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Introduction: Automated manual lymphatic drainage therapy (AMLDT) is available for home use in the form of a pneumatic mat of 16 pressurized air channels that inflate and deflate to mimic the stretch and release action of manual lymphatic drainage therapy. Four cases (a patient with complex regional pain syndrome and lymphedema, a healthy patient, a breast cancer survivor with chronic pain, and a patient with a history of abdominal surgery) underwent near-infrared fluorescence lymphatic imaging (NIRFLI) with AMLDT to evaluate the effect of AMLDT on lymphatic pumping and pain., Methods: Each patient received 32-36 injections of 25 μg indocyanine green (ICG) on the anterior and posterior sides of their body and underwent 1 h of NIRFLI to assess the drainage of ICG laden lymph toward regional nodal basins at baseline. Each patient lay supine on the mat for 1 h of AMLDT with NIRFLI to assess lymphatic flow during treatment. A final NIFRFLI assessment was done 30-60 min posttreatment with the patient in the supine and prone position. Patients reported baseline and posttreatment pain using the Visual Analogue Scale. An imager analyzed NIRFLI images using ImageJ (US National Institutes of Health). Using time stamps of the first and last images to determine time lapsed and the number of pulses observed in a timeframe, pulsing frequency (pulses/min) was obtained to assess lymphatic function., Results: All 4 cases completed the NIRFLI and AMLDT without complications; all 3 patients with baseline pain reported reduced pain posttreatment. AMLDT appeared to alter lymphatic contractility, with both increased and decreased pulsing frequencies observed, including in nonaffected limbs. Pulsing frequencies were very heterogeneous among patients and varied within anatomic regions of the same patient., Discussion: This proof-of-concept study suggests that AMLDT may impact lymphatic contractility. Further research on its effect on lymphatic function is warranted., Competing Interests: CF was employed by Intellicure, LLC. JR and CF are listed as inventors on patents related to NIRFLI and may receive future financial benefit from its commercialization. MM is Chief Medical Officer for Eva Medtec. FA had no conflict of interest to declare during the study but is now an advisor to Eva Medtec. KE received consulting fees from Strategic Solutions, Inc., which was funded by Eva Medtec, for her participation in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Aldrich, Rasmussen, Karni, Fife, Aviles, Eckert and Melin.)

The role of the lymphatics in the clearance of cerebrospinal fluid (CSF) from the brain has been implicated in multiple neurodegenerative conditions. In premature infants, intraventricular hemorrhage causes increased CSF production and, if clearance is impeded, hydrocephalus and severe developmental disabilities can result. In this work, we developed and deployed near-infrared fluorescence (NIRF) tomography and imaging to assess CSF ventricular dynamics and extracranial outflow in similarly sized, intact non-human primates (NHP) following microdose of indocyanine green (ICG) administered to the right lateral ventricle. Fluorescence optical tomography measurements were made by delivering ~10 mW of 785 nm light to the scalp by sequential illumination of 8 fiber optics and imaging the 830 nm emission light collected from 22 fibers using a gallium arsenide intensified, charge coupled device. Acquisition times were 16 seconds. Image reconstruction used the diffusion approximation and hard-priors obtained from MRI to enable dynamic mapping of ICG-laden CSF ventricular dynamics and drainage into the subarachnoid space (SAS) of NHPs. Subsequent, planar NIRF imaging of the scalp confirmed extracranial efflux into SAS and abdominal imaging showed ICG clearance through the hepatobiliary system. Necropsy confirmed imaging results and showed that deep cervical lymph nodes were the routes of extracranial CSF egress. The results confirm the ability to use trace doses of ICG to monitor ventricular CSF dynamics and extracranial outflow in NHP. The techniques may also be feasible for similarly-sized infants and children who may suffer impairment of CSF outflow due to intraventricular hemorrhage.

The lymphatics play important roles in chronic diseases/conditions that comprise the bulk of healthcare worldwide. Yet the ability to routinely image and diagnose lymphatic dysfunction, using commonly available clinical imaging modalities, has been lacking and as a result, the development of effective treatment strategies suffers. Nearly two decades ago, investigational near-infrared fluorescence lymphatic imaging and ICG lymphography were developed as routine diagnostic for clinically evaluating, quantifying, and treating lymphatic dysfunction in cancer-related and primary lymphedema, chronic venous disease, and more recently, autoimmune and neurodegenerative disorders. In this review, we provide an overview of what these non-invasive technologies have taught us about lymphatic (dys) function and anatomy in human studies and in corollary animal studies of human disease. We summarize by commenting on new impactful clinical frontiers in lymphatic science that remain to be facilitated by imaging., Competing Interests: ES-M, JR, and CF have financial interests in a University of Texas Health Science Center start-up, Lymphatic Science, which seeks to commercialize near-infrared fluorescence lymphatic imaging devices and the approaches described herein. The conflict of interest on the part of ES-M and JR is managed by the University Research Conflict of Interest Committee., (Copyright © 2023 Sevick-Muraca, Fife and Rasmussen.)

Breast cancer-related lymphedema (BCRL) occurs in ~ 40% of patients after axillary lymph node dissection (ALND), radiation therapy (RT), or chemotherapy. First-line palliative treatment utilizes compression garments and specialized massage. Reparative microsurgeries have emerged as a second-line treatment, yet both compression and surgical therapy are most effective at early stages of LE development. Identifying patients at the highest risk for BCRL would allow earlier, more effective treatment. Perometric arm volume measurements, near-infrared fluorescent lymphatic imaging (NIRF-LI) data, and blood were collected between 2016 and 2021 for 40 study subjects undergoing treatment for breast cancer. Plasma samples were evaluated using MILLIPLEX human cytokine/chemokine panels at pre-ALND and at 12 months post-RT. A Mann-Whitney t -test showed that G-CSF, GM-CSF, IFN-2α, IL-10, IL-12p40, IL-15, IL-17A, IL-1β, IL-2, IL-3, IL-6, and MIP-1β were significantly higher at pre-ALND in those presenting with BCRL at 12 months post-RT. MIP-1β and IL-6 were significantly higher at pre-ALND in those who developed dermal backflow, but no BCRL, at 12 months post-RT. Plasma IL-15, IL-3, and MIP-1β were elevated at 12 months after RT in those with clinical BCRL. These findings establish BCRL as a perpetual inflammatory disorder, and suggest the use of plasma cytokine/chemokine levels to predict those at highest risk.

Purpose: Mild breast cancer-related lymphedema (BCRL) is clinically diagnosed as a 5%-10% increase in arm volume, typically measured no earlier than 3-6 months after locoregional treatment. Early BCRL treatment is associated with better outcomes, yet amid increasing evidence that lymphedema exists in a latent form, treatment is typically delayed until arm swelling is obvious. In this study, we investigated whether near-infrared fluorescence lymphatic imaging (NIRF-LI) surveillance could characterize early onset of peripheral lymphatic dysfunction as a predictor of BCRL., Methods: In a prospective, longitudinal cohort/observational study (NCT02949726), subjects with locally advanced breast cancer who received axillary lymph node dissection and regional nodal radiotherapy (RT) were followed serially, between 2016 and 2021, before surgery, 4-8 weeks after surgery, and 6, 12, and 18 months after RT. Arm volume was measured by perometry, and lymphatic (dys) function was assessed by NIRF-LI., Results: By 18 months after RT, 30 of 42 study subjects (71%) developed mild-moderate BCRL (i.e., ≥ 5% arm swelling relative to baseline), all manifested by "dermal backflow" of lymph into lymphatic capillaries or interstitial spaces. Dermal backflow had an 83% positive predictive value and 86% negative predictive value for BCRL, with a sensitivity of 97%, specificity of 50%, accuracy of 83%, positive likelihood ratio of 1.93, negative likelihood ratio of 0.07, and odds ratio of 29.00. Dermal backflow appeared on average 8.3 months, but up to 23 months, before the onset of mild BCRL., Conclusion: BCRL can be predicted by dermal backflow, which often appears months before arm swelling, enabling early treatment before the onset of edema and irreversible tissue changes., (© 2022. The Author(s).)

Objective: Lipedema is an inflammatory subcutaneous adipose tissue disease that develops in women and may progress to lipolymphedema, a condition similar to lymphedema, in which lymphatic dysfunction results in irresolvable edema. Because it has been shown that dilated lymphatic vessels, impaired pumping, and dermal backflow are associated with presymptomatic, cancer-acquired lymphedema, this study sought to understand whether these abnormal lymphatic characteristics also characterize early stages of lipedema prior to lipolymphedema development., Methods: In a pilot study of 20 individuals with Stage I or II lipedema who had not progressed to lipolymphedema, lymphatic vessel anatomy and function in upper and lower extremities were assessed by near-infrared fluorescence lymphatic imaging and compared with that of a control population of similar age and BMI., Results: These studies showed that, although lower extremity lymphatic vessels were dilated and showed intravascular pooling, the propulsion rates significantly exceeded those of control individuals. Upper extremity lymphatics of individuals with lipedema were unremarkable. In contrast to individuals with lymphedema, individuals with Stage I and II lipedema did not exhibit dermal backflow., Conclusions: These results suggest that, despite the confusion in the diagnoses between lymphedema and lipedema, their etiologies differ, with lipedema associated with lymphatic vessel dilation but not lymphatic dysfunction., (© 2022 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).)

Objective: We used near-infrared fluorescence lymphatic imaging in a pilot study to assess the lymphatics in preulcerative (C2-C4) venous insufficiency and determine whether involvement and/or degradation of lymphatic anatomy or function could play a role in the progression of chronic venous insufficiency. We also explored the role of lymphatics in early peripheral arterial disease., Methods: After informed consent and intradermal injections of indocyanine green for rapid lymphatic uptake, near-infrared fluorescence lymphatic imaging was used to assess the lymphatic anatomic structure and quantify the lymphatic propulsion rates in subjects with early venous insufficiency. The anatomic observations included interstitial backflow, characterized by the abnormal spreading of indocyanine green from the injection site primarily into the surrounding interstitial tissues; dermal backflow, characterized by the retrograde movement of dye-laden lymph from collecting lymphatics into the lymphatic capillaries; and lymphatic vessel segmentation and dilation., Results: Ten subjects with venous insufficiency were enrolled, resulting in two legs with C2 disease, nine legs with C3 disease, eight legs with C4 disease, and one leg with C5 disease. Interstitial and/or dermal backflow were observed in 25%, 33%, and 41% of the injection sites in each limb with C2, C3, and C4 disease, respectively. Distinct vessel segmentation and dilation were observed in limbs with a C3 and higher classification, and dermal backflow proximal to the injection sites was observed in two legs with C4 disease and in the inguinal region of the C5 study subject. The overall average lymph propulsion rates were 1.3 ± 0.4, 1.2 ± 0.7, and 0.8 ± 0.5 contractile events/min for limbs with C2, C3, and C4 disease, respectively. One subject with peripheral arterial disease, who had previously undergone bypass surgery, presented with extensive dermal backflow and lymphatic reflux., Conclusions: Near-infrared fluorescence lymphatic imaging demonstrated that, compared with normal health subjects, the lymphatic anatomy and contractile function generally degrade with the severity of venous insufficiency. Lymphatic abnormalities mimic those in early cancer-acquired lymphedema subjects, as previously observed by us and others. Additional studies are needed to decipher the relationship, including any causality, between lymphatic dysfunction and peripheral vascular disease and venous insufficiency., (Copyright © 2020 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Cutaneous tuberculosis secondary to skin inoculation of Mycobacterium tuberculosis is uncommon but it can occur in the health care settings. Herein, we report an unusual case of primary cutaneous tuberculosis of the thumb following a needlestick injury. The infection progressed with a necrotic granuloma, lymphatic dysfunction as visualized by near-infrared fluorescence lymphatic imaging, and the development of an axillary web syndrome., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Chylothorax is a rare complication of pediatric cardiac operations that occurs more frequently in children with Noonan syndrome, a genetic disorder associated with cardiac defects and lymphatic anomalies., Case Presentation: We report a case of postoperative chylothorax in a 6-month-old infant with Noonan syndrome where multimodality lymphatic imaging guided management was followed. Drainage patterns of the lymphatic capillaries in the lower and upper extremities were visualized during near-infrared fluorescence lymphatic imaging (NIRFLI). Dynamic magnetic resonance lymphangiography (MRL) further identified the site of leakage in the thoracic duct and subsequently guided surgical intervention., Conclusions: Application of multimodality imaging allows for greater individualization of treatment and should be considered in patients with complex cases such as those with syndromes associated with a higher incidence of chylothorax. IRB Number: HSC-MS-13-0754, December 10, 2013.

Breast-cancer-acquired lymphedema is routinely diagnosed from the appearance of irreversible swelling that occurs as a result of lymphatic dysfunction. Yet in head and neck cancer survivors, lymphatic dysfunction may not always result in clinically overt swelling, but instead contribute to debilitating functional outcomes. In this review, we describe how cancer metastasis, lymph node dissection, and radiation therapy alter lymphatic function, as visualized by near-infrared fluorescence lymphatic imaging. Using custom gallium arsenide (GaAs)-intensified systems capable of detecting trace amounts of indocyanine green administered repeatedly as lymphatic contrast for longitudinal clinical imaging, we show that lymphatic dysfunction occurs with cancer progression and treatment and is an early, sub-clinical indicator of cancer-acquired lymphedema. We show that early treatment of lymphedema can restore lymphatic function in breast cancer and head and neck cancer patients and survivors. The compilation of these studies provides insights to the critical role that the lymphatics and the immune system play in the etiology of lymphedema and associated co-morbidities.

Recently, fluorescence imaging using shortwave infrared light (SWIR, 1,000-2,000 nm) has been proposed as having advantage over conventional near-infrared fluorescence (NIRF) imaging due to the reduced tissue scattering, negligible autofluorescence, comparable tissue absorption, and the discovery that indocyanine green (ICG), used clinically as a NIRF contrast agent, also has fluorescence emission in SWIR regime. Images of ICG in small animals acquired by commercial Si-based and InGaAs-based imaging cameras have been qualitatively compared, however the lack of working standards to quantify performance of these imaging systems limits quantitative comparison. Without quantification using a traceable in vitro test, clinical adoption of rapidly evolving advances in both NIRF and SWIR imaging devices will become limited. In this work, we developed an ICG based fluorescent solid working standard calibrated with SI units (mW [Formula: see text]cm [Formula: see text]sr -1 ) for quantification of measurement sensitivity of Si, GaAs-intensified Si, and InGaAs based camera systems, their signal-to-noise ratio (SNR), and contrast in non-clinical tests. In addition, we present small animal and large animal imaging with ICG for qualitative comparison of the same SWIR fluorescence and NIRF imaging systems. Results suggest that SWIR fluorescence imaging of ICG may have superior resolution in small animal imaging compared to NIRF imaging, but lack of measurement sensitivity, SNR, contrast, as well as water absorption limits deep penetration in large animals.

Evidence overwhelmingly suggests that the lymphatics play a critical role in the clearance of cerebrospinal fluid (CSF) from the cranial space. Impairment of CSF outflow into the lymphatics is associated with a number of pathological conditions including spaceflight-associated neuro-ocular syndrome (SANS), a problem that limits long-duration spaceflight. We used near-infrared fluorescence lymphatic imaging (NIRFLI) to dynamically visualize the deep lymphatic drainage pathways shared by CSF outflow and disrupted during head-down tilt (HDT), a method used to mimic the cephalad fluid shift that occurs in microgravity. After validating CSF clearance into the lymph nodes of the neck in swine, a pilot study was conducted in human volunteers to evaluate the effect of gravity on the flow of lymph through these deep cervical lymphatics. Injected into the palatine tonsils, ICG was imaged draining into deep jugular lymphatic vessels and subsequent cervical lymph nodes. NIRFLI was performed under HDT, sitting, and supine positions. NIRFLI shows that lymphatic drainage through pathways shared by CSF outflow are dependent upon gravity and are impaired under short-term HDT. In addition, lymphatic contractile rates were evaluated from NIRFLI following intradermal ICG injections of the lower extremities. Lymphatic contractile activity in the legs was slowed in the gravity neutral, supine position, but increased under the influence of gravity regardless of whether its force direction opposed (sitting) or favored (HDT) lymphatic flow toward the heart. These studies evidence the role of a lymphatic contribution in SANS., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Rationale : Cytotoxic T-lymphocyte-associated antigen 4 (CTLA - 4) is a co-inhibitory checkpoint receptor that is expressed by naïve T-cells in lymph nodes (LNs) to inhibit activation against "self" antigens (Ags). In cancer, anti-CTLA-4 blocks inhibitory action, enabling robust activation of T-cells against tumor Ags presented in tumor draining LNs (TDLNs) . However, anti-CTLA-4 is administered intravenously with limited exposure within TDLNs and immune related adverse events (irAEs) are associated with over-stimulation of the immune system. Methods : Herein, we first deliver anti-CTLA-4 in an orthotopic mammary carcinoma murine model using a nanotopographical microneedle-array device to compare its anti-tumor response to that from systemic administration. Additionally, to demonstrate the feasibility of lymphatic delivery in humans using the device, we use near-infrared fluorescence imaging to image delivery of ICG to LNs. Results : Our data show that lymphatic infusion results in more effective tumor growth inhibition, arrest of metastases, increased tumor infiltrating lymphocytes and complete responses when compared to conventional systemic administration. In clinical studies, we demonstrate for the first time that nanotopographic infusion can deliver ICG through the lymphatics directly to the axilla and inguinal LNs of healthy human volunteers. Conclusion : Taken together, these results suggest that regional delivery using a nanotopography-based microneedle array could revolutionize checkpoint blockade immunotherapy by reducing systemic drug exposure and maximizing drug delivery to TDLNs where tumor Ags present. Future work is needed to determine whether lymphatic delivery of anti-CTLA-4 can alleviate irAEs that occur with systemic dosing., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Ten head and neck cancer survivors diagnosed with head and neck lymphedema (HNL) were imaged using near-infrared fluorescence lymphatic imaging (NIRFLI) prior to and immediately after an initial advance pneumatic compression device treatment and again after 2 weeks of daily at-home use. Images assessed the impact of pneumatic compression therapy on lymphatic drainage. Facial composite measurement scores assessed reduction/increase in external swelling, and survey results were obtained. After a single pneumatic compression treatment, NIRFLI showed enhanced lymphatic uptake and drainage in all subjects. After 2 weeks of daily treatment, areas of dermal backflow disappeared or were reduced in 6 of 8 subjects presenting with backflow. In general, reductions in facial composite measurement scores tracked with reductions in backflow and subject-reported improvements; however, studies are needed to determine whether longer treatment durations can be impactful and whether advanced pneumatic compression can be used to ameliorate backflow characteristic of HNL.

The relationship between lymphatic and venous malformations in Klippel-Trénaunay syndrome is difficult to assess. Herein the authors describe near-infrared fluorescence lymphatic imaging to assess the lymphatics of a subject with a large port-wine stain and right leg edema. Although lymphatic vessels in the medial, affected knee appeared dilated and perhaps tortuous, no definitive abnormal lymphatic pooling or propulsion was observed. The lymphatics in the affected limb were well defined but less numerous than in the contralateral limb, and active, contractile function was observed in all vessels. As demonstrated, near-infrared fluorescence lymphatic imaging enables the clinical assessment of lymphatics in lymphovenous malformations., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Background: The lymphatic vasculature provides a route for cancer metastases, and its dysfunction after cancer treatment can result in lymphedema. However, changes in the lymphatics before, during, and after surgery and radiation remain unclear., Methods: Near-infrared fluorescence lymphatic imaging was performed before and after lymph node dissection and fractionated radiotherapy to assess changes in external lymphatic function., Results: Patients who underwent both lymph node dissection and radiotherapy developed lymphatic dermal backflow on treated sides ranging from days after the start of radiotherapy to weeks after its completion, whereas contralateral regions that were not associated with lymph node dissection but also treated with radiotherapy experienced no such changes in external lymphatic anatomies., Conclusion: The external lymphatics undergo transient changes during and weeks after lymph node dissection and radiotherapy. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1177-1188, 2017., (© 2017 Wiley Periodicals, Inc.)

Background: Evidence suggests lymphatic function mediates local rheumatoid arthritis (RA) flares. Yet biologics that target the immune system are dosed systemically via the subcutaneous (SC) administration route, thereby inefficiently reaching local lymphatic compartments. Nanotopography has previously been shown to disrupt tight cellular junctions, potentially enhancing local lymphatic delivery and potentially improving overall therapeutic efficacy., Method: We first characterized nanotopography (SOFUSA™) delivery of an anti-TNF drug, etanercept, by comparing pharmacokinetic profiles to those obtained by conventional SC, intravenous (IV), and intradermal (ID) routes of administration, and assessed uptake of radiolabeled etanercept in draining lymph nodes (LNs) in single dosing studies. We then compared etanercept efficacy in a progressive rat model of collagen-induced arthritis (CIA), administered systemically via SC route of administration; via the regional lymphatics through ID delivery; or through a nanotopography (SOFUSA™) device at 10, 12, and 14 days post CIA induction. Measurements of hind limb swelling and near-infrared fluorescence (NIRF) imaging of afferent lymph pumping function and reflux were conducted on days 11, 13, and 18 post CIA induction and compared to untreated CIA animals. Univariate and multivariate analysis of variance were used to compare the group differences for percentage swelling and lymphatic contractile activity., Results: Even though all three modes of administration delivered an equal amount of etanercept, SOFUSA™ delivery resulted in increased lymphatic pumping and significantly reduced swelling as compared to untreated, ID, and SC groups. Pharmacokinetic profiles in serum and LN uptake studies showed that using the nanotopography device resulted in the greatest uptake and retention in draining LNs., Conclusions: Locoregional lymphatic delivery of biologics that target the immune system may have more favorable pharmacodynamics than SC or IV administration. Nanotopography may provide a more efficient method for delivery of anti-TNF drugs to reverse impairment of lymphatic function and reduce swelling associated with RA flares.

Primary lymphedema in the pediatric population remains poorly diagnosed and misunderstood due to a lack of information on the causation and underlying anatomy of the lymphatic system. Consequently, therapeutic protocols for pediatric patients remain sparse and with little evidence to support them. In an effort to better understand the causation of primary pediatric lymphedema and to better inform clinical care, we report the use of near-infrared fluorescence lymphatic imaging on the extremities of an alert, 21-month-old boy who presented with unilateral right arm and hand lymphedema at birth. The imaging results indicated an intact, apparently normal lymphatic anatomy with no obvious malformation, but with decreased lymphatic contractile function of the affected upper extremity relative to the contralateral and lower extremities. We hypothesized that the lack of contraction of the lymphatic vessels rather than an anatomic malformation was the source of the unilateral extremity swelling, and that compression and manual lymphatic drainage could be effective treatments., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2017 by the American Academy of Pediatrics.)

Objective: Currently, lymphedema (LED) is typically diagnosed clinically on the basis of a patient's history and characteristic physical findings. Whereas the diagnosis of LED is sometimes confirmed by lymphoscintigraphy (LSG), the technique is limited in both its ability to identify disease and to guide therapy. Recent advancements provide opportunities for new imaging techniques not only to assist in the diagnosis of LED, based on anatomic changes, but also to assess contractile function and to guide therapeutic intervention. The purpose of this contribution was to review these imaging techniques., Methods: Literature for each technique is reviewed and discussed, and the evidence for each of these new diagnostic techniques was assessed on several criteria to determine if each could (1) establish whether disease is present, (2) determine the severity of the disease process, (3) define the pathophysiologic mechanism of the disease process, (4) demonstrate whether intervention is possible as well as what type, and (5) objectively grade the response to therapy., Results: LSG is currently the standard test to confirm LED. Duplex ultrasound (DUS) is a simple, readily available noninvasive test that can identify LED by specific tissue characteristics as well as the response to therapy. Magnetic resonance imaging and computed tomography scans similarly demonstrate the alterations in epidermal and subcutaneous tissue, but the latter can also detect obstructing neoplasms as a cause of secondary LED. Moreover, magnetic resonance lymphangiography details lymphatic vessels and nodes and their function. Newer fluorescence imaging techniques provide opportunities to image lymphatic anatomy and function. Visible microlymphangiography by fluorescein sodium is limited by tissue light absorption to imaging depths of 200 μm. Near-infrared fluorescence lymphatic imaging, a newer test using intradermal injection of indocyanine green, can penetrate several centimeters of tissue and can visualize the initial and conducting lymphatics, the lymph node basins, and the active function of lymphangions (the key module) in exquisite detail., Conclusions: The availability and the noninvasive nature of DUS should make this modality the first choice for establishing the diagnosis of LED based on tissue changes. Further studies comparing DUS with LSG, however, are needed. The costs of magnetic resonance imaging and computed tomography limit their adoption as a means to regularly assess the lymphatics. Whereas lymphatic truncal anatomy and transit times can be delineated by the older technique of LSG, near-infrared fluorescence lymphatic imaging is rapid, highly sensitive, and repeatable and provides exquisite detail for lymphatic vessel anatomy and function of the lymphangions as well as the response to therapy., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown cost effectiveness and quality-of-life benefit of pneumatic compression therapy (PCT) for lymphedema. Insurers, such as the Centers for Medicare/Medicaid (CMS), however, desire visual proof that PCT moves lymph. Near-infrared fluorescence lymphatic imaging (NIRFLI) was used to visualize lymphatic anatomy and function in four subjects with primary and cancer treatment-related lymphedema (LE) of the lower extremities before, during, and after pneumatic compression therapy (PCT). Optically transparent and windowed PCT garments allowed visualization of lymph movement during single, one-hour PCT treatment sessions. Visualization revealed significant extravascular and lymphatic vascular movement of intradermally injected dye in all subjects. In one subject with sufficient patent lymphatic vessels to allow quantification of lymph pumping velocities and frequencies, these values were significantly increased during and after PCT as compared to pre-treatment values. Lymphatic contractile activity in patent lymphatic vessels occurred in concert with the sequential cycling of PCT. Direct visualization revealed increased lymphatic function, during and after PCT therapy, in all lymphedema-affected extremities. Further studies are warranted to assess the effects of PCT pressure and sequences on lymph uptake and movement.

To date, no emerging preclinical or clinical near-infrared fluorescence (NIRF) imaging devices for noninvasive and/or surgical guidance have their performances validated on working standards with SI units of radiance that enable comparison or quantitative quality assurance. In this work, we developed and deployed a methodology to calibrate a stable, solid phantom for emission radiance with International System of Units (SI) units of mW ·sr(-1) ·cm(-2) for use in characterizing the measurement sensitivity of ICCD and IsCMOS detection, signal-to-noise ratio, and contrast. In addition, at calibrated radiances, we assess transverse and lateral resolution of ICCD and IsCMOS camera systems. The methodology allowed demonstration of superior SNR of the ICCD over the IsCMOS technology and superior resolution of the IsCMOS over the ICCD approach. Contrast depended upon the camera settings (binning and integration time) and gain of intensifier. Finally, because the architecture of CMOS and CCD camera systems results in vastly different performance, we comment on the utility of these technologies for small animal imaging as well as clinical applications for noninvasive and surgical guidance.

Background: Recent advancements in near-infrared fluorescence lymphatic imaging (NIRFLI) technology provide opportunities for non-invasive, real-time assessment of lymphatic contribution in the etiology and treatment of ulcers. The objective of this study was to assess lymphatics in subjects with venous leg ulcers using NIRFLI and to assess lymphatic impact of a single session of sequential pneumatic compression (SPC)., Methods: Following intradermal microdoses of indocyanine green (ICG) as a lymphatic contrast agent, NIRFLI was used in a pilot study to image the lymphatics of 12 subjects with active venous leg ulcers (Clinical, Etiologic, Anatomic, and Pathophysiologic [CEAP] C6). The lymphatics were imaged before and after a single session of SPC to assess impact on lymphatic function., Results: Baseline imaging showed impaired lymphatic function and bilateral dermal backflow in all subjects with chronic venous insufficiency, even those without ulcer formation in the contralateral limb (C0 and C4 disease). SPC therapy caused proximal movement of ICG away from the active wound in 9 of 12 subjects, as indicated by newly recruited functional lymphatic vessels, emptying of distal lymphatic vessels, or proximal movement of extravascular fluid. Subjects with the longest duration of active ulcers had few visible lymphatic vessels, and proximal movement of ICG was not detected after SPC therapy., Conclusions: This study provides visible confirmation of lymphatic dysfunction at an early stage in the etiology of venous ulcer formation and demonstrates the potential therapeutic mechanism of SPC therapy in removing excess fluid. The ability of SPC therapy to restore fluid balance through proximal movement of lymph and interstitial fluid may explain its value in hastening venous ulcer healing. Anatomical differences between the lymphatics of longstanding and more recent venous ulcers may have important therapeutic implications., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Although lower extremity edema/lymphedema can result from venous and/or lymphatic abnormalities, effective treatment depends upon understanding their relative contributions to the condition. Herein we use near-infrared fluorescence lymphatic imaging in a 16 year-old female diagnosed with unilateral lymphedema of the right leg and previously treated with left iliac vein stenting in an attempt to alleviate lymphedema. The imaging shows that abnormal lymphatic anatomy, rather than venous occlusion, was likely responsible for unilateral swelling., Competing Interests: Conflict of Interest CEF, JCR and EMS are listed as inventors on patents related to near-infrared fluorescence lymphatic imaging. JCR has received fees for consulting from NIRF Imaging, Inc., a UTHSCH start-up company seeking to commercialize the imaging technology. JCR and EMS may receive future financial benefit from NIRF Imaging, Inc. The other authors declare no potential conflict of interest.

Background: Lymphangiomatosis is a rare disorder of the lymphatic system that can impact the dermis, soft tissue, bone, and viscera and can be characterized by lymphangiomas, swelling, and chylous discharge. Whether disordered lymphangiogenesis in lymphangiomatosis affects the function and anatomy of the entire systemic lymphatic circulation or is localized to specific sites is not fully known., Methods and Results: A 35-year-old Caucasian female diagnosed with whole-body lymphangiomatosis at 2 months of age and who continues to present with progressive disease was imaged with near-infrared fluorescence lymphatic imaging. While the peripheral lymphatics in the extremities appeared largely normal compared to prior studies, we observed tortuous lymphatic vessels, fluorescence drainage from the peripheral lymphatics into lymphangiomas, and extensive dermal lymphatics in the left thigh and inguinal regions where the subject had previously had surgical assaults, potentially indicating defective systemic lymphangiogenesis., Conclusions: Further research into anatomical and functional lymphatic changes associated with the progression and treatment of lymphangiomatosis could aid in understanding the pathophysiology of the disease as well as point to treatment strategies.

The performance evaluation of a variety of small animal tomography measurement approaches and algorithms for recovery of fluorescent absorption cross section has not been conducted. Herein, we employed an intensified CCD system installed in a commercial small animal CT (Computed Tomography) scanner to compare image reconstructions from time-independent, continuous wave (CW) measurements and from time-dependent, frequency domain (FD) measurements in a series of physical phantoms specifically designed for evaluation. Comparisons were performed as a function of (1) number of projections, (2) the level of preprocessing filters used to improve the signal-to-noise ratio (SNR), (3) endogenous heterogeneity of optical properties, as well as in the cases of (4) two fluorescent targets and (5) a mouse-shaped phantom. Assessment of quantitative recovery of fluorescence absorption cross section was performed using a fully parallel, regularization-free, linear reconstruction algorithm with diffusion approximation (DA) and high order simplified spherical harmonics ( SPN) approximation to the radiative transport equation (RTE). The results show that while FD measurements may result in superior image reconstructions over CW measurements, data acquisition times are significantly longer, necessitating further development of multiple detector/source configurations, improved data read-out rates, and detector technology. FD measurements with SP3 reconstructions enabled better quantitative recovery of fluorescent target strength, but required increased computational expense. Despite the developed parallel reconstruction framework being able to achieve more than 60 times speed increase over sequential implementation, further development in faster parallel acceleration strategies for near-real time and real-time image recovery and more precise forward solution is necessary.

This article provides an overview of the recent developments in the diagnosis, treatment, and prevention of cancer-related lymphedema. Lymphedema incidence by tumor site is evaluated. Measurement techniques and trends in patient education and treatment are also summarized to include current trends in therapeutic and surgical treatment options as well as longer-term management. Finally, an overview of the policies related to insurance coverage and reimbursement will give the clinician an overview of important trends in the diagnosis, treatment, and management of cancer-related lymphedema., (© 2014 American Cancer Society.)

The lymphatic vasculature plays a critical role in a number of disease conditions of increasing prevalence, such as autoimmune disorders, obesity, blood vascular diseases, and cancer metastases. Yet, unlike the blood vasculature, the tools available to interrogate the molecular basis of lymphatic dysfunction/disease have been lacking. More recently, investigators have reported that dysregulation of the PI3K pathway is involved in syndromic human diseases that involve abnormal lymphatic vasculatures, but there have been few compelling results that show the direct association of this molecular pathway with lymphatic dysfunction in humans. Using near-infrared fluorescence lymphatic imaging (NIRFLI) to phenotype and next generation sequencing (NGS) for unbiased genetic discovery in a family with non-syndromic lymphatic disease, we discovered a rare, novel mutation in INPPL1 that encodes the protein SHIP2, which is a negative regulator of the PI3K pathway, to be associated with lymphatic dysfunction in the family. In vitro interrogation shows that SHIP2 is directly associated with impairment of normal lymphatic endothelial cell (LEC) behavior and that SHIP2 associates with receptors that are associated in lymphedema, implicating its direct involvement in the lymphatic vasculature.

Objective: Investigational, near-infrared fluorescence (NIRF) lymphatic imaging was used to assess lymphatic architecture and contractile function in participants diagnosed with Dercum's disease, a rare, poorly understood disorder characterized by painful lipomas in subcutaneous adipose tissues., Methods: After informed consent and as part of an FDA-approved feasibility study to evaluate lymphatics in diseases in which their contribution has been implicated, three women diagnosed with Dercum's disease and four control subjects were imaged. Each participant received multiple intradermal and subcutaneous injections of indocyanine green (ICG, total dose ≤400 µg) in arms, legs, and/or trunk. Immediately after injection, ICG was taken up by the lymphatics and NIRF imaging was conducted., Results: The lymphatics in the participants with Dercum's disease were intact and dilated, yet sluggishly propelled lymph when compared to control lymphatics. Palpation of regions containing fluorescent lymphatic pathways revealed tender, fibrotic, tubular structures within the subcutaneous adipose tissue that were associated with painful nodules, and, in some cases, masses of fluorescent tissue indicating that some lipomas may represent tertiary lymphoid tissues., Conclusions: These data support the hypothesis that Dercum's disease may be a lymphovascular disorder and suggest a possible association between abnormal adipose tissue deposition and abnormal lymphatic structure and function., (Copyright © 2014 The Obesity Society.)

Chylothorax is a rare but serious complication in children who undergo heart surgery. Its pathogenesis is poorly understood, and invasive surgical treatments are considered only after conservative management fails. Current diagnostic imaging techniques, which could aid decision making for earlier surgical intervention, are difficult to apply. Herein, we deployed near-infrared fluorescence (NIRF) lymphatic imaging to allow the visualization of abnormal lymphatic drainage in an infant with postoperative chylothorax to guide the choice of surgical management. A 5-week-old male infant, who developed chylothoraces after undergoing Norwood surgery for hypoplastic left heart syndrome, was intradermally administered trace doses of indocyanine green in both feet and the left hand. NIRF imaging was then performed at the bedside to visualize lymphatic drainage patterns. Imaging results indicated impeded lymphatic drainage from the feet toward the trunk with no fluorescence in the chest indicating no leakage of peripheral lymph at the thoracic duct. Instead, lymph drainage occurred from the axilla directly into the pleural cavity. As a result of imaging, left pleurodesis was performed to stop the pleural effusion with the result of temporary decrease of left chest tube drainage. Although additional studies are required to understand normal and abnormal lymphatic drainage patterns in infants, we showed the potential of using NIRF lymphatic imaging at the bedside to visualize the lymphatic drainage pathway to guide therapy. Timely management of chylothorax may be improved by using NIRF imaging to understand lymphatic drainage pathways.

The lymphatic circulatory system has diverse functions in lipid absorption, fluid homeostasis, and immune surveillance and responds dynamically when presented with infection, inflammation, altered hemodynamics, and cancer. Visualization of these dynamic processes in human disease and animal models of disease is key to understanding the contributory role of the lymphatic circulatory system in disease and to devising effective therapeutic strategies. Longitudinal, non-destructive, and repeated imaging is necessary to expand our understanding of disease progression and regression in basic science and clinical investigations. Herein we summarize recent advances in in vivo lymphatic imaging employing magnetic resonance, computed tomography, lymphoscintigraphy, and emerging optical techniques with respect to their contributory roles in both basic science and clinical research investigations.

Purpose: Although fluorescence molecular imaging is rapidly evolving as a new combinational drug/device technology platform for molecularly guided surgery and noninvasive imaging, there remains no performance standards for efficient translation of "first-in-humans" fluorescent imaging agents using these devices., Methods: The authors employed a stable, solid phantom designed to exaggerate the confounding effects of tissue light scattering and to mimic low concentrations (nM-pM) of near-infrared fluorescent dyes expected clinically for molecular imaging in order to evaluate and compare the commonly used charge coupled device (CCD) camera systems employed in preclinical studies and in human investigational studies., Results: The results show that intensified CCD systems offer greater contrast with larger signal-to-noise ratios in comparison to their unintensified CCD systems operated at clinically reasonable, subsecond acquisition times., Conclusions: Camera imaging performance could impact the success of future "first-in-humans" near-infrared fluorescence imaging agent studies.

One limitation of fluorescence molecular imaging that can limit clinical implementation and hamper small animal imaging is the inability to eliminate ambient light. Herein, we demonstrate the ability to conduct rapid non-invasive, far-red and near-infrared fluorescence imaging in living animals and a phantom under ambient light conditions using a modulated image intensified CCD (ICCD) and a laser diode operated in homodyne detection. By mapping AC amplitude from three planar images at varying phase delays, we show improvement in target-to-background ratios (TBR) and reasonable signal-to-noise ratios (SNR) over continuous wave measurements. The rapid approach can be used to accurately collect fluorescence in situations where ambient light cannot be spectrally conditioned or controlled, such as in the case of fluorescent molecular image-guided surgery.

We experimentally and numerically investigated the characteristics of 128 Gb/s dual polarization - quadrature phase shift keying signals received with two types of nonlinear equalizers (NLEs) followed by soft-decision (SD) low-density parity-check (LDPC) forward error correction (FEC). Successful co-operation among SD-FEC and NLEs over various nonlinear transmissions were demonstrated by optimization of parameters for NLEs.

As part of a proof-of-concept study for future delivery of targeted near-infrared fluorescent (NIRF) tracers, we sought to assess the delivery of micrograms of indocyanine green to all the axillary lymph nodes following intraparenchymal breast injections and intradermal arm injections in 20 subjects with advanced breast carcinoma and undergoing complete axillary lymph node dissection. Lymphatic vessels and nodes were assessed in vivo. Ex vivo images demonstrated that 87% of excised lymph nodes, including 81% of tumor-positive lymph nodes, were fluorescent. Future clinical studies using microdose amounts of tumor-targeting NIRF contrast agents may demonstrate improved surgical intervention with reduced morbidity.

Fluorescence gene reporters have recently become available for excitation at far-red wavelengths, enabling opportunities for small animal in vivo gene reporter fluorescence tomography (GRFT). We employed multiple projections of the far-red fluorescence gene reporters IFP1.4 and iRFP, excited by a point source in transillumination geometry in order to reconstruct the location of orthotopically implanted human prostate cancer (PC3), which stably expresses the reporter. Reconstruction was performed using a linear radiative-transfer-based regularization-free tomographic method. Positron emission tomography (PET) imaging of a radiolabeled antibody-based agent that targeted epithelial cell adhesion molecule overexpressed on PC3 cells was used to confirm in vivo GRFT results. Validation of GRFT results was also conducted from ex vivo fluorescence imaging of resected prostate tumor. In addition, in mice with large primary prostate tumors, a combination of GRFT and PET showed that the radiolabeled antibody did not penetrate the tumor, consistent with known tumor transport limitations of large (∼150  kDa) molecules. These results represent the first tomography of a living animal using far-red gene reporters.

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging using indocyanine green (ICG), a dye that has been used for 50 years in humans, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 μl) of ICG (645 μM) or cABD-IRDye800 (200 μM) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 μg/kg for ICG or 113-1,700 μg/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

The work presented herein describes the system design and performance evaluation of a miniaturized near-infrared fluorescence (NIRF) frequency-domain photon migration (FDPM) system with non-contact excitation and homodyne detection capability for small animal fluorescence tomography. The FDPM system was developed specifically for incorporation into a Siemens micro positron emission tomography/computed tomography (microPET/CT) commercial scanner for hybrid small animal imaging, but could be adapted to other systems. Operating at 100 MHz, the system noise was minimized and the associated amplitude and phase errors were characterized to be ±0.7% and ±0.3°, respectively. To demonstrate the tomographic ability, a commercial mouse-shaped phantom with 50 µM IRDye800CW and ⁶⁸Ga containing inclusion was used to associate PET and NIRF tomography. Three-dimensional mesh generation and anatomical referencing was accomplished through CT. A third-order simplified spherical harmonics approximation (SP₃) algorithm, for efficient prediction of light propagation in small animals, was tailored to incorporate the FDPM approach. Finally, the PET-NIRF target co-localization accuracy was analyzed in vivo with a dual-labeled imaging agent targeting orthotopic growth of human prostate cancer. The obtained results validate the integration of time-dependent fluorescence tomography system within a commercial microPET/CT scanner for multimodality small animal imaging.

ALFIA (Automated Lymphatic Function Imaging Analysis), an algorithm providing quantitative analysis of investigational near-infrared fluorescence lymphatic images, is described. Images from nine human subjects were analyzed for apparent lymphatic propagation velocities and propulsion periods using manual analysis and ALFIA. While lymphatic propulsion was more easily detected using ALFIA than with manual analysis, statistical analyses indicate no significant difference in the apparent lymphatic velocities although ALFIA tended to calculate longer propulsion periods. With the base ALFIA algorithms validated, further automation can now proceed to provide a clinically relevant analytic tool for quantitatively assessing lymphatic function in humans.

Current treatment of unilateral breast cancer-related lymphedema (BCRL) is only directed to the afflicted arm. Near-infrared fluorescent imaging (NIRF) of arm lymphatic vessel architecture and function in BCRL and control subjects revealed a trend of increased lymphatic abnormalities in both the afflicted and unafflicted arms with increasing time after lymphedema onset. These pilot results show that BCRL may progress to affect the clinically "normal" arm, and suggest that cancer-related lymphedema may become a systemic, rather than local, malady. These findings support further study to understand the etiology of cancer-related lymphedema and lead to better diagnostics and therapeutics directed to the systemic lymphatic system.

Background: Lymphedema is a complication that may occur after surgical resection and radiation treatment in a number of cancer types and is especially debilitating in regions where treatment options are limited. Although upper and lower extremity lymphedema may be effectively treated with manual lymphatic drainage (MLD) therapies and devices that use compression to direct proximal flow of lymph fluids, head and neck lymphedema is more challenging., Methods and Results: Herein, we describe the compassionate use of an investigatory technique of near-infrared (NIR) fluorescence imaging to understand the lymphatic anatomy and function, help direct MLD, and use 3-dimensional (3D) surface profilometry to monitor response to therapy in a patient with head and neck lymphedema after surgery and radiation treatment., Conclusion: NIR fluorescence imaging provides a mapping of functional lymph vessels for direction of efficient MLD therapy in the head and neck. Additional studies are needed to assess the efficacy of MLD therapy when directed by NIR fluorescence imaging., (Copyright © 2010 Wiley Periodicals, Inc.)

With the aid of indocyanine green (ICG), lymphatic architecture and function in both mice and humans has been successfully imaged non-invasively using near-infrared (NIR) fluorescence imaging devices. Maximal measurement sensitivity of NIR fluorescence imaging devices is needed for "first-in-humans" molecularly targeting NIR fluorescence agents that are brighter than non-specific ICG. In this study, we developed a solid phantom and measurement approach for the quantification of excitation light leakage and measurement sensitivity of NIR fluorescence imaging devices. The constructed solid phantom, consisting of quantum dots impregnated onto specularly reflective surface, shows long-term stability and can be used as a traceable fluorescence standard. With the constructed solid phantom, the intensified CCD (ICCD)-based device demonstrated more than 300% higher measurement sensitivity compared to the Electron Multiplying CCD (EMCCD) based device when integration time was maintained less than 1.0 s.

The lymphatic system is the secondary circulatory system responsible for fluid homeostasis and protein transport in the body. In addition, because the lymphatic system provides a primary pathway for cancer metastasis, lymph node involvement is routinely used as a determinant in cancer staging. Despite their importance, the lymphatics remain poorly understood, in part because of the historic lack of imaging modalities with sufficient spatial and/or temporal resolution to visualize the fine lymphatic structure and subtle contractile function. In recent years, near-infrared fluorescence (NIRF) imaging has emerged as a new imaging modality to non-invasively visualize the lymphatics and assess contractile lymphatic function in humans following administration of microdose amounts of a NIRF contrast agent. In this contribution, we first review NIRF imaging and its clinical application in sentinel lymph node mapping, intraoperative guidance, and assessing the architecture and contractile function of the lymphatics in health and in cancer-related lymphedema. We then present recent NIRF lymphatic imaging for non-invasive assessment of lymphatics both in preclinical melanoma models and in human subjects with melanoma.

The goal of preclinical fluorescence-enhanced optical tomography (FEOT) is to provide three-dimensional fluorophore distribution for a myriad of drug and disease discovery studies in small animals. Effective measurements, as well as fast and robust image reconstruction, are necessary for extensive applications. Compared to bioluminescence tomography (BLT), FEOT may result in improved image quality through higher detected photon count rates. However, background signals that arise from excitation illumination affect the reconstruction quality, especially when tissue fluorophore concentration is low and/or fluorescent target is located deeply in tissues. We show that near-infrared fluorescence (NIRF) imaging with an optimized filter configuration significantly reduces the background noise. Model-based reconstruction with a high-order approximation to the radiative transfer equation further improves the reconstruction quality compared to the diffusion approximation. Improvements in FEOT are demonstrated experimentally using a mouse-shaped phantom with targets of pico- and subpico-mole NIR fluorescent dye.

Objective: To investigate the feasibility of assessing the efficacy of manual lymphatic drainage (MLD), a method for lymphedema (LE) management, by using near-infrared (NIR) fluorescence imaging., Design: Exploratory pilot study., Setting: Primary care unit., Participants: Subjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y)., Intervention: Indocyanine green (25 μg in 0.1 mL each) was injected intradermally in bilateral arms or legs of subjects. Diffused excitation light illuminated the limbs, and NIR fluorescence images were collected by using custom-built imaging systems. Subjects received MLD therapy, and imaging was performed pre- and posttherapy., Main Outcome Measures: Apparent lymph velocities and periods between lymphatic propulsion events were computed from fluorescence images. Data collected pre- and post-MLD were compared and evaluated for differences., Results: By comparing pre-MLD lymphatic contractile function against post-MLD lymphatic function, results showed that average apparent lymph velocity increased in both the symptomatic (+23%) and asymptomatic (+25%) limbs of subjects with LE and control limbs (+28%) of healthy subjects. The average lymphatic propulsion period decreased in symptomatic (-9%) and asymptomatic (-20%) limbs of subjects with LE, as well as in control limbs (-23%)., Conclusions: We showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD., (Copyright © 2011 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Although the importance of lymphatic function is well recognized, the lack of real-time imaging modalities limits our understanding of its role in many diseases. In a phase 0 exploratory study, we used dynamic, near-infrared (NIR) fluorescence imaging to assess the extremes of lymphatic architecture and transport in healthy human subjects and in subjects clinically diagnosed with unilateral lymphedema (LE), a disease that can be prevalent in cancer survivors., Methods and Results: Active lymphatic propulsion was imaged after intradermal injections of 25 µg of indocyanine green (total maximum dose ≤400 µg) bilaterally in the arms or legs of control and subjects. Images show well-defined lymphatic structures with propulsive dye transport in limbs of healthy subjects. In LE subjects, we observed extravascular dye accumulation, networks of fluorescent lymphatic capillaries, and/or tortuous lymphatic vessels in all symptomatic and some asymptomatic limbs. Statistical models indicate that disease status and/or limb significantly affect parameters of apparent lymph propagation velocity and contractile frequency., Conclusions: These clinical research studies demonstrate the potential of NIR fluorescence imaging as a diagnostic measure of functional lymphatics and as a new tool in translational research studies to decipher the role of the lymphatic system in cancer and other diseases.