Challenges and Opportunities for Intravital Near-Infrared Fluorescence

Oct 15, 2018 - CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and i-Lab, CAS Center for Excellence in Brain Science, Suzhou ...
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Challenges and Opportunities for Intravital Near-Infrared Fluorescence Imaging Technology in the Second Transparency Window ACS Nano Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 10/15/18. For personal use only.

Chunyan Li and Qiangbin Wang* CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and i-Lab, CAS Center for Excellence in Brain Science, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 China School of Nano Technology and Nano Bionics, University of Science and Technology of China, Hefei 230026 China ABSTRACT: The past decade has witnessed rapid technological development on nanoscale probes and imaging optics in the second near-infrared transparency window (NIR-II, 1000−1700 nm). These methods hold great promise for biomedical applications due to their deep penetration through tissues and high fidelity of images. However, applications of these techniques in biomedical research and translational medicine will require a number of issues to be addressed. In this Perspective, we examine the technical challenges for intravital NIR-II fluorescence imaging technology and discuss where the development of this cutting-edge technique fits in the future.

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infrared window, NIR-II window; Figure 1a); the NIR-II window achieves unprecedented penetration depth and resolution compared to that of the visible and NIR-I regions3 due to less scattering by tissue and negligible autofluorescence in this region. As shown in Figure 1b, although the absorption of water is somewhat stronger in the NIR-II than in the visible and NIR-I regions (Figure 1b), the scattering effect is significantly inhibited (Figure 1c).4 In addition, the autofluorescence originating from endogenous molecules such as flavins, porphyrins, collagens, and exogenous food is mainly located in the visible and NIR-I regions with negligible effect on NIR-II imaging (Figure 1d). Benefiting from these merits, bioimaging in the NIR-II window is expected to be the nextgeneration intravital fluorescence imaging technique. Increasing attention has been focused on the development of novel NIR-II fluorescence nanoprobes, imaging systems, and bioapplications. Single-walled carbon nanotubes (SWCNTs) were first reported as a NIR-II nanoprobe for intravital fluorescence imaging by the Dai group after surface phospholipid− polyethylene glycol functionalization,5 leading to an appreciation of the deeper tissue imaging and higher spatial resolution in comparison with previous fluorescence imaging methods.6−9 However, SWCNTs as NIR-II fluorescence probes for biomedical applications suffer from some inevitable drawbacks: (1) Polydisperse size and morphology. The wide distribution of SWCNTs spanning hundreds of nanometers leads to

n the past few decades, we have witnessed the rapid development of new technologies in biomedical imaging for the early diagnosis, treatment, and prognosis evaluation of diseases. Among various imaging modalities, fluorescence imaging has unique advantages for visualizing the anatomy and function of tissues and organs in living organisms with fast feedback, multiple optical channels, high sensitivity and resolution, and absence of ionizing radiation. However, fluorescence imaging faces a critical challenge in in vivo imaging due to its poor tissue penetration depth.1 Typically, fluorophores with emissions located in the visible range (400− 650 nm) and the first near-infrared (NIR-I) window (650−950 nm) can only realize tissue penetration depths of several millimeters. The propagation of photons in biological tissue needs to overcome absorption and scattering induced by various biological molecules including hemoglobin, fats, and water, which can lead to attenuation of the signal proportional to the depth of the feature of interest, especially in the visible region. The transport mean free path (TMFP) can then be defined as TMFP = 1/[μs × (1 − g ) + μa ] = 1/(μs ′ + μa )

where μs is the tissue’s scattering coefficient, μa is the tissue’s absorption coefficient, g is the anisotropy function defining the degree of forward scattering, and μs′ is the tissue’s reduced scattering coefficient (μs′ = μs × (1 − g)), which depends on multiple scattering processes of photons.2 Based on optical simulations and empirical derivation, a new “biological-tissue transparency window” was found to be located at 1000−1700 nm (denoted as the second near© XXXX American Chemical Society

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Figure 1. (a) Effective attenuation coefficient of various biological components including oxygenated blood, deoxygenated blood, skin, and fatty tissue.3 Reprinted with permission from ref 3. Copyright 2009 Springer Nature. (b) Absorption coefficient of water (μa) in the range of 400−1800 nm. (c) Reduced scattering coefficient (μs′) of different biological tissues and intralipid solution in the range of 400−1700 nm. (d) Autofluorescence spectra of ex vivo mouse liver (black), spleen (red), and heart tissue (blue) under 808 nm excitation.4 Images (b−d) reprinted with permission from ref 4. Copyright 2017 Springer Nature.

diverse pharmacokinetics in vivo, and the needle-like structure causes potential damage to cells and tissues. (2) Broad emission spectrum. Single-walled carbon nanotubes exhibit a broad emission spectrum covering the whole NIR-II region, which prevents multiplex fluorescence imaging without signal overlap. (3) Low quantum yield (QY). The QY of SWCNTs is less than 3% in aqueous solution, which greatly impedes highfidelity imaging from being obtained with high spatiotemporal resolution.10 Thus, it is urgent to develop new NIR-II-emitting nanoprobes for a wide variety of biomedical imaging applications.

CdSe decreases the strain between the core of InAs and the outer shell of ZnSe and therefore improves its QY.11 Jin et al. reported the aqueous synthesis of PbS QDs with glutathione (GSH) as the surface capping ligand by reacting Pb(CH3COO)2 with Na2S in the presence of GSH, exhibiting tunable fluorescence emission from 1000 to 1400 nm.12 Other QDs, including PbSe, InAs, and HgTe, have also been documented as having NIR-II fluorescence emission properties. Note that the above-mentioned NIR-II-emitting QDs usually contain highly toxic components such as Hg, Pb, etc., which significantly limit their practical biological applications. In 2010, our group reported NIR-II-emissive Ag2S QDs by thermal decomposing a single-source precursor of Ag(DDTC) [(C2H5)2NCS2Ag].13 Further, we optimized the solution chemistry by regulating the reactant chemicals, reaction temperature, and time and successfully obtained a series of high-quality Ag2S QDs with QY close to 20% and tunable emission wavelengths from 900 to 1200 nm.14 Because it contains no toxic components and exhibits high chemical stability with a solubility product constant of ∼10−50, Ag2S QDs show reasonable biocompatibility and long-term photostability.15,16 Afterward, using Ag2S QDs as the NIR-II fluorescence probe, we executed extensive biomedical studies, including high signal-to-noise ratio detection of tumors in vivo, dynamic monitoring of tumor growth and real-time evaluation of therapeutic effects, in situ tracking of transplanted stem cells and their regenerative function in vivo, and imaging-guided precise operation of glioma.17−21 The advantages of NIR-II imaging are inspiring increasing research efforts in the NIR-II window. Recently, more NIR-II fluorescence nanoprobes, such as rare-earth nanoparticles,22−24 polymer nanomaterials,25,26 and aggregation-induced-emission (AIE) dots,27 have been reported. After a decade of

The NIR-II window achieves unprecedented penetration depth and resolution compared to that of the visible and NIR-I regions due to less scattering by tissue and negligible autofluorescence in this region. One of the most important developments in NIR-II fluorescence probes has been the use of inorganic semiconductor nanocrystals (also called quantum dots; QDs), which exhibit advantageous optical properties for bioimaging, such as large absorption coefficients across a wide spectral range, narrow-band emissions, enhanced photostability, and high QY. More importantly, these materials possess size- and composition-dependent fluorescence emission properties that can be finely tuned. Banin et al. reported the synthesis of InAs/ CdSe/ZnSe core/shell1/shell2 (CSS) QDs with emission ranging from 885 to 1425 nm, where the intermediate layer of B

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maximum (fwhm) of the nanoprobe. In addition, the present NIR-II nanoprobes are mostly excited in the NIR-I window, which fails to activate nanoprobes in deep tissue imaging due to poor penetration depth. Therefore, a nanoprobe with excitation and emission in the NIR-II window would be beneficial. More attention should be paid to the rational design of nanoprobes and precise modulation of the excitation and emission spectra. (4) Feasible functionalization. For intravital applications of NIR-II fluorescence nanoprobes, good water solubility is important. Although many reports have successfully shown increased solubility by hydrophilic functionalization, the nanoprobe usually loses its original fluorescence intensity due to photon traps introduced in the functionalization process. In addition, the exogenous nanomaterials can easily be recognized by the mononuclear phagocytic system (MPS), which results in unspecific uptake in liver and spleen, etc. Therefore, functionalizing nanoprobes with stealth molecules such as poly(ethylene glycol) to avoid nonspecific uptake in MPS should be taken into account. In addition, a specific biomolecule is required to realize the target recognition for in vivo imaging. Hence, a facile strategy to endow the nanoprobe with water solubility and targeting functionality while maintaining its fluorescence properties is important. In Vivo Second Near-Infrared Window Imaging System. As an emerging technology, the application of NIRII imaging in small animal models suffers from the absence of a commercially available NIR-II imaging system and thus remains at an early stage. Classical fluorescence imaging takes advantages of emission wavelengths from 400 to 900 nm, and the detector used for in vivo imaging systems is a siliconbased charge-coupled device (CCD) or complementary metal oxide semiconductors (CMOS) camera, which is unusable for NIR-II imaging studies.29 The emergence of shortwave infrared (SWIR) cameras based on semiconductor alloys with narrower band gaps such as InGaAs and HgCdTe, however, has enabled NIR-II fluorescence imaging technology. The Dai group developed a simple NIR-II fluorescence imaging facility placed on an optical platform. A liquidnitrogen-cooled, 320 × 256 pixel two-dimensional InGaAs array was employed to collect photons in NIR-II and acquire fluorescence images. The excitation light was provided by an 808 nm diode laser coupled to a 4.5 mm focal length collimator and filtered by an 850 nm short-pass filter and a 1000 nm short-pass filter. Compared with other fluorescent imaging systems, this custom-built system provides a powerful tool with deep penetration and high resolution due to greatly reduced scattering by tissues and low autofluorescence in the NIR-II region. Subsequently, for individual studies, some groups also attempted to set up their own experimental apparatuses, which were frequently thwarted due to the lack of instrumental knowledge and optical expertise. A commercial NIR-II imaging instrument would be welcomed by the NIR-II fluorescence research community. As a successful trial for the commercialization of NIR-II imaging technology, Suzhou NIROptics Technologies Co., Ltd., in China offers affordable instruments for NIR-II imaging studies and has greatly promoted the booming development in this field. However, current NIR-II imaging systems for biomedical applications still face technical challenges. First, obtaining a large-area, highquality chip of SWIR focal plane array (FPA) remains difficult. A 640 × 512 pixel FPA remains the most popular detector available today, which limits high-resolution imaging. Second,

One of the most important developments in NIR-II fluorescence probes has been the use of inorganic semiconductor nanocrystals, which exhibit advantageous optical properties for bioimaging. development, scientists have now developed a multitude of intravital NIR-II fluorescence imaging technologies for diversified research, and some important biomedical applications have been achieved. However, NIR-II fluorescence imaging technology is still in its infancy. In this Perspective, we discuss some important issues surrounding the promise of more powerful NIR-II imaging technologies for more advanced translational medicine applications.

TECHNICAL CHALLENGES Design and Optimization of Novel Nanoprobes Emitting in the Second Near-Infrared Window. Numerous fluorescent materials, including inorganic and organic nanomaterials, have been developed as nanoprobes for biological labeling and imaging. However, most of these materials have fluorescence emissions below 900 nm. To date, only a handful of NIR-II fluorescent nanomaterials have been developed, such as SWCNTs, Ag2S QDs, Ag2Se QDs, rareearth doped NPs, etc. To expand the present arsenal for further biomedical applications, nanoprobes should meet the following requirements: (1) Biocompatibility. The first and foremost issue that hinders clinical applications of NIR-II fluorescence nanoprobes is concerns about their toxicity. Although a number of existing NIR-II nanoprobes exhibit outstanding imaging performance, the biocompatibility issues including the chemical components, chemical stability, and morphology features of the nanoprobes should be carefully considered in further preclinical imaging studies and clinical utility. Therefore, when designing new nanoprobes, researchers should strive for the nanoprobes to contain no heavy-metal elements, have high chemical stability, and have explicit pharmacokinetics. (2) High fluorescence quantum yield and high photostability. According to the Rose criterion,28 high fluorescence QY can greatly improve the signal-to-noise ratio (SNR) and temporal resolution of in vivo imaging, avoiding the loss of dynamic bioinformation, as well as reduce the dosage of the nanoprobes administrated in vivo. To date, most of the reported NIR-II fluorescence probes have a QY below 20%, leaving plenty of room for improvement. Resistance to photobleaching and blinking capabilities will also affect the imaging quality and data accuracy, especially in the cases of continuous long-term imaging. Thus, it is an urgent need to develop new NIR-II fluorescence nanoprobes with high fluorescence QY and photostability. (3) Tunable excitation and emission wavelengths. Due to the complexity of living organisms, real-time monitoring or evaluation of multiplexed events in vivo is more important than single-function analysis. Thus, multiplexed fluorescence imaging using different nanoprobes with distinguishable fluorescence emission is necessary. Crosstalk between various emission spectra can be avoided with a large Stokes shift and a sharp emission spectrum with narrow full width at halfC

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Figure 2. Conceptual NIR-II fluorescence imaging facility for future biomedical applications.

the readout circuit (ROIC) is a key part of the FPA. For higher-quality NIR-II images, a ROIC with smaller size, higher electron injection efficiency, and higher frame frequency output is needed. However, the electronic and mechanical connection between the chip and ROIC is another technical difficulty. Third, current imaging systems mainly use highpower lasers as excitation sources. The question of how to convert the Gaussian beam to the flattened beam for even illumination of the whole animal (or a significant fraction thereof) is another important issue. In addition, difficulty in obtaining high cutoff filters and high numerical aperture nearinfrared focusing lenses should be considered. For more advanced applications, new imaging equipment should be further exploited: (1) intravital microscopic imaging such as NIR-II fluorescence endoscopy and fiber-optic confocal laserscanning microendoscopy for highly sensitive and specific imaging of the digestive tract or cardiovascular system; (2) intravital NIR-II fluorescence tomography imaging for accurate positioning and quantitative analysis; (3) high-speed NIR-II fluorescence imaging for in vivo real-time dynamic monitoring of important physiological and pathological processes; (4) NIR-II fluorescence-based multiple-modality imaging for simultaneous acquisition of multisource information.

Taking advantage of the imaging penetration depth and clarity in the NIR-II window will provide useful insight into how exogenous and endogenous stem cells participate in the therapeutic process and further facilitate the clinical applications of stem cells. attention for its potential to treat numerous previously incurable diseases. The fate of exogenous stem cells after transplantation, including their distribution, viability, differentiation, and cell clearance, is not well understood; understanding the processes and underlying mechanisms of regeneration will be critical for improving therapeutic efficacy. For endogenous stem cell-based therapy, however, implanting advanced, three-dimensional scaffolds to provide appropriate niches for stem cell recruitment may further aid regenerative medicine. In vivo monitoring of the recruitment behavior of the scaffolds will provide important guidance for endogenous stem cell therapy. Taking advantage of the imaging penetration depth and clarity in the NIR-II window will provide useful insight into how exogenous and endogenous stem cells participate in the therapeutic process and further facilitate the clinical applications of stem cells. In Vivo Sensing. Fluorescence nanoprobes have been executed as biosensors for in vivo tumor detection, tumor microenvironment sensing, and so on. However, the fluorescence “always on” property introduces unspecific signals in the areas through which the nanoprobes travel, which sacrifices the detection precision of target analytes. Therefore, it is desirable to design and to develop new kinds of “off-to-on” nanoprobes for highly specific intravital biosensing. The nanoprobes would begin in the “off” state without fluorescence and then would light up to the “on” state when interacting with the target species; such off-on fluorescence would offer higher specificity and precision in vivo. In addition, the high spectral resolution of NIR-II fluorescence provides extensive opportunities for simultaneous multiplexed detection of different targets in vivo. In Vivo Drug Screening. Drug screening in small animal models is a key step in sophisticated drug discovery. Traditional methods for drug screening depend on the analysis of blood samples and ex vivo organs and tissues, which cannot provide complete information on candidate drugs in vivo due

FUTURE APPLICATIONS FOR SECOND NEAR-INFRARED WINDOW FLUORESCENCE IMAGING TECHNOLOGY Intravital NIR-II fluorescence imaging has opened possibilities for insights into the mysteries of living bodies and mechanisms of diseases. At present, the potential of this novel technology is still at an early stage. With the many above-mentioned technical hurdles to be settled, we envisage the future of intravital NIR-II fluorescence imaging technology and outline some potential applications using this technological marvel (Figure 2). Brain Science Studies. Mapping brain activity is important for understanding how the brain works and the fundamental mechanisms of brain-related diseases. We propose to couple NIR-II fluorescence nanoprobes with membrane protein-based electron acceptors to develop voltage-sensitive fluorescent nanoprobes for simultaneous readouts of neuronal activities, which could record neurons as they operate, with high temporal precision in large quantity. Another direction to be considered is the noninvasive, remote stimulation of specific neurons deep in the brain to alleviate and to treat specific neurological symptoms or diseases. Stem-Cell-Based Regenerative Medicine Studies. Stem-cell-based regenerative medicine has attracted extensive D

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(8) Iverson, N. M.; Barone, P. W.; Shandell, M.; Trudel, L. J.; Sen, S.; Sen, F.; Ivanov, V.; Atolia, E.; Farias, E.; McNicholas, T. P.; Reuel, N.; Parry, N. M. A.; Wogan, G. N.; Strano, M. S. In Vivo Biosensing via Tissue-Localizable Near-Infrared-Fluorescent Single-Walled Carbon Nanotubes. Nat. Nanotechnol. 2013, 8, 873−880. (9) Ghosh, D.; Bagley, A. F.; Na, Y. J.; Birrer, M. J.; Bhatia, S. N.; Belcher, A. M. Deep, Noninvasive Imaging and Surgical Guidance of Submillimeter Tumors Using Targeted M13-Stabilized Single-Walled Carbon Nanotubes. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13948− 13953. (10) Diao, S.; Hong, G. S.; Robinson, J. T.; Jiao, L. Y.; Antaris, A. L.; Wu, J. Z.; Choi, C. L.; Dai, H. J. Chirality Enriched (12,1) and (11,3) Single-Walled Carbon Nanotubes for Biological Imaging. J. Am. Chem. Soc. 2012, 134, 16971−16974. (11) Aharoni, A.; Mokari, T.; Popov, I.; Banin, U. Synthesis of InAs/ CdSe/ZnSe Core/Shell1/Shell2 Structures with Bright and Stable Near-Infrared Fluorescence. J. Am. Chem. Soc. 2006, 128, 257−264. (12) Nakane, Y.; Tsukasaki, Y.; Sakata, T.; Yasuda, H.; Jin, T. Aqueous Synthesis of Glutathione-Coated PbS Quantum Dots with Tunable Emission for Non-Invasive Fluorescence Imaging in the Second Near-Infrared Biological Window (1000−1400 nm). Chem. Commun. 2013, 49, 7584−7586. (13) Du, Y. P.; Xu, B.; Fu, T.; Cai, M.; Li, F.; Zhang, Y.; Wang, Q. B. Near-Infrared Photoluminescent Ag2S Quantum Dots from a Single Source Precursor. J. Am. Chem. Soc. 2010, 132, 1470−1471. (14) Zhang, Y. J.; Liu, Y. S.; Li, C. Y.; Chen, X. Y.; Wang, Q. B. Controlled Synthesis of Ag2S Quantum Dots and Experimental Determination of the Exciton Bohr Radius. J. Phys. Chem. C 2014, 118, 4918−4923. (15) Zhang, Y.; Hong, G. S.; Zhang, Y. J.; Chen, G. C.; Li, F.; Dai, H. J.; Wang, Q. B. Ag2S Quantum Dot: A Bright and Biocompatible Fluorescent Nanoprobe in the Second Near-Infrared Window. ACS Nano 2012, 6, 3695−3702. (16) Zhang, Y.; Zhang, Y. J.; Hong, G. S.; He, W.; Zhou, K.; Yang, K.; Li, F.; Chen, G. C.; Liu, Z.; Dai, H. J.; Wang, Q. B. Biodistribution, Pharmacokinetics and Toxicology of Ag2S Near-Infrared Quantum Dots in Mice. Biomaterials 2013, 34, 3639−3646. (17) Hong, G. S.; Robinson, J. T.; Zhang, Y. J.; Diao, S.; Antaris, A. L.; Wang, Q. B.; Dai, H. J. In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared Region. Angew. Chem., Int. Ed. 2012, 51, 9818−9821. (18) Li, C. Y.; Zhang, Y. J.; Wang, M.; Zhang, Y.; Chen, G. C.; Li, L.; Wu, D. M.; Wang, Q. B. In Vivo Real-Time Visualization of Tissue Blood Flow and Angiogenesis using Ag2S Quantum Dots in the NIRII Window. Biomaterials 2014, 35, 393−400. (19) Hu, F.; Li, C. Y.; Zhang, Y. J.; Wang, M.; Wu, D. M.; Wang, Q. B. Real-Time in Vivo Visualization of Tumor Therapy by a NearInfrared-II Ag2S Quantum Dot-Based Theranostic Nanoplatform. Nano Res. 2015, 8, 1637−1647. (20) Chen, G. C.; Tian, F.; Zhang, Y.; Zhang, Y. J.; Li, C. Y.; Wang, Q. B. Tracking of Transplanted Human Mesenchymal Stem Cells in Living Mice using Near-Infrared Ag2S Quantum Dots. Adv. Funct. Mater. 2014, 24, 2481−2488. (21) Li, C. Y.; Cao, L. M.; Zhang, Y. J.; Yi, P. W.; Wang, M.; Tan, B.; Deng, Z. W.; Wu, D. M.; Wang, Q. B. Preoperative Detection and Intraoperative Visualization of Brain Tumors for More Precise Surgery: A New Dual-Modality MRI and NIR Nanoprobe. Small 2015, 11, 4517−4525. (22) Naczynski, D. J.; Tan, M. C.; Zevon, M.; Wall, B.; Kohl, J.; Kulesa, A.; Chen, S.; Roth, C. M.; Riman, R. E.; Moghe, P. V. RareEarth-Doped Biological Composites as in Vivo Shortwave Infrared Reporters. Nat. Commun. 2013, 4, 2199. (23) Wang, R.; Li, X. M.; Zhou, L.; Zhang, F. Epitaxial Seeded Growth of Rare-Earth Nanocrystals with Efficient 800 nm NearInfrared to 1525 nm Short-Wavelength Infrared Downconversion Photoluminescence for In Vivo Bioimaging. Angew. Chem., Int. Ed. 2014, 53, 12086−12090. (24) Fan, Y.; Wang, P. Y.; Lu, Y. Q.; Wang, R.; Zhou, L.; Zheng, X. L.; Li, X. M.; Piper, J. A.; Zhang, F. Lifetime-Engineered NIR-II

to limited samples, statically acquired data, and different analytical methods. Moreover, traditional methods usually sacrifice the model animals at various time points, meaning that the evaluation of the candidate drugs is performed on different animals and introduces individual variations. In contrast, NIR-II fluorescence imaging technology offers an unprecedented, in situ, and real-time way to collect the spatiotemporal distribution, pharmacokinetics, and pharmacodynamics information on candidate drugs, in which no sacrifice of the animals is required and, thus, individual differences are eliminated. This technology will open a new avenue for highly efficient drug screening at the model-animal level and will facilitate the drugs to clinical practices. In summary, NIR-II fluorescence imaging technology has already become an interdisciplinary research tool for chemistry, materials science, biomedicine, and physics. With the rapid development of this field, we envisage that important and exciting new directions will emerge well beyond the limited pathways described herein.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Qiangbin Wang: 0000-0001-6589-6328 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (2016YFA0101503, 2017YFA0205503), National Natural Science Foundation of China (Nos. 21671198, 21425103, 21501192, 21703282), Natural Science Foundation of Jiangsu Province (BK20170066, BE2016682), Youth Innovation Promotion Association of Chinese Academy of Sciences (CAS), and Open Research Fund Program of the State Key Laboratory of Virology of China (No. 2016IOV004). The authors thank Prof. Guosong Hong at Stanford University for helpful discussions. REFERENCES (1) Weissleder, R.; Pittet, M. J. Imaging in the Era of Molecular Oncology. Nature 2008, 452, 580−589. (2) Ntziachristos, V. Going Deeper than Microscopy: The Optical Imaging Frontier in Biology. Nat. Methods 2010, 7, 603−614. (3) Smith, A. M.; Mancini, M. C.; Nie, S. M. Second Window for in Vivo Imaging. Nat. Nanotechnol. 2009, 4, 710−711. (4) Hong, G. S.; Antaris, A. L.; Dai, H. J. Near-Infrared Fluorophores for Biomedical Imaging. Nat. Biomed. Eng. 2017, 1, 0010. (5) Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H. J. A Route to Brightly Fluorescent Carbon Nanotubes for Near-Infrared Imaging in Mice. Nat. Nanotechnol. 2009, 4, 773−780. (6) Hong, G.; Lee, J. C.; Robinson, J. T.; Raaz, U.; Xie, L.; Huang, N. F.; Cooke, J. P.; Dai, H. Multifunctional in Vivo Vascular Imaging Using Near-Infrared II Fluorescence. Nat. Med. 2012, 18, 1841−1846. (7) Robinson, J. T.; Hong, G. S.; Liang, Y. Y.; Zhang, B.; Yaghi, O. K.; Dai, H. J. In Vivo Fluorescence Imaging in the Second NearInfrared Window with Long Circulating Carbon Nanotubes Capable of Ultrahigh Tumor Uptake. J. Am. Chem. Soc. 2012, 134, 10664− 10669. E

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