Non-invasive Near-infrared Fluorescence Imaging Research Articles

Constructing Oxygen-related Defects in Carbon Nanodots with Janus Optical Properties: Noninvasive NIR Fluorescent Imaging and Effective Photocatalytic Therapy.

Noninvasive fluorescence (FL) imaging and high-performance photocatalytic therapy (PCT) are opposing optical properties that are difficult to combine in a single material system. Herein, we report a facile approach to introducing oxygen-related defects in carbon dots (CDs) via post-oxidation with 2-iodoxybenzoic acid (IBX), in which some nitrogen atoms were substituted by oxygen atoms. Unpaired electrons in these oxygen-related defects rearranged the electronic structure of the oxidized carbon dots (ox-CDs), resulting in an emerging near-infrared (NIR) absorption band. These defects not only contributed to enhanced NIR bandgap emission but also acted as trappers for photoexcited electrons to promote efficient charge separation on the surface, leading to abundant photo-generated holes on the ox-CDs surface under visible-light irradiation. Under white LED torch irradiation, the photo-generated holes oxidized hydroxide to hydroxyl radicals in the acidification of the aqueous solution. In contrast, no hydroxyl radicals were detected in the ox-CDs aqueous solution under 730nm laser irradiation, indicating noninvasive NIR FL imaging potential. Utilizing the Janus optical properties of the ox-CDs, the in vivo NIR FL imaging of sentinel lymph nodes around tumors and efficient photothermal enhanced tumor PCT were demonstrated. This article is protected by copyright. All rights reserved.

Spatio-temporally deciphering peripheral nerve regeneration in vivo after extracellular vesicle therapy under NIR-II fluorescence imaging.

Extracellular vesicles (EVs) show potential as a therapeutic tool for peripheral nerve injury (PNI), promoting neurological regeneration. However, there are limited data on the in vivo spatio-temporal trafficking and biodistribution of EVs. In this study, we introduce a new non-invasive near-infrared fluorescence imaging strategy based on glucose-conjugated quantum dot (QDs-Glu) labeling to target and track EVs in a sciatic nerve injury rat model in real-time. Our results demonstrate that the injected EVs migrated from the uninjured site to the injured site of the nerve, with an increase in fluorescence signals detected from 4 to 7 days post-injection, indicating the release of contents from the EVs with therapeutic effects. Immunofluorescence and behavioral tests revealed that the EV therapy promoted nerve regeneration and functional recovery at 28 days post-injection. We also found a relationship between functional recovery and the NIR-II fluorescence intensity change pattern, providing novel evidence for the therapeutic effects of EV therapy using real-time NIR-II imaging at the live animal level. This approach initiates a new path for monitoring EVs in treating PNI under in vivo NIR-II imaging, enhancing our understanding of the efficacy of EV therapy on peripheral nerve regeneration and its mechanisms.

In vivo tracking of bioorthogonally labeled T-cells for predicting therapeutic efficacy of adoptive T-cell therapy

Non-invasive tracking of T-cells may help to predict the patient responsiveness and therapeutic outcome. Herein, we developed bioorthogonal T-cell labeling and tracking strategy using bioorthogonal click chemistry. First, ovalbumin (OVA) antigen-specific cytotoxic T-cells (CTLs) were incubated with N-azidoacetyl-D-mannosamine-tetraacylated (Ac4ManNAz) for incorporating azide (N3) groups on the surface of CTLs via metabolic glycoengineering. Subsequently, azide groups on the CTLs were chemically labeled with near infrared fluorescence (NIRF) dye, Cy5.5, conjugated dibenzylcyclooctyne (DBCO-Cy5.5) via bioorthogonal click chemistry, resulting in Cy5.5-labeled CTLs (Cy5.5-CTLs). The labeling efficiency of Cy5.5-CTLs could be readily controlled by changing concentrations of Ac4ManNAz and DBCO-Cy5.5 in cultured cells. Importantly, Cy5.5-CTLs presented the strong NIRF signals in vitro and they showed no significant changes in the functional properties, such as cell viability, proliferation, and antigen-specific cytolytic activity. In ovalbumin (OVA)-expressing E.G-7 tumor-bearing immune-deficient mice, intravenously injected Cy5.5-CTLs were clearly observed at targeted solid tumors via non-invasive NIRF imaging. Moreover, tumor growth inhibition of E.G-7 tumors was closely correlated with the intensity of NIRF signals from Cy5.5-CTLs at tumors after 2–3 days post-injection. The Cy5.5-CTLs showed different therapeutic responses in E.G-7 tumor-bearing immune-competent mice, in which they were divided by their tumor growth efficacy as ‘high therapeutic response (TR (+))’ and ‘low therapeutic response (TR (-))’. These different therapeutic responses of Cy5.5-CTLs were highly correlated with the NIRF signals of Cy5.5-CTLs at targeted tumor tissues in the early stage. Therefore, non-invasive tracking of T-cells can be able to predict and elicit therapeutic responses in the adoptive T-cell therapy.

Noninvasive Near-Infrared Fluorescence Imaging of the Ureter During Robotic Surgery: A Demonstration in a Porcine Model.

Background: Iatrogenic ureteral injury is one of the feared complications during intrapelvic surgery. There are limited data on the use of novel near-infrared fluorescence (NIRF) imaging dyes for the purpose of noninvasive ureteral visualization in robot-assisted laparoscopic surgery (RALS). In this study, we evaluated the feasibility of NIRF imaging of the ureter using the IRDye® 800BK dye as the fluorescence dye and a robotic platform with Firefly™ technology as an imaging system. Materials and Methods: An intravenous dose of 0.15 mg/kg was administered in 3 pigs and NIRF imaging was performed for a total duration of 60 minutes. The intraoperative video recordings were analyzed to determine fluorescence intensities and the target-to-background ratio (TBR). Results: In all included animals, a clear delineation of the ureter was achieved from 5 minutes after dye administration until the end of the study. During this time period, the ureter was clearly distinguishable from its surroundings and no statistical differences in TBR were observed. Conclusion: The IRDye 800BK dye, a novel NIRF dye currently undergoing clinical translation, is a promising contrast agent used for noninvasive ureteral imaging, which has the potential to be valuable during RALS.

Fluorescence imaging of lymphatic outflow of cerebrospinal fluid in mice

Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non-invasive near-infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG-laden CSF outflow into the lymphatics following intrathecal lumbar administration. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. This drainage pattern was also visualized by NIRFI following intrathecal thoracic injection. In situ intravital imaging following intrathecal lumbar injection of blue dye shows similar distributions to that seen in vivo with ICG. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics.

Early Detection of Acute Drug-Induced Liver Injury in Mice by Noninvasive Near-Infrared Fluorescence Imaging

Hepatocellular and cholestatic forms of drug-induced liver injury (DILI) are major reasons for late-stage termination of small-molecule drug discovery research projects. Biochemical serum markers are limited in their ability to sensitively and specifically detect both of these common DILI forms in preclinical models, and tissue-specific approaches to assessing this are labor intensive, requiring extensive animal dosing, tissue preparation, and pathology assessment. In vivo fluorescent imaging offers noninvasive detection of biologic changes detected directly in the livers of living animals. Three different near-infrared fluorescent imaging probes, specific for cell death (Annexin-Vivo 750), matrix metalloproteases (MMPSense 750 FAST), and transferrin receptor (Transferrin-Vivo 750) were used to measure the effects of single bolus intraperitoneal doses of four different chemical agents known to induce liver injury. Hepatocellular injury–inducing agents, thioacetamide and acetaminophen, showed optimal injury detection with probe injection at 18–24 hours, the liver cholestasis-inducing drug rifampicin required early probe injection (2 hours), and chlorpromazine, which induces mixed hepatocellular/cholestatic injury, showed injury with both early and late injection. Different patterns of liver responses were seen among these different imaging probes, and no one probe detected injury by all four compounds. By using a cocktail of these three near-infrared fluorescent imaging probes, all labeled with 750-nm fluorophores, each of the four different DILI agents induced comparable tissue injury within the liver region, as assessed by epifluorescence imaging. A strategy of probe cocktail injection in separate cohorts at 2 hours and at 20–24 hours allowed the effective detection of drugs with either early- or late-onset injury.

Effect of lidocaine with and without epinephrine on lymphatic contractile activity in mice in vivo.

A local anesthetic, lidocaine, is known to affect cutaneous blood flow when injected into the skin. However, it is unknown if dermal lymphatic function can also be affected. Therefore, we characterized lymphatic function in response to administration of lidocaine with and without epinephrine. Non-invasive near-infrared fluorescence imaging (NIRFI) with intradermal injection of indocyanine green (ICG) was used to characterize the lymphatic "pumping" function in mice after subcutaneous injection of 2% lidocaine with and without 1:100,000 epinephrine or saline. NIRFI was performed for 10-20min immediately after and 1, 3, and 5h after these interventions. Lymphatic contraction frequencies significantly decreased 10min after subcutaneous injection of lidocaine and remained plateaued for another 5min, before returning to baseline. However, addition of 1:100,000 epinephrine to 2% lidocaine rapidly increased lymphatic contraction frequencies at 5min post-injection, which returned to baseline levels 15min later. Injection of saline also increased lymphatic contraction frequency 5min after injection, which returned to baseline 10min post-injection. Although lidocaine administration showed a decrease in lymphatic function, the combination of epinephrine with lidocaine resulted in a predominant net effect of increased contractile activity.

(Invited) Applications of Highly Bright PbS Quantum Dots to Non-Invasive Near-Infrared Fluorescence Imaging in the Second Optical Window

Near-infrared (NIR) fluorescence imaging in the wavelength of 1000-1400 nm (2nd-NIR optical window) is expected to offer better spatiotemporal deep-tissue imaging due to the lower autofluorescence and scattering than that in the 1st-NIR optical window (700-1000 nm). During the past decade considerable progress has been made in the development of NIR fluorescent materials that emit over 1000 nm for non-invasive fluorescence imaging. Although single-walled carbon nanotube (SWNT), rare-earth metal ion doped nanomaterials and silver sulfide (Ag2S) quantum dots (QDs) have been reported as fluorescent probes in the 2nd-NIR optical window, their fluorescence brightness is relatively low, with quantum yields (QY) less than 6 %. To achieve deep-tissue imaging with high spatiotemporal resolution, we have developed fluorescent lead sulfide (PbS) QDs with high quantum yields (QY >10 %) in the 2nd-NIR region. We present the synthesis of 2nd-NIR emitting PbS QDs and their applications to the non-invasive fluorescence imaging of lymph nodes, cerebral blood vessels, breast tumors, and phagocytic cells in mice.

Near-Infrared Emitting PbS Quantum Dots for in Vivo Fluorescence Imaging of the Thrombotic State in Septic Mouse Brain.

Near-infrared (NIR) fluorescent imaging is a powerful tool for the non-invasive visualization of the inner structure of living organisms. Recently, NIR fluorescence imaging at 1000–1400 nm (second optical window) has been shown to offer better spatial resolution compared with conventional NIR fluorescence imaging at 700–900 nm (first optical window). Here we report lead sulfide (PbS) quantum dots (QDs) and their use for in vivo NIR fluorescence imaging of cerebral venous thrombosis in septic mice. Highly fluorescent PbS QDs with a 1100 nm emission peak (QD1100) were prepared from lead acetate and hexamethyldisilathiane, and the surface of QD1100 was coated with mercaptoundecanoic acid so as to be soluble in water. NIR fluorescence imaging of the cerebral vessels of living mice was performed after intravascular injection (200–300 μL) of QD1100 (3 μM) from a caudal vein. By detecting the NIR fluorescence of QD1100, we achieved non-invasive NIR fluorescence imaging of cerebral blood vessels through the scalp and skull. We also achieved NIR fluorescence imaging of cerebral venous thrombosis in septic mice induced by the administration of lipopolysaccharide (LPS). From the NIR fluorescence imaging, we found that the number of thrombi in septic mice was significantly increased by the administration of LPS. The formation of thrombi in cerebral blood vessels in septic mice was confirmed by enzyme-linked immunosorbent assay (ELISA). We also found that the number of thrombi significantly decreased after the administration of heparin, an inhibitor of blood coagulation. These results show that NIR fluorescence imaging with QD1100 is useful for the evaluation of the pathological state of cerebral blood vessels in septic mice.

Synthesis and evaluation of Cy5.5-Rit tracer for specific near-infrared fluorescence imaging of sentinel lymph node

Sentinel lymph nodes biopsy (SLNB) is a critically important technique to determine the metastatic status of primary breast cancer. Here we reported the new use of a conjugate, Cy5.5-Rituximab (Cy5.5-Rit), which is specific toward the CD20 receptor, as an imaging agent for non-invasive near-infrared fluorescence (NIRF) imaging of sentinel lymph nodes (SLN). The conjugate, Cy5.5-Rit, was synthesized with the ratio of dye to Rituximab close to 1.0. Both gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of Cy5.5-Rit. For in vivo NIRF imaging, the conjugate stayed in SLN as long as 9days post injection. The in vivo imaging results of SLN targeting were also confirmed by in vitro HE stain. In addition, the direct fluorescence signal of dissected lymph node demonstrated that Cy5.5-Rit binds to the surface of lymph node cells. All of these results showed that the conjugate Cy5.5-Rit has potential for non-invasive optical imaging of SLN.

Biocompatibility of a Self-Assembled Crosslinkable Hyaluronic Acid Nanogel

Hyaluronic acid nanogel (HyA-AT) is a redox sensitive crosslinkable nanogel, obtained through the conjugation of a thiolated hydrophobic molecule to the hyaluronic acid chain. Engineered nanogel was studied for its biocompatibility, including immunocompatibility and hemocompatability. The nanogel did not compromise the metabolic activity or cellular membrane integrity of 3T3, microvascular endothelial cells, and RAW 264.7 cell lines, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase release assays. Also, we didn't observe any apoptotic effect on these cell lines through the Annexin V-FITC test. Furthermore, the nanogel cell internalization was analyzed using murine bone marrow derived macrophages, and the in vivo and ex vivo biodistribution of the Cy5.5 labeled nanogel was monitored using a non-invasive near-infrared fluorescence imaging system. The HyA-AT nanogel exhibits fairly a long half-live in the blood stream, thus showing potential for drug delivery applications.

Tracking the degradation of polysaccharide hydrogels by non-invasive near-infrared fluorescence imaging

Tracking the degradation of polysaccharide hydrogels by non-invasive near-infrared fluorescence imaging

Glycan multivalency effects toward albumin enable N-glycan-dependent tumor targeting

Multivalent interactions play an essential role in molecular recognition in living systems. These effects were employed to target tumor cells using albumin clusters bearing ∼10 molecules of asparagine-linked glycans (N-glycans). Noninvasive near-infrared fluorescence imaging clearly revealed A431 tumors implanted in BALB/cA-nu/nu mice after 1h in an N-glycan structure-dependent manner, thereby demonstrating the efficient use of glycan multivalency effects for tumor targeting in vivo.

Non-invasive near-infrared fluorescence imaging of the neutrophil response in a mouse model of transient cerebral ischaemia

Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia.

Applications of Highly Bright PbS Quantum Dots to Non-Invasive Near-Infrared Fluorescence Imaging in the Second Optical Window

Near-infrared (NIR) fluorescence imaging in the wavelength of 1000–1400 nm (2nd-NIR optical window) is expected to offer better spatiotemporal deep-tissue imaging due to the lower autofluorescence and scattering than that in the 1st-NIR optical window (700–1000 nm). During the past decade considerable progress has been made in the development of NIR fluorescent materials that emit over 1000 nm for non-invasive fluorescence imaging. Although single-walled carbon nanotube (SWNT), rare-earth metal ion doped nanomaterials and silver sulfide (Ag2S) quantum dots (QDs) have been reported as fluorescent probes in the 2nd-NIR optical window, their fluorescence brightness is relatively low, with quantum yields (QY) less than 6%. To achieve deep-tissue imaging with high spatiotemporal resolution, we have developed fluorescent lead sulfide (PbS) QDs with high quantum yields (QY >10%) in the 2nd-NIR region. This paper summarize the synthesis of 2nd-NIR emitting PbS QDs and their applications to the non-invasive fluorescence imaging of lymph nodes, cerebral blood vessels, breast tumors, and phagocytic cells in mice.

Platinum-Incorporating Poly(N-vinylpyrrolidone)-poly(aspartic acid) Pseudoblock Copolymer Nanoparticles for Drug Delivery.

Cisplatin-incorporating pseudoblock copolymer nanoparticles with high drug loading efficiency (ca. 50%) were prepared built on host-guest inclusion complexation between β-cyclodextrin end-capped poly(N-vinylpyrrolidone) block and admantyl end-capped poly(aspartic acid) block, followed by the coordination between cisplatin and carboxyl groups in poly(aspartic acid). The host-guest interaction between the two polymer blocks was examined by two-dimensional nuclear overhauser effect spectroscopy. The size and morphology of nanoparticles formed were characterized by dynamic light scattering, zeta potential, transmission electron microscopy, and atomic force microscopy. The size control of nanoparticles was carried out by varying the ratio of poly(N-vinylpyrrolidone) to poly(aspartic acid). The nanoparticles were stable in the aqueous medium with different pH values but disintegrated in the medium containing Cl(-) ions. The in vitro and in vivo antitumor effects of cisplatin-loaded nanoparticles were evaluated. The biodistribution of the nanoparticles in vivo was studied by noninvasive near-infrared fluorescence imaging and ion-coupled plasma mass spectrometry. It was found that cisplatin-loaded nanoparticles could effectively accumulate in the tumor site and exhibited significant superior in vivo antitumor activity to the commercially available free cisplatin by combining the tumor volume, body weight, and survival rate measurements.

生体の第2光学窓における非侵襲近赤外蛍光イメージング

We describe non-invasive near-infrared fluorescence imaging of deep tissues in the wavelength of 1000-1400 nm, which is called as the second optical window. Compared with the first optical window (700-1000 nm), this optical window makes it possible to achieve deep tissue imaging with a high spatial resolution due to the lower autofluorescence and scattering of near-infrared light. Here, we report near-infrared fluorescent probes and their application to the non-invasive fluorescence imaging of mouse lymph nodes, brain blood vessels, breast tumors, and immune cells in the second optical window.

In vivo lymphatic imaging of a human inflammatory breast cancer model.

Background: Inflammatory breast cancer (IBC) remains the most aggressive type of breast cancer with the greatest potential for metastasis and as a result, the highest mortality rate. IBC cells invade and metastasize through dermal lymphatic vessels; however, it is unknown how lymphatic drainage patterns change during IBC growth and metastasis. Herein, we non-invasively and longitudinally imaged lymphatics in an animal model of IBC using near-infrared fluorescence (NIRF) imaging.Materials and methods: Mice were imaged in vivo prior to, and up to 11 weeks after subcutaneous or orthotopic inoculation of human IBC SUM149 cells, which were stably transfected with infrared fluorescence protein (iRFP) gene reporter (SUM149-iRFP), following intradermal (i.d.) injection of indocyanine green (ICG).Results: Fluorescence images showed well-defined lymphatic vessels prior to SUM149-iRFP inoculation. However, altered lymphatic drainage patterns including rerouting of lymphatic drainage were detected in mice with SUM149-iRFP, due to lymphatic obstruction of normal lymphatic drainages caused by tumor growth. In addition, we observed tortuous lymphatic vessels and extravasation of ICG-laden lymph in mice with SUM149-iRFP. We also observed increased and dilated fluorescent lymphatic vessels in the tumor periphery, which was confirmed by ex vivo immunohistochemical staining of lymphatic vessels.Conclusions: Our pre-clinical studies demonstrate that non-invasive NIRF imaging can provide a method to assess changes in lymphatic drainage patterns during IBC growth and metastasis.

Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging

The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression due in part to the lack of imaging techniques currently available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal aspect of the left hindpaw for baseline data or directly to the popliteal lymph node (PLN) and until 21 days post-implantation (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were also detected at 21 days p.i. in mice with tumor in the paw and at 11 days p.i. in mice with tumor in the PLN, due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.

Abstract A43: Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging

Abstract The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression, due in part to the lack of imaging techniques available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using dynamic near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal foot for baseline data and until 21 days post-implant (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). In addition, intravital color images using a stereomicroscope were acquired after a skin incision. Then, the PLNs and inguinal LNs (ILNs) were harvested for ex vivo NIRF and intravital color imaging. We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were detected at 21 days p.i., due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Magnified fluorescent images of tumor-draining lymphatic vessels showed numerous fluorescent lymphatic vessels at day 21 p.i. as compared to well-defined fluorescent vessels from a baseline image. Our CT data showed enlarged tumor-draining PLNs at 21 days p.i. and intravital images confirmed the PLN harbored melanoma cells. Overlay of white light and fluorescent images of dissected LNs showed strong ICG fluorescence in the ILN and to less extent in the PLN on the tumor bearing side, indicating significantly reduced lymph flow to the PLN due to blockage of normal lymph flow by tumor in the PLN. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action. Citation Format: Sunkuk Kwon, Germaine Agollah, Grace Wu, Sevick Sevick-Muraca. Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A43.