Near-infrared Fluorescence Emission Research Articles

Abstract 4117: Imaging hypoxia: Development of a PET-optical smart probe

Abstract Hypoxia is a characteristic trait of many aggressive tumors. Identification of hypoxic tissues is important for establishing patient treatment plans and prognoses. We show progress towards the development of a hypoxia-activated molecular probe that combines near-infrared (NIR) fluorescence and positron emission tomography (PET) imaging. The fluorescence emission enables facile scrutiny of probe function in vitro and positron emission facilitates translation for clinical use. The probe is a small molecule that possesses a nitro-aromatic group which is sensitive to hypoxic environments. The caged smart probe is non-fluorescent due to photo-induced electron transfer from the nitro-aromatic group. Nitroreductase, an enzyme which is upregulated in hypoxic environments, activates the fluorescence by reducing the nitro group, thereby disabling the quenching effect. Wet chemistry techniques were used to develop the probe which was analyzed by UV/Vis and fluorescence spectroscopies. In vitro and in vivo imaging experiments were also performed. The probe demonstrated membrane permeability and lysosomal accumulation in glioblastoma cells. Additional favorable characteristics include NIR emission and low toxicity to cells. The fluorescence was detected in subcutaneous murine cancer models providing evidence of potential clinical translation toward applications like image-guided surgery. In short, the probe was synthesized in good yield from commercially-available materials and showed promising optical characteristics in cells and animal models. Future work involves F-18 radiolabeling of the probe followed by microPET imaging. Citation Format: Jessica L. Klockow, Kenneth S. Hettie, Frederick T. Chin. Imaging hypoxia: Development of a PET-optical smart probe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4117.

Charge compensation effects of Yb3+ on the Bi+: near-infrared emission in PbF2 crystal.

The use of Yb3+ codoping for enhancing the near-infrared (NIR) emission in Bi, Yb codoped PbF2 crystal was investigated for the first time, to the best of our knowledge. The NIR and visible fluorescence emission properties of the as-grown crystals were investigated in detail. It was found that the Yb3+ ion can act as an effective charge compensated ion to bring about the conversion from Bi2+ to Bi+, enhancing the NIR fluorescence emission in Bi:PbF2 crystal. Moreover, the NIR fluorescence emission peaking at around 1090 and 1485nm was demonstrated to be two different Bi+ related centers, Bi(I) and Bi(II), respectively. These results suggest that Bi, Yb codoped PbF2 crystal may become an attractive gain material for developing NIR broadband lasers under pumps of different wavelengths.

Aggregation-Induced Emission-Active Near-Infrared Fluorescent Organic Nanoparticles for Noninvasive Long-Term Monitoring of Tumor Growth.

Effective long-term monitoring of tumor growth is significant for the evaluation of cancer therapy. Aggregation-induced emission-active near-infrared (NIR) fluorescent organic nanoparticles (TPFE-Rho dots) are designed and synthesized for long-term in vitro cell tracking and in vivo monitoring of tumor growth. TPFE-Rho dots display the advantages of NIR fluorescent emission, large Stokes shift (∼180 nm), good biocompatibility, and high photostability. In vitro cell tracing studies demonstrate that TPFE-Rho dots can track SK-Hep-1 cells over 11 generations. In vivo optical imaging results confirm that TPFE-Rho dots can monitor tumor growth for more than 19 days in a real-time manner. This work indicates that TPFE-Rho dots could act as NIR fluorescent nanoprobes for real-time long-term in situ in vivo monitoring of tumor growth.

Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors.

For the highly sensitive near-infrared (NIR) optical detection of epidermal growth factor receptors (EGFRs) expressed on cancer cells, bioluminescence resonance energy transfer (BRET) coupled NIR quantum dots (QDs) are prepared by direct conjugation of his-tagged Renilla luciferase (RLuc) recombinant protein (HisRLuc·GB1) to glutathione-coated CdSeTe/CdS QDs (GSH-QDs). The recombinant protein has two functional groups consisting of a luciferase enzyme and an immunoglobulin binding domain (GB1) of protein G. Recombinant protein (HisRLuc·GB1) conjugated QDs (GB1·RLuc-QDs) show BRET-coupled NIR emission, which results from energy transfer from luciferin to QDs with a high BRET efficiency of ca. 50%. Since the GB1·RLuc-QDs have the GB1 domain at their surface, the QDs have an ability to bind the Fc moiety of immunoglobulin G (IgG). The resulting IgG bound QDs can be used as a molecular imaging probe with NIR fluorescence and BRET-coupled NIR emission. For NIR optical detection of EGFRs on cancer cells, we conjugated anti-EGFR monoclonal antibody to the GB1·RLuc-QDs. Herein, we show that the detection sensitivity of EGFRs by BRET-coupled NIR emission of GB1·RLuc-QDs is at least three times higher than that of the NIR fluorescence of the QDs. The conjugates between anti-EGFR antibody and GB1·RLuc-QDs make it possible to perform BRET-based highly sensitive NIR imaging of EGFRs in living cells.

Fine tuning the emission wavelengths of the 7-hydroxy-1-indanone based nano-structure dyes: Near-infrared (NIR) dual emission generation with large stokes shifts

Near-infrared (NIR) fluorescent dyes have recently gained special attention due to their applications to use as molecular probes for imaging of biological targets and sensitive determination. In this study, photophysical properties of the 7-hydroxy-1-indanone based fluorophors A1, A2, A3, B1, B2 and 3R-B2 (R=CF3, NH2, NO2 and OMe) in the gas and three solution phases were probed using TD-DFT method at PBE0/6-311++G(d,p) and M06-2X/6-311++G(d,p) levels of theory. In addition to structural and photophysical properties as well as ESIPT mechanism of all mentioned molecules, the FC and relaxed potential energy surfaces of B2 and 3R-B2 (R=CF3 and NH2) molecules were explored in gas phase and acetonitrile, cyclohexane and water solvents. It is predicted that the A1, A3 and 3R-B2 chromophores afford normal (615–670nm) and NIR fluorescence emissions (770–940nm; biological window) with the large Stokes shifts of >160 and >300nm, respectively. A good aggrement was found between theoretical and experimental results. In sum, these new types of dyes may render the new approaches for the development of the most efficient NIR fluorescent probes for enhanced image contrast and optimal apparent brightness in biological applications.

Pr3+/Er3+ co-doped tellurite glass with ultra-broadband near-infrared fluorescence emission

Pr3+/Er3+ co-doped tellurite glass with composition 80TeO2-10Na2O-5WO3-5Nb2O5 was synthesized by the conventional melt-quenching technique, and the obtained glass was characterized by the UV/Vis/NIR absorption spectrum, near-infrared fluorescence spectrum, differential scanning calorimeter (DSC) curve, X-Ray diffraction (XRD) pattern and Raman spectrum. Under the 488nm excitation, it was found that Pr3+/Er3+ co-doped tellurite glass could emit an ultra-broadband near-infrared fluorescence which extended from 1200 to 1650nm with full-width at half-maximum (FWHM) of about 236nm, and this ultra-broadband fluorescence was contributed by the multichannel emissions of Pr3+ and 1.53µm band emission of Er3+. The luminescent mechanism and energy transfer processes between Pr3+ and Er3+ ions which are responsible for the observed ultra-broadband fluorescence were analyzed. Meanwhile, optical absorption bands that are assigned with corresponding electronic transitions with respect to Pr3+ and Er3+ ions were identified from the measured absorption spectrum, and based on the optical absorption data, the gain coefficient and important spectroscopic parameters like Judd-Ofelt intensity parameter, spontaneous radiative transition probability, radiative lifetime and branching ratio were calculated to elucidate the radiative properties of doped rare-earth ions. In addition, the DSC curve exhibited the good thermal stability of glass host with ΔT >150°C, Raman spectral study displayed the presence of different vibrational groups and XRD pattern confirmed the amorphous structural nature of the prepared glass. The present results indicate that Pr3+/Er3+ co-doped tellurite glass is promising for the ultra-broadband near-infrared band fiber amplifiers covering the expanded low-loss communication window.

Real-Time Monitoring of Endogenous Cysteine Levels In Vivo by near-Infrared Turn-on Fluorescent Probe with Large Stokes Shift.

Cysteine (Cys), as an important biothiol, plays a major role in many physiological processes like protein synthesis, detoxification and metabolism, and also is closely associated with a variety of diseases; thus the design of novel highly selective and sensitive near-infrared (NIR) fluorescent probes for Cys detection in vivo is of great significance. Herein, we report a selective and sensitive NIR turn-on fluorescent probe (CP-NIR) with large Stokes shift for detecting Cys in vivo. Upon addition of Cys to the solution of the probe, it is absorption wavelength shifts from 550 to 600 nm, accompanying with an obvious enhancement of NIR fluorescence emission centering around 760 nm. This Michael-addition reaction-based probe shows a large Stokes shift (160 nm), low detection limit (48 nM), fast response time, and low toxicity. Moreover, this novel NIR probe with good cell permeability was successfully applied to monitoring endogenous Cys in living cells and in a mouse model.

Chemotherapy-Induced Macrophage Infiltration into Tumors Enhances Nanographene-Based Photodynamic Therapy.

Increased recruitment of tumor-associated macrophages (TAM) to tumors following chemotherapy promotes tumor resistance and recurrence and correlates with poor prognosis. TAM depletion suppresses tumor growth, but is not highly effective due to the effects of tumorigenic mediators from other stromal sources. Here, we report that adoptive macrophage transfer led to a dramatically enhanced photodynamic therapy (PDT) effect of 2-(1-hexyloxyethyl)-2-devinyl pyropheophor-bide-alpha (HPPH)-coated polyethylene glycosylated nanographene oxide [GO(HPPH)-PEG] by increasing its tumor accumulation. Moreover, tumor treatment with commonly used chemotherapeutic drugs induced an increase in macrophage infiltration into tumors, which also enhanced tumor uptake and the PDT effects of GO(HPPH)-PEG, resulting in tumor eradication. Macrophage recruitment to tumors after chemotherapy was visualized noninvasively by near-infrared fluorescence and single-photon emission CT imaging using F4/80-specific imaging probes. Our results demonstrate that chemotherapy combined with GO(HPPH)-PEG PDT is a promising strategy for the treatment of tumors, especially those resistant to chemotherapy. Furthermore, TAM-targeted molecular imaging could potentially be used to predict the efficacy of combination therapy and select patients who would most benefit from this treatment approach. Cancer Res; 77(21); 6021-32. ©2017 AACR.

Benzoyl Peroxide Detection in Real Samples and Zebrafish Imaging by a Designed Near-Infrared Fluorescent Probe

A novel near-infrared fluorescence off-on probe, (E)-3,3-dimethyl-1-propyl-2-(2-(6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3H-indolium (1), is developed and applied to benzoyl peroxide (BPO) detection in real samples and fluorescence imaging in living cells and zebrafish. When arylboronate as the recognition unit is connected to a stable hemicyanine skeleton, the probe is readily prepared, which exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm and high sensitivity with a low detection limit of 47 nM. Upon reaction with BPO, phenylboronic acid pinacol ester is oxidized, followed by hydrolysis and 1,4-elimination of o-quinone methide to release fluorophore. In addtion, the probe displays high selectivity toward BPO over other common substances, which makes it of great potential use in quantitative and simple detection of BPO in wheat flour and antimicrobial agent. More importantly, the probe has been successfully demonstrated for monitoring BPO in living HeLa cells and zebrafish. The probe with superior properties could be of great potential use in other biosystems and in vivo studies.

Near-Infrared Emissive Discrete Platinum(II) Metallacycles: Synthesis and Application in Ammonia Detection.

Two novel discrete organoplatinum(II) metallacycles are prepared by means of coordination-driven self-assembly of a 90° organoplatinum(II) acceptor, cis-(PEt3)2Pt(OTf)2, with two donors, a pyridyl donor, 9,10-di(4-pyridylvinyl)anthracene, and one of two dicarboxylate ligands. Both metallacycles display aggregation-induced emission as well as solvatochromism. More interestingly, both metallacycles exhibit near-infrared fluorescent emission in the solid state and can be used to detect ammonia gas.

The enhanced and broadband near-infrared emission in Pr3+/Nd3+ co-doped tellurite glass

This paper reports an enhanced and broadband near-infrared fluorescence emission in the Pr3+/Nd3+ co-doped tellurite glass, which was prepared using melt-quenching technique. Under the excitation of 488 nm laser diode (LD), three near-infrared emission bands at around 0.9, 1.04 and 1.30 μm from 3P1,0 → 1G4, 1G4→3H4 and 1G4→3H5 radiative transitions respectively were observed in the Pr3+ single-doped glass, and the fluorescence intensities increased further with the introduction of Nd3+ ions, which is mainly attributed to the energy transfers from Nd3+ to Pr3+ emissions. Meanwhile, the spectral overlapping of Pr3+:1G4→3H4 and Nd3+:4F3/2 → 4I11/2 radiative transitions resulted in a broadband emission ranging from 1000 to 1100 nm, whose full-width at half-maximum (FWHM) reached about 66 nm. Additionally, the spectroscopic properties of Nd3+ and Pr3+ ions were analyzed using Judd-Ofelt theory and the thermal stability property of prepared glass was characterized by the differential scanning calorimeter (DSC) measurement, and larger than 134 °C for the difference ΔT(=Tx−Tg) was observed, which indicates its feasibility for later fiber drawing. The enhanced fluorescence and broadband emission indicate that Pr3+/Nd3+ co-doped tellurite glass can be applied in the near-infrared band tunable lasers and broadband optical amplifiers.

Real-time tracking and selective visualization of exogenous and endogenous hydrogen sulfide by a near-infrared fluorescent probe

Rapid capture and selective visualization of hydrogen sulfide (H2S) in living systems has remained challenging. In this work, by employing ortho-aldehyde assisted thiolysis of 2,4-dinitrophenyl (DNP) ether approach and utilizing the dicyanomethylene-4H-chromene as the fluorophore, we developed a new fluorescent probe (E)-2-(2-(4-(2,4-dinitrophenoxy)-3-formylstyryl)-4H-chromen-4-ylidene) malo- nonitrile (2-CHO-OH) for this purpose with improved recognition performances. Probe 2-CHO-OH showed the advantage of quick reaction (8min) with H2S, resulting in a strong near-infrared fluorescence emission (32-fold enhancement) and large Stokes shift (137nm). More importantly, the probe could highly selectively detect H2S from other biological related species including cysteine, homocysteine and glutathione with distinct dual colorimetric and fluorescent signal changes. The detection limit of H2S was estimated as 8.3×10−8M, which was much lower than the intracellular concentration. Thanks to these unique features, 2-CHO-OH has been successfully applied for NIR fluorescence imaging of both the exogenous and endogenous H2S in living cells, demonstrating its value of practical application in biology.

Enhanced broadband near-infrared luminescence from Pr3+-doped tellurite glass with silver nanoparticles

Pr3+-doped tellurite glasses containing metallic silver NPs were synthesized by the conventional melt-quenching technique. Structural, thermal and optical properties of the synthesized glass samples were characterized by X-Ray diffraction (XRD) curves, Raman spectra, differential scanning calorimeter (DSC) curves, transmission electron microscopy (TEM) images, UV/Vis/NIR absorption and near-infrared fluorescence emission spectra. The XRD curves confirmed the amorphous structural nature of the synthesized glasses, the Raman spectra identified the presence of different vibrational groups, the DSC curves verified the good thermal stability, and the TEM images revealed the nucleated silver NPs with average diameter about 10 nm dispersed in the glass matrix and its surface Plasmon resonance (SPR) absorption band was located at around 510 nm. Besides, Judd-Ofelt intensity parameters Ωt(t = 2, 4, 6) and other important spectroscopic parameters like transition probability, radiative lifetime, branching ratio were calculated to evaluate the radiative properties of Pr3+ levels from the measured optical absorption spectra. It was found that Pr3+-doped tellurite glasses could emit an ultra-broadband fluorescence extending from 1250 to 1650 nm under the 488 nm excitation, and this fluorescence emission increased further with the introduction of silver NPs. The enhanced fluorescence was mainly attributed to the increased local electric field around Pr3+ induced by silver NPs. The present results demonstrate that Pr3+-Ag codoped tellurite glass is a promising candidate for the near-infrared band ultra-broadband fiber amplifiers covering the expanded low-loss communication window.

Enzyme-sensitive gemcitabine conjugated albumin nanoparticles as a versatile theranostic nanoplatform for pancreatic cancer treatment

Development of gemcitabine (GEM) nanocarriers as theranostic agents for pancreatic cancer chemotherapy has received extensive attention in recent years. A novel enzyme-sensitive albumin-based GEM delivery nanoplatform was developed in this research by simple conjugation of GEM to human serum albumin (HSA) via cathepsin B cleavable peptide GFLG and then complexing with near-infrared (NIR) dye IR780, forming a HSA-GEM/IR780 complex. The successful preparation of HSA-GEM/IR780 complex was confirmed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS), UV–vis–NIR absorption spectra and fluorescent emission spectra. The in vivo performance of HSA-GEM/IR780 complex was carried out on BxPC-3 pancreatic tumor xenografted mice. As revealed by in vivo NIR imaging, HSA-GEM/IR780 exhibited enhanced accumulation and long-term retention in tumor tissues compared to free IR780. Meanwhile, compared to free GEM, the deamination of GEM nanovectors into inactive 2′,2′-difluorodeoxyuridine (dFdU) can be greatly suppressed, while the concentration of the activated form of GEM (gemcitabine triphosphate, dFdCTP) was significantly increased in tumor tissue, thus exhibiting superior tumor inhibition activity with minimal side effects.

Optical and structural properties of Nd:MgO:LiNbO3 crystal irradiated by 2.8-MeV He ions

We report the optical and structural properties of helium-implanted optical waveguides in Nd:MgO:LiNbO3 laser crystals. The prism-coupling method is used to investigate the dark-mode properties at the wavelength of 632.8 nm. The spontaneous generation of ultraviolet, blue, red, and near-infrared fluorescence emissions is demonstrated under excitation with an 808-nm laser diode. The effects of ion irradiation on the structural properties are characterized using the high-resolution X-ray diffraction technique. The results show that the initial luminescence properties of Nd:MgO:LiNbO3 crystals are slightly modified by irradiation with 2.8 MeV He ions at fluences of 1.5 × 1016 ions/cm2 at room temperature.

Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off–On Probe

A new near-infrared fluorescence off-on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off-on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10-3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.

Mono-dispersed Ba2 +-doped Nano-hydroxyapatite conjugated with near-infrared Cu-doped CdS quantum dots for CT/fluorescence bimodal targeting cell imaging

Nano-hydroxyapatite particles (Ca10(PO4)6(OH)2, nano-HAp), the main inorganic components of human bone and teeth, has attracted much attentions as a kind of biocompatible carrier to construct dual/multi modal bioimaging probes in recent years. However, the currently developed HAp-based probes still have some shortcomings, such as poor mono-dispersion, potential toxicity and none near-infrared emission, which hinder their further applications in the field of bioimaging. In order to solve the above problems and to combine the high space resolution of CT and high sensitivity of fluorescence together, here we developed a novel dual-modal (CT/fluorescence) bioimaging probe based on nano-HAp. Ba2+ as a CT contrast agent was doped into the lattice of nano-HAp, and Cu-doped quantum dots with near-infrared fluorescent emission and hyaluronic acid (HA) were conjugated with the HAp as a fluorescent agent and tumor-targeting ligand individually to realize its near-infrared imaging and tumor-targeting. The as-received Ba2+-doped HAp-QDs-HA conjugates not only well maintained the good dispersion of nano-HAp, but also possessed near-infrared emission at about 700nm, good biocompatibility and photostability. Furthermore, the in-vitro CT and fluorescence cell imaging of the conjugates also exhibited good dual-modal bioimaging and tumor-targeting ability for potential applications in the field of early diagnosis and therapy of cancer.

Interaction of Single-Walled Carbon Nanotubes (SWCNTs) with Photosynthetic Organisms

For years, the distinctive optical and structural properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of promising applications in the field of cell nanobiotechnology. In particular, the synergistic interaction of SWCNTs with photosynthetic organisms has the potential to enhance the native performance of biological machineries, paving the way for novel, renewable, and low cost solutions for light-harvesting, energy conversion, subcellular sensing, and DNA delivery. Recent studies have largely focused on enabling cellular uptake of SWCNTs by engineering the SWCNT surface through non-covalent side-wall functionalizations. Non-covalent functionalization with a rich variety of biomolecules and polymers has been shown to potentially increase SWCNT solubility and membrane translocation while endowing these nanostructures with enhanced biocompatibility. We performed a systematic investigation of the effect of SWCNT functionalization on membrane penetration properties using a novel biological host. A custom-built optical setup was used for the unprecedented characterization of near-infrared fluorescence emissions of internalized SWCNTs. These results were used to inform fuctionalization design rules for SWCNT internalization, and more broadly, present a versatile approach towards imaging internalized SWCNTs in biological systems.

A Novel Near-Infrared Fluorescence Sensor for H<sub>2</sub>O<sub>2</sub> Based on N-Acetyl-L-Cysteine-Capped Gold Nanoparticles

A novel near-infrared fluorescence quenching method has been developed for the determination of hydrogen peroxide based on N-acetyl-L-cysteine-capped gold nanoparticles (NAC-AuNPs) as a fluorescence probe. The prepared gold nanoparticles with the size of about 1.91 nm exhibited strong near-infrared fluorescence emission at 693 nm with excitation wavelength at 450 nm in aqueous solution. The fluorescence intensity of NAC-AuNPs was quenched dramatically by adding hydrogen peroxide. Therefore, it could be used to detect hydrogen peroxide based on the fluorescence quenching intensity was linear with the concentration of hydrogen peroxide. Under the optimal experimental conditions, the linear range and detection limit were 1.0×10-6 –3.0×10-2 mol/L and 1.0×10-7 mol/L, respectively. The possible quenching mechanism was investigated by time-resolved fluorescence spectroscopy. The proposed method was simple, sensitive and showed good repeatability and stability.

A Novel Modality for Functional Imaging in Acute Intervertebral Disk Herniation via Tracking Leukocyte Infiltration.

Inflammation plays a key role in the progression of intervertebral disk (IVD) herniation and associated low back pain. However, real-time spatial diagnosis of inflammation associated with acute disk herniation has not been investigated. We sought to detect local neutrophil and macrophage infiltration near disk herniation via the formyl peptide receptor 1 (FPR1)-mediated molecular imaging in a disk puncture mouse model to elucidate pathophysiological process of disk herniation. Disk herniation was induced in mouse with an established needle puncture procedure. Degenerative change of disk and infiltration of neutrophils and macrophages were detected with Safranin-O, hematoxylin and eosin (H&E), and immunohistochemical staining after injury. FPR1-specific imaging probes cFLFLF-PEG-Cy7 and [99mTc]HYNIC-PEG-cFLFLF were administered systemically to sham and disk injury mice. Leukocyte infiltration was tracked by in vivo near-infrared fluorescence (NIRF) and single-photon emission tomography (SPECT) imaging. The peptide-receptor binding specificity was further investigated with FPR1-/- mice via ex vivo NIRF scan and in vitro binding assays. Safranin-O staining exhibited disorganized disk structure and loss of proteoglycan after puncture. Massive inflammatory cells were observed in the anterior region of punctured annulus in the injury group. The majority of neutrophils were detected at 1 through 3days, while infiltration of macrophages appeared the most at 7days after injury. NIRF and SPECT images revealed preferential accumulation of cFLFLF probes in herniation site in wild-type mice but not in FPR1-/- mice. Binding of the cFLFLF peptide to FPR1 was also observed in RAW 267.4 cells and macrophages isolated from wild-type mice, whereas much less signal was observed in macrophages from FPR1-/- mice. The presence of macrophage infiltration was also detected in human-herniated disk samples by immunohistochemistry. For the first time, leukocyte infiltration around acute disk herniation site was detected directly and non-invasively in a timely fashion using FPR1-targeted molecular imaging modalities. Such functional imaging of disk herniation via infiltrated leukocytes would advance the understanding of etiology and facilitate drug delivery and treatment monitoring of disk herniation.