stable neutral luminescent radicals with unpaired electrons exhibit unique spin-allowed doublet-doublet transitions,which has attracted significant attention.Although they are pure organic molecules without metal ions thus thought to have low biological toxicity,the application of luminescent radicals to bioimaging has rarely been reported.Here,a stable radical with efficient near-infrared(NIR)emission and good photostability was designed and synthesized.After being wrapped into nanoparticles,it was applied to cell fluorescence imaging.The cytotoxicity experiments suggested that the nanoparticles have remarkable biocompatibility and excellent stability.An NIR fluorescent signal was successfully observed in the cytoplasm of HCT116 cells.The experimental results gave the first example of NIR emitting radical nanoparticles for cell fluorescence imaging and proved the feasibility of the application of luminescent radicals to fluorescence imaging.

Application of near-infrared fluorescence imaging in theranostics of gastrointestinal tumors

Gastrointestinal cancers have become an important cause of cancer-related death in humans.Improving the early diagnosis rate of gastrointestinal tumors and improving the effect of surgical treatment can significantly improve the survival rate of patients.The conventional diagnostic method is high-definition white-light endoscopy,which often leads to missed diagnosis.For surgical treatment,intraoperative tumor localization and post-operative anastomotic state evaluation play important roles in the effect of surgical treatment.As a new imaging method,near-infrared fluorescence imaging(NIRFI)has its unique advantages in the diagnosis and auxiliary surgical treatment of gastrointestinal tumors due to its high sensitivity and the ability to image deep tissues.In this review,we focus on the latest advances of NIRFI technology applied in early diagnosis of gastrointestinal tumors,identification of tumor margins,identification of lymph nodes,and assessment of anastomotic leakage.In addition,we summarize the advances of NIRFI systems such as macro imaging and micro imaging systems,and also clearly describe the application process of NIRFI from system to clinical application,and look into the prospect of NIRFI applied in the theranostics of gastrointestinal tumors.

Progress and Challenges of Water-soluble NIR-II Organic Fluorophores for Fluorescence Imaging In vivo

The small-molecule fluorophores for the second near-infrared(NIR-II,1000–1700 nm)window have attracted increasing attention in basic scientific research and preclinical practice owing to their deep-photo penetration,minimal physiological toxicity and simplicity of chemical modification.However,most of the reported small-molecule NIR-II fluorophores suffered from poor water solubility,which can easily cause organ toxicity.In addition,the aggregation caused by their poor water solubility in the aqueous solution would also result in weak fluorescence of these NIR-II fluorophores.Thus,it is highly desirable and valuable to develop water-soluble small-molecule NIR-II fluorophores with excellent photophysical properties for high-contrast in vivo imaging.In this review,we summarize the recent research advances in water-soluble small-molecule NIR-II fluorophores and highlight the representative bioimaging applications.Moreover,the potential challenges and perspectives of water-soluble small-molecule NIR-II fluorophores are discussed as well.We anticipate this review can help researchers to grab the latest information of water-soluble small-molecule fluorophores for NIR-II imaging,sequentially boosting their further development.

Palladium-free chemoselective probe for in vivo fluorescence imaging of carbon monoxide

Carbon monoxide(CO)is a vital intracellular gas messenger known for its cytoprotective and homeostatic properties.It plays a pivotal role in a myriad of biological processes.Therefore,the precise detection of CO is of paramount importance in unraveling the intricacies of pathological mechanisms and advancing the development of disease diagnosis.We herein introduce NFCOP,a state-of-the-art near-infrared(NIR)turn-on fluorescence(FL)probe that has been meticulously designed for highly sensitive,swift and selective imaging of CO.The NFCOP response occurred rapidly with CO,within just 10 s,and the calculated detection limit for CO was determined to be 0.32μmol/L.Further investigations conducted at the cellular level and in vivo demonstrated that NFCOP possesses high sensitivity and selectivity for imaging CO.

Rational design of biodegradable semiconducting polymer nanoparticles for NIR-II fluorescence imaging-guided photodynamic therapy

Semiconducting polymer nanoparticles(SPNs)have shown great promise in second near-infrared window(NIR-II)phototheranostics.However,the issue of long metabolic time significantly restricts the clinical application of SPNs.In this study,we rationally designed a biodegradable SPN(BSPN50)for NIR-II fluorescence imaging-guided photodynamic therapy(PDT).BSPN50 is prepared by encapsulating a biodegradable SP(BSP50)with an amphiphilic copolymer F-127.BSP50 is composed of NIR-II fluorescent diketopyrrolopyrrole(DPP)segment and degradable poly(phenylenevinylene)(PPV)segment with the ratio of 50/50.BSPN50 has both satisfactory degradability under myeloperoxidase(MPO)/hydrogen peroxide(H_(2)O_(2))and NIR-II fluorescence emission upon 808 nm laser excitation.Furthermore,BSPN50 shows good photodynamic efficacy under 808 nm laser irradiation.BSPN50 shows a faster degradation rate than BSPN100 which has no PPV segment both in vitro and in vivo.In addition,BSPN50 can effectively diagnose tumor via NIR-II fluorescence imaging and inhibit the tumor growth by PDT.Thus,our study provides a rational approach to construct biodegradable nanoplatforms for efficient tumor NIR-II phototheranostics.

Near-infrared fluorescent probes for imaging of amyloid plaques in Alzheimer's disease

One of the early pathological hallmarks of Alzheimer's disease(AD) is the deposition of amyloid-β(Aβ) plaques in the brain. There has been a tremendous interest in the development of Aβ plaques imaging probes for early diagnosis of AD in the past decades. Optical imaging, particularly near-infrared fluorescence(NIRF) imaging, has emerged as a safe, low cost, real-time, and widely available technique, providing an attractive approach for in vivo detection of Aβ plaques among many different imaging techniques. In this review,we provide a brief overview of the state-of-the-art development of NIRF Aβ probes and their in vitro and in vivo applications with special focus on design strategies and optical, binding, and brain-kinetic properties.

Emerging applications of near-infrared fluorescent metal nanoclusters for biological imaging

Fluorescent metal nanoclusters（MNCs） have recently emerged as a novel kind of promising fluorescent probes for biological imaging because of their ultrasmall core size（〈2 nm）, strong photoluminescence,facile availability and good biocompatibility. In this review, we provide an update on recent advances in the development of near infrared（NIR）-emitting MNCs in terms of synthesis strategies and bioimaging applications. We mainly focus on the utilization of NIR-emitting MNCs（including Au, Ag, Cu and alloy NCs） either as single modal imaging（fluorescence intensity-based imaging, fluorescence lifetime imaging, two-photon imaging） probes or as multimodal imaging（such as NIR fluorescence/X-ray computed tomography/magnetic resonance imaging, NIR fluorescence/photoacoustic imaging/magnetic resonance imaging, NIR fluorescence/single photon emission computed tomography） probes in biological cells and tissues. Finally, we give a brief outlook on the future challenges and prospects of developing NIR-emitting MNCs for bioimaging.

3D fluorescence emission difference microscopy based on spatial light modulator

We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2
vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid,doughnut and z-axis hollow excitation spot,respectively.Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors.Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED.Also,the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads.The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.