Lymphatic Imaging Research Articles

Optimizing quantitative in vivo fluorescence imaging with near‐infrared quantum dots

Quantum dots (QDs) are fluorescent nanoparticles with broad excitation and narrow, wavelength-tunable emission spectra. They are used extensively for in vitro fluorescence imaging studies and more recently for in vivo small animal and pre-clinical studies. To date there has been little concern about the selection of QD size (and thus emission wavelength peak) and excitation wavelengths, as they have little relevance to the results of in vitro studies. In vivo imaging, however, poses additional constraints, such as the scattering and absorption by tissue, which may influence the signal intensity at the body surface. Here, we demonstrate that longer-wavelength excitation and emission yield less quantization error in measured relative fluorescence intensity, using three near-infrared QDs (QD655, QD705 and QD800) applied to in vivo lymphatic imaging, and a range of excitation wavelengths from the blue to the red. Statistically significant differences in quantization error were observed between nearly all pairs of excitation wavelengths (445-490, 503-555, 575-605, 615-665 and 671-705 nm). Similarly, quantization error decreased with longer emission wavelengths (655, 705 and 800 nm). Light absorbance and scattering were demonstrated to be more potent factors than absorbance efficiency of QDs in producing quantization error in the measured fluorescence intensity. As a result, while wavelengths can be adjusted for qualitative experiments, the longest possible wavelengths should be used if quantification is desired during QD imaging experiments.

Preclinical Lymphatic Imaging

Noninvasive in vivo imaging of lymphatic vessels and lymphatic nodes is expected to fulfill the purpose of analyzing lymphatic vessels and their function, understanding molecular mechanisms of lymphangiogenesis and lymphatic spread of tumors, and utilizing lymphatic molecular markers as a prognostic or diagnostic indicator. In this review, we provide a comprehensive summary of in vivo imaging modalities for detecting lymphatic vessels, lymphatic drainage, and lymphatic nodes, which include conventional lymphatic imaging techniques such as dyes and radionuclide scintigraphy as well as novel techniques for lymphatic imaging such as optical imaging, computed tomography, magnetic resonance imaging, ultrasound, positron emission tomography using lymphatic biomarkers, photoacoustic imaging, and combinations of multiple modalities. The field of lymphatic imaging is ever evolving, and technological advances, combined with the development of new contrast agents, continue to improve the research of lymphatic vascular system in health and disease states as well as to improve the accuracy of diagnosis in the relevant diseases.

Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema.

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.

Functional lymphatic imaging in tumor-bearing mice

The lymphatic system provides a route for dissemination of metastatic cancer cells. Yet to date transient changes in lymphatic drainage pathways and function as a result of tumor growth and metastasis have not been completely elucidated. Herein, we non-invasively imaged functional and architectural lymphatic changes in mice with regional, palpable lymph node (LN) involvement using dynamic near-infrared (NIR) fluorescence imaging with intradermal injection of indocyanine green (ICG) to both tumor-free mice and mice bearing C6/LacZ rat glioma tumors in the tail or hindlimb. We found that lymphatic drainage pathways were transiently altered and the contractile function of regional conducting lymphatic vessels was reduced or lost with progressive disease. Therefore, transient changes in the regional lymphatic architecture and function that occur with progressive disease, can be imaged using NIR fluorescence, and may provide a new method to stage disease.

Near‐infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice

To develop an in vivo imaging method to assess lymphatic draining function in the K/BxN mouse model of inflammatory arthritis. Indocyanine green, a near-infrared fluorescent dye, was injected intradermally into the footpads of wild-type mice, mouse limbs were illuminated with an 806-nm near-infrared laser, and the movement of indocyanine green from the injection site to the draining popliteal lymph node (LN) was recorded with a CCD camera. Indocyanine green near-infrared images were analyzed to obtain 5 measures of lymphatic function across time. Images of K/BxN arthritic mice and control nonarthritic littermates were obtained at 1 month of age, when acute joint inflammation commenced, and again at 3 months of age, when joint inflammation became chronic. Lymphangiogenesis in popliteal LNs was assessed by immunochemistry. Indocyanine green and its transport within lymphatic vessels were readily visualized, and quantitative measures were derived. During the acute phase of arthritis, the lymphatic vessels were dilated, with increased indocyanine green signal intensity and lymphatic pulses, and popliteal LNs became fluorescent quickly. During the chronic phase, new lymphatic vessels were present near the foot. However, the appearance of indocyanine green in lymphatic vessels was delayed. The size and area of popliteal LN lymphatic sinuses progressively increased in the K/BxN mice. Our findings indicate that indocyanine green near-infrared lymphatic imaging is a valuable method for assessing the lymphatic draining function in mice with inflammatory arthritis. Indocyanine green-near-infrared imaging of K/BxN mice identified 2 distinct lymphatic phenotypes during the acute and chronic phase of inflammation. This technique can be used to assess new therapies for lymphatic disorders.

Indocyanine Green and Lymphatic Imaging: Current Problems

Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has recently been presented as a comparatively easy and informative technique to image lymphatic channels in vivo. However, no data or references have been provided concerning the impact of ICG application on normal lymphatic contractility and lymph transport. Thus, the imaging agent and/or the method of administration may introduce a significant artifact. Standard pharmacological tests were performed to investigate the influence of ICG on the spontaneous contractility of isolated, cannulated, and pressurized rat mesenteric lymphatic vessels. The data demonstrate that non-irradiated ICG dramatically and dynamically influences the contractility of rat lymphatic vessels in both a dose- and diluent-dependent manner with low ICG concentrations principally altering contractile frequency and higher ICG concentrations completely blocking lymphatic contractility. Currently, both researchers and doctors should exercise caution in extrapolating the data obtained with ICG imaging to normal lymphatic function regardless of whether it was obtained in mice, pigs, or humans. Careful and extended pharmacological tests must be performed to evaluate the mechanism of action of ICG on the contractility and physiology of lymphatic vessels with consideration of dose, diluent, and duration of irradiation.

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

Nanotechnology in Thoracic Surgery

Nanotechnology is an exciting and rapidly progressing field offering potential solutions to multiple challenges in the diagnosis and treatment of lung cancer, with the potential for improving imaging and mapping techniques, drug delivery, and ablative therapy. With promising preclinical results in many applications directly applicable to thoracic oncology, it is possible that the frontiers of minimally invasive thoracic surgery will eventually be explored at the nanoscale.

Abstract 5238: Near-infrared fluorescence imaging of human lymphatics in healthy subjects and in cancer survivors

Abstract Near-infrared (NIR) fluorescence imaging was used to non-invasively image the (dys)function of the lymphatics in healthy control subjects and in subjects clinically diagnosed with unilateral lymphedema, a little understood disease characterized by chronic swelling and commonly observed in cancer survivors following therapeutic, complete nodal dissection. As part of an FDA approved feasibility trial and after informed consent, 24 control and 20 lymphedema (both hereditary and acquired) subjects received multiple intradermal injections of 25 µg of indocyanine green (total dose ≤ 400 µg) in bilateral arms or legs. The limbs were illuminated with NIR excitation light and the resulting fluorescent signal was imaged using custom, intensified CCD cameras. Lymphatic architecture and propulsive lymphatic flow from the injection sites to the nodal basin was observed. The apparent lymph velocities and propulsion periods were determined for the control subjects arms and legs as well as for the asymptomatic and symptomatic limbs of the lymphedema subjects. While the lymphatics of the control subjects consisted of well-defined vessels which propelled fluid from the injection sites to the regional nodal basin, diseased lymphatics exhibited distinct architectural malformations, such as (i) extravascular fluorescence in which the ICG diffused away from the injection site or leaked out of the lymphatic vessels, (ii) dense networks of lymphatic capillaries, (iii) tortuous lymphatic vessels, and (iv) lymphatic backflow and/or reflux. Statistical analyses of control subjects found subtle but significant differences in the left and right velocities in both arms (0.84 cm/s and 0.76 cm/s, p=8.1e-5) and legs (0.99 cm/s and 0.87 cm/s, p=0.032) but not in the periods of contralateral limbs. The difference in velocity in control arms (0.79 cm/s) and legs (0.94 cm/s) is small but significant (p=1.22e-7) while the difference in period is not. Significant differences in period exist between control (arms: 48.2 s; legs: 52.2 s) and both asymptomatic (arms: 39.6 s, p=2.68e-6; legs: 65.3 s, p=0.0022) and symptomatic (arms: 33.2 s, p=4.14e-5; legs: 72.1 s, p=0.00014) limbs. Significant differences were also found between the velocities in the control (0.94 cm/s) and both asymptomatic (0.83 cm/s, p=0.0084) and symptomatic (0.78 cm/s, p=0.0036) legs but not in the arms of control and lymphedema subjects. Results demonstrate that non-invasive NIR fluorescence imaging of microgram amounts of fluorophore can detect architectural and functional lymphatic abnormalities. The lack of significant differences between the velocities and periods of the asymptomatic and symptomatic limbs suggests that lymphedema may be a systemic disease regardless of onset etiology. Supported in parts by the American Cancer Society and the Longaberger Foundation (RSG-06-213-01-LR) and the National Institutes of Health (R01 HL092923 and U54 CA136404). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5238.

Abstract 1567: The multi-targeted kinase inhibitor Sunitinib blocks VEGF-C signaling pathway and lymphangiogenesis

Abstract Metastasis is the main cause of therapeutic failure and death in cancer patient. Tumor cells disseminate to distant organs through lymphatic and blood vessels. The degree of metastasis in regional lymph nodes is a prognostic factor and determines treatment course of patients. Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) family regulate angiogenesis and lymphangiogenesis. Several clinical studies have shown that the expression of VEGF-C is associated with lymphangiogenesis and lymph node metastasis in many different types of cancer. Sunitinib is a tyrosine kinase inhibitor that inhibits VEGFR, PDGFR, KIT, FLT3, RET, and CSF1R. The main mechanism for tumor growth inhibition is thought to be anti-angiogenic effect by inhibiting VEGFR-2 kinase activity. However, the effect of sunitinib on VEGF-C/VEGFR-3 pathway and lymphangiogenesis remain unclear. Therefore, we evaluated the effect of sunitinib on lymphangiogenesis with using lymphatic endothelial cell (LEC) and imaging breast cancer model. Sunitinib inhibited VEGF-C-induced phosphorylation of VEGFR-3, Akt and ERK1/2 at submicromolar level in LEC. Additionally, the growth and tube formation were also blocked by sunitinib. Furthermore, sunitinib treatment significantly suppressed tumor lymphangiogenesis and axillary lymph node metastasis as evaluated by photon counting method. These results suggest that Sunitinib may be beneficial for breast cancer patients by inhibiting lymph node metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1567.

Abstract 4332: Ultra-low background optical sentinel lymph node imaging using two unique nano-particles

Abstract Word limit: 2600 characters, including abstract body and title. Optical imaging has the potential to become a robust tool in intraoperative sentinel lymph node mapping because of its portability, high spatial resolution, and absence of radiation. Most preclinical and clinical studies have used conventional fluorescent agents, which need excitation light to emit fluorescence. However, excitation light also induces autofluorescence (background signal) from intrinsic fluorophores in the normal tissue, lowering the target-to-background ratio. We developed two new methods for optical sentinel lymph node mapping to eliminate background signals using two unique nano-particles; an upconverting nano-particle (UCNP) and a self-illuminating bioluminescence resonance energy transfer quantum-dot (BRET-QD) nano-particle. UCNPs are unique nano-sized particles that emit light at shorter wavelengths with excitation in the near infrared (NIR). Since fluorophores typically emit light at longer wavelengths than the excitation light, there is no autofluorescence. Moreover, by changing the composition of the doping metals, UCNPs can be designed to emit at specific wavelengths. When UCNPs were injected into the chin of anesthetized mice, and excited at 950-nm, superficial neck lymph nodes were successfully depicted with no background signals in the visible-range (peak 550 nm) or NIR-range (peak 800 nm) in all mice. Furthermore, with serial injection of the visible and NIR-range UCNPs, simultaneous lymphatic imaging was achieved in two colors. The BRET-QD is self-illuminating and employs the substrate, coelenterazine, which reacts with luciferase conjugated on the BRET-QD. The bioluminescent light in blue efficiently excites the NIR QD, and emits photons in the NIR, therefore, there is no autofluorophore excitation. Moreover, by changing the QDs within BRET-QD, it is possible to alter their emission wavelength. BRET-QD655, (QD655 in its core), was injected at different sites (chin, ear, forepaws, and hind paws) of anesthetized mice. After BRET-QD655 injection, coelenterazine was intravenously injected and imaging was performed without excitation light. In all mice, sentinel lymph nodes in each lymphatic basin were clearly visualized with ultra-low background signals, and lasted at least 30 min after coelenterazine injections. In conclusion, we demonstrate that optical imaging using two unique nano-particles were able to depict sentinel lymph nodes with minimal autofluorescence. This enables the direct, real time sentinel lymph node mapping without extensive post processing of the images. These two novel nano-particles have the potential to be robust tools for sentinel lymph node detection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4332.

Medical and Surgical Management of Chylothorax and Associated Outcomes

Chylothorax is an uncommon form of pleural effusion that can be associated with traumatic and nontraumatic causes. Optimal management and outcome for patients with chylothorax remain unclear. This retrospective single-center study assessed the modes of management for chylothorax in 74 adult patients (≥ 18years old) and associated outcomes. The role of lymphangiographic imaging was also evaluated. Initial treatment approach was nonsurgical in 57 patients (77%) but a surgical procedure (pleurodesis, thoracic duct ligation, and/or surgical repair) was eventually performed in 44 patients (59%). The rate of resolution with initial treatment measures was significantly worse for patients with nontraumatic chylothorax compared with those with traumatic causes (27% versus 50%, P=0.048). Even after additional therapeutic maneuvers including surgery, chylous effusion recurred more commonly in nontraumatic chylothorax when compared with the traumatic group (50% versus 13%, respectively, P<0.001). Lymphatic imaging did not seem to materially influence management. Nonsurgical approaches may lead to resolution of the chylothorax in nearly one half of patients with traumatic chylothorax but in only a minority of those with nontraumatic chylothorax. The majority of patients with nontraumatic chylothorax will eventually require surgical maneuvers, but one third of such patients still fail to resolve their chylothorax.

T1619: Gastric Lymphatic Mapping With Infra-Red Endoscopic Imaging (IRE): Dose Response Study With Indocyanine Green (ICG) in an ex vivo Porcine Model

Endoscopic resection is limited to mucosal cancer as it cannot assess nodal disease. ICG, which absorbs infra-red rays, is used with IRE to guide sentinel node biopsy in cancer surgery. Its use in endoscopic nodal detection has not been described. This is a dose response study to find the minimum ICG concentration for lymphatic imaging.

Tumor Microvasculature and Microenvironment: Novel Insights Through Intravital Imaging in Pre-Clinical Models

Intravital imaging techniques have provided unprecedented insight into tumor microcirculation and microenvironment. For example, these techniques allowed quantitative evaluations of tumor blood vasculature to uncover its abnormal organization, structure and function (e.g., hyper-permeability, heterogeneous and compromised blood flow). Similarly, imaging of functional lymphatics has documented their absence inside tumors. These abnormalities result in elevated interstitial fluid pressure and hinder the delivery of therapeutic agents to tumors. In addition, they induce a hostile microenvironment characterized by hypoxia and acidosis, as documented by intravital imaging. The abnormal microenvironment further lowers the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition to these mechanistic insights, intravital imaging may also offer new opportunities to improve therapy. For example, tumor angiogenesis results in immature, dysfunctional vessels--primarily caused by an imbalance in production of pro- and anti-angiogenic factors by the tumors. Restoring the balance of pro- and anti-angiogenic signaling in tumors can "normalize" tumor vasculature and thus, improve its function, as demonstrated by intravital imaging studies in preclinical models and in cancer patients. Administration of cytotoxic therapy during periods of vascular normalization has the potential to enhance treatment efficacy.

Folate-PEG-CKK2-DTPA, A Potential Carrier for Lymph-Metastasized Tumor Targeting

A novel conjugate, Folate-PEG-CKK(2)-DTPA, was designed and prepared as a carrier for lymphatic metastasized tumor imaging diagnosis and targeting therapy. Folate-PEG-CKK(2)-DTPA was synthesized and characterized by analysis High Performance Liquid Chromatography, Size Exclusive Chromatography and (1)H-NMR. (99m)Tc-labeled conjugation was prepared, and in vivo quantitative biodistribution and SPECT imaging were studied after subcutaneously injected into the rats and rabbits, respectively. Cell uptake study was carried in a KB cell line using fluorescent methods. In vivo and ex vivo fluorescent imaging study was carried in tumor-bearing nude mouse to evaluate its targeting ability. Folate-PEG-CKK(2)-DTPA was synthesized with high purity. Both in vivo biodistribution study and SPECT imaging study show the rapid direction and high distribution of the conjugation to the lymph nodes. The uptake of fluorescence-labeled Folate-PEG-CKK(2)-DTPA in human oral epidermis carcinoma cells was observed. In vivo and ex vivo fluorescent imaging study indicated it could accumulate in tumor region after vein tail injection in nude mouse. All these findings suggested Folate-PEG-CKK(2)-DTPA as a novel and dependable carrier for tumor diagnosis and therapy, especially for lymph-metastasized tumors.

In Vivo Real-Time, Multicolor, Quantum Dot Lymphatic Imaging

The lymphatic network is complex and difficult to visualize in real-time in vivo. Moreover, the direction of flow within lymphatic networks is often unpredictable especially in areas with well-developed "watershed" or overlapping lymphatics. Herein, we report a method of in vivo real-time multicolor lymphatic imaging using cadmium-selenium quantum dots (Qdots) with a fluorescence imaging system that enables the simultaneous visualization of up to five distinct lymphatic basins in real-time. Five visually well-distinguishable carboxyl-Qdots (Qdot 545, 565, 585, 605, and 655) were selected and injected subdermally into mice at five different sites, and serially imaged in vivo or in situ under surgery with real-time multicolor lymphatic imaging. In all seven mice, in vivo lymphatic images successfully distinguished all five lymphatic basins with different colors in real-time. These visualizations of lymph node lasted up to at least 7 days. This method could have a considerable potential in lymphatic research for studying the anatomy and flow within the lymphatic system as well as in some limited clinical settings where real-time visible fluorescence could facilitate procedures under surgery or endoscopy.

New Approaches to Lymphatic Imaging

Accurate imaging of the lymphatic system can aid in cancer staging, optimize surgical procedures to reduce lymphedema, and may one day be a means of delivering intralymphatic therapy. The Sentinel Lymph Node (SLN) concept has been pivotal in driving new imaging techniques. Metastasis to a SLN is a critical indicator of advanced disease. However, presently, few tools are available for imaging the lymphatics, and even fewer are available for locating the SLN for biopsy. Recently, new macromolecular agents, including gadolinium-labeled dendrimers, fluorescent quantum dots, and fluorescently-labeled immunoglobins, have been used to image the lymphatics and SLN with MRI and optical techniques, and new fluorescent nanoparticles such as upconverting nanocrystals are potential future agents. Additionally, multi-modality probes combining two modalities such as optical/MR dendrimers have been designed to provide both preoperative imaging, and intraoperative guidance during lymph node resections. These probes can map the lymphatic system for maximal therapeutic benefit while minimizing complications such as lymphedema. Advances in the understanding of the molecular mechanisms of lymphangiogenesis and lymphatic spread of tumors offer the opportunity for more targeted imaging of the lymphatic system. Additionally, these imaging agents could be used as powerful research tools for tracking immunological cells and monitoring the immune response as well as providing the means to deliver lymphatic-targeted therapies. The future holds great promise for the translation of these methods to the clinic.

Clinical Implications of Near-Infrared Fluorescence Imaging in Cancer

Near-infrared (NIR) fluorescence cancer imaging is a growing field for both preclinical and clinical application to the clinical management for cancer patients due to its advantageous features, including a high spatial resolution, portability, real-time display and detailed molecular profiling with the multiplexed use of fluorescent probes. In this review, we present a basic concept of NIR fluorescence imaging and overview its potential clinical applications for in vivo cancer imaging, including cancer detection/characterization, lymphatic imaging (sentinel lymph node detection) and surgical/endoscopic guidance. NIR fluorescence imaging can compensate some limitations of conventional imaging modalities, and thus it could play an important role for cancer imaging combined with other modalities in clinical practice.

Preoperative localization of sentinel lymph nodes using intradermal microbubbles and contrast-enhanced ultrasonography in patients with breast cancer

Sentinel lymph node (SLN) biopsy is the standard procedure for axillary staging in early breast cancer. Lymphatic imaging after intraparenchymal microbubble injection has been reported in animal models. The aim of this study was to identify and localize SLNs before surgery by contrast-enhanced ultrasonography after intradermal injection of microbubbles in patients with breast cancer. Fifty-four consecutive, consenting patients with primary breast cancer were recruited. Patients received a periareolar intradermal injection of microbubble contrast agent. Breast lymphatics were visualized by ultrasonography and followed to identify putative axillary SLNs. A guidewire was deployed to localize the SLN. The next day, patients underwent standard tumour excision and SLN biopsy. SLNs were identified and guidewires inserted in 48 patients. In these patients operative findings confirmed that the wired lymph nodes were SLNs. The sensitivity of SLN detection, compared with radioisotope and blue dye, was 89 per cent. Five patients were found to have metastases in SLNs. In these patients the SLNs were identified correctly and localized before surgery with guidewires. SLNs may be identified and localized before surgery using contrast-enhanced ultrasonography after injection of microbubbles.

Nanoparticles in sentinel lymph node mapping

The lymph nodes and lymphatic vessels are more difficult to access than most vascular structures. Interstitial injection of imaging agents is often necessary to opacify the lymphatics. Traditionally, radionuclide methods of sentinel node imaging have dominated this field, however, limitations in resolution and exposure to radiation have encouraged the development of newer imaging methods. Among these are magnetic resonance lymphography in which a Gadolinium labeled nanoparticle is injected and imaged providing superior anatomic resolution and assessment of lymphatic dynamics. Optical imaging employing various nanoparticles including quantum dots also provide the capability of mapping each lymphatic basin in another "color". Taken together this "toolbox" of lymphatic imaging agents is poised to improve our understanding of the lymphatic system.