Fluorescence Sentinel Lymph Node Mapping Research Articles

Comparison of indirect computed tomographic lymphography and near-infrared fluorescence sentinel lymph node mapping for integumentary canine mast cell tumors.

To compare the independent and combined use of indirect computed tomographic lymphography (ICTL) and near-infrared fluorescence (NIRF) for sentinel lymph node (SLN) mapping in dogs with integumentary mast cell tumors (MCT) and report the metastatic LN rate. Prospective clinical study. Twenty client-owned dogs. Dogs underwent preoperative ICTL, then intraoperative NIRF SLN mapping and excision of the anatomic lymph node (ALN) and/or SLN, and primary MCT. Technique agreement was complete if the same SLN was detected, and partial if the same SLN was detected along with additional SLN. No agreement occurred if the techniques detected different or no SLN. MCT were graded using two- and three-tier schemes, and LN were graded from HN0-3; HN2-3 were considered metastatic. Complete, partial, and no agreement between ICTL and NIRF was seen in 8/20 (40%), 8/20 (40%), and 4/20 (20%) dogs, respectively. Detection of ICTL-SLN and NIRF-SLN failed in 1/20 (5%) and 4/20 (20%), respectively. Tumors were grade II/low-grade in 19/20 (95%) and grade III/high-grade in 1/20 (5%) dogs. Nineteen out of 20 (95%) dogs had HN2-3 LN. Technique agreement of at least one SLN was seen in 16/20 (80%) dogs. Although most MCT were classified as intermediate to low grade, LN metastases were commonly detected. Combining ICTL and NIRF for MCT SLN mapping yields high SLN detection rates. Lymph node metastasis may be more common than previously reported for intermediate to low grade MCT.

Fluorescent sentinel lymph node mapping with icg in endometrial cancer. An initial experience in Argentina

Background: Near-infrared fluorescence-based sentinel lymph node (SLN) mapping technique with indocyanine green (ICG) has been found a useful surgical modality for sentinel node detection and disease staging in endometrial cancer (EC) worldwide. Objective: We aimed to introduce the SNL mapping technology using ICG and Near Infrared (NIR) fluorescence in Argentina. We aimed to assess the overall and bilateral detection rate of ICG based SNL mapping along with verifying its feasibility and effectiveness in surgical staging. We also aimed to discover the perceptions of surgeons regarding the use of ICG as a tracer and NIR as light-medium in SNL mapping. Method: ICG SLNs identification was evaluated retrospectively. This study was performed at the Hospital Aleman de Buenos Aires in September, 2017The detection rates were calculated along with the analysis of the number of SNL observed and operative time, and time taken by overall surgery. Subsequently, the surgeons were interviewed to ascertain their opinion of using ICG SNL mapping in endometrial cancer staging. Results: Four patients with endometrial cancer were included for analysis. The overall and bilateral detection rates have been 100% and 75%, respectively. While, under white light, no SLNs were identified. The average operative time of nodes resection was estimated 2.15 minutes. The fluorescence has been used for 2.2 minutes and overall time taken by surgery was estimated 71.5 minutes. The estimated blood loss (EBL) has been lower than 50 ml. The responses of surgeons’ from the interview have been 100% positive. The cost of the procedure based on the amount of dye used was 25 dollars per patient. Conclusion: Laparoscopic ICG SLN resulted to be feasible, practical and no expensive surgical modality for uterine cancer triage.

Robotic single site sentinel lymph node fluorescence detection

: In the last decades, new technologies such as robotics, single-site surgery, and near infrared fluorescence sentinel lymph node mapping has reduced invasiveness in the surgical management of oncological cancer patients. We present our surgical technique to treat low risk endometrial cancer with total hysterectomy and fluorescence sentinel lymph node mapping using robotic single site approach. The Da Vinci® Si System Single port platform, with 3D 8.5 mm firefly endoscope, semi rigid monopolar hook, bipolar forceps and wristed needle holder, has been used. Robotic single-site approach with sentinel lymph node fluorescence detection is feasible and applicable for the treatment of low-risk endometrial cancer.

Fluorescence sentinel lymph node mapping in patients with cervical cancer

Objective:to assess the efficacy of fluorescence sentinel lymph node (SLN) detection with indocyanine green (ICG) in patients with cervical cancer.Materials and methods.We analyzed the results of examination and treatment in 80 patients with morphologically verified stage IA1–IIA1 cervical cancer. All patients underwent laparoscopic surgery with fluorescence SLN detection with ICG using a high-resolution infrared camera.Results.SLNs were detected in 78 out of 80 patients (97.5 %); the rate of bilateral SLN detection was 80.0 % (64 out of 80). SLNs were not identified in 2 patients only (2.5 %). The majority of SLNs (99 out of 176; 56.28 %) were located in the internal group of lymph nodes, near to the common iliac artery bifurcation. Eleven out of 80 patients (13.75 %) were found to have pelvic lymph node metastasis. SLN metastatic lesions were observed in 10 out of 80 patients (12.5 %), whereas only one participant had non-SLN metastasis. Therefore, the sensitivity of the fluorescence method with ICG was 98.75 %.Conclusion.Fluorescence SLN mapping with ICG in patients with stage IA1–IIA1 cervical cancer is an effective diagnostic method allowing SLN topical diagnosis, intraoperative restaging, and correction of the treatment strategy.

Real-Time Fluorescent Sentinel Lymph Node Mapping with Indocyanine Green in Women with Previous Conization Undergoing Laparoscopic Surgery for Early Invasive Cervical Cancer: Comparison with Radiotracer ± Blue Dye

Study ObjectiveTo evaluate the added value of the fluorescence dye indocyanine green (ICG) for sentinel lymph node (SLN) mapping in women with cervical cancer who had undergone previous conization (stage 1A-1B1) by comparing ICG versus Tc99m radiotracer + blue dye (BD). DesignRetrospective study (Canadian Task Force classification II-2). SettingTwo European academic medical centers, San Gerardo Hospital, Italy and University of Berne, Switzerland. PatientsSixty-five women with early stage (IA-IB1) cervical cancer who had undergone previous conization and who underwent SLN mapping with Tc99m ± BD (n = 23) or ICG (n = 42) followed by pelvic lymphadenectomy and fertility-sparing surgery or hysterectomy were included in this analysis. InterventionOverall detection rate and bilateral SLN mapping rates of ICG were compared with those obtained using the standard Tc99m radiocolloid and BD. Measurement and Main ResultsOverall, 220 SLNs were detected. The median number of SLNs per patient in the Tc99m ± BD group was 2 (range, 1–5) and in the ICG group, 3 (range, 2–15). The detection rate of SLNs was 95.7% in Tc99m ± BD group and 100% in the ICG group (p = .354). The women injected with ICG had a higher rate of bilateral mapping of the SLNs as compared with the Tc99m ± BD group (95.2% vs 69.6%, p = .016%). Only 12% of the patients (8/65) presented metastatic nodes, 2 in the Tc99m ± BD group and 6 in the ICG group. ConclusionIn early-stage cervical cancer patients conization had no significant impact on the SLN detection rate using both techniques (ICG and radiotracer ± BD). In this scenario a higher bilateral mapping rate was confirmed using the fluorescent dye ICG rather than the standard techniques.

Indocyanine Green versus Radiotracer with or without Blue Dye for Sentinel Lymph Node Mapping in Stage >IB1 Cervical Cancer (>2 cm)

Study ObjectiveTo compare sentinel lymph node (SLN) mapping in women with cervical cancer stage >IB1 (tumor size >2 cm) using indocyanine green (ICG) versus the standard technique using radioisotope technetium 99m radiocolloid (Tc99m) radiotracer with or without blue dye. DesignEuropean multicenter, retrospective observational study (Canadian Task Force classification II-2). SettingFour academic medical centers. PatientsNinety-five women with stage IB1 cervical cancer (>2 cm) who underwent SLN mapping with Tc99m with or without blue dye or ICG and radical hysterectomy. InterventionThe detection rate and bilateral mapping rate were compared between ICG and standard Tc99m radiotracer with or without blue dye. Lymphadenectomy was performed, and the false-negative rate was assessed. Measurements and Main ResultsForty-seven patients underwent SLN mapping with Tc99m with or without blue dye, and 48 did so with ICG. All patients underwent radical hysterectomy with or without bilateral salpingo-oophorectomy between 2008 and 2016. The overall detection rate of SLN mapping was 91.5% for Tc99m with or without blue dye and 100% for ICG. A 91.7% rate of bilateral migration was achieved for ICG, significantly higher than the 66% obtained with Tc99m with or without blue dye (p = .025). Nine of the 23 SLN-positive patients (39.1%) were diagnosed exclusively as a result of the ultrastaging used to identify micrometastases or isolated tumor cells only. ConclusionsIn advanced cervical cancer (stage IB1 >2 cm), the detection rate and bilateral migration rate on real-time fluorescent SLN mapping were higher with ICG than with Tc99m radiotracer with or without blue dye. SLN mapping and ultrastaging can provide additional information for nodal staging in advanced cervical cancer. In this setting, ICG is a promising tool for mapping, appearing less affected by higher disease stage compared with traditional methods.

Indocyanine green fluorescence imaging of lymph nodes during robotic-assisted laparoscopic operation for endometrial cancer. A prospective validation study using a sentinel lymph node surgical algorithm

ObjectiveA sentinel lymph node (SLN) strategy may have particular value in endometrial cancer (EC) because a therapeutic effect of lymphadenectomy per se is unproven. The aim was to evaluate indocyanine green (ICG) and near-infrared (NIR) fluorescence mapping using a surgical algorithm. MethodsFrom November 2012 through December 2015, women with apparently early stage EC underwent robot-assisted laparoscopic hysterectomy including ICG fluorescence SLN mapping following the Memorial Sloane Kettering Cancer Center (MSKCC) surgical algorithm. ResultsAmong 108 patients included, ≥1 SLNs was identified in 104 (96%), bilaterally in 84 (78%) and unilaterally in 20 patients (18%). Four patients failed SLN mapping. All SLN-positive patients had pelvic SLNs. Median number of nodes were 4.0 and 6.0 (p<0.001), when SLNs only and SLNs plus non-SLNs were removed, respectively. Lymph node metastases were detected in 17 patients (16%). One patient who failed SLN mapping had a non-SLN metastasis. The remaining 16 patients had metastases in SLNs, 12 in SLNs only and four in both SLNs and non-SLNs. Routine pathology detected 75% of patients with cancer positive SLNs while 25% were based on extended pathology. Lymph node metastases were found among 9% with low-, 11% with intermediate- and 32% with high-risk profiles, respectively. ConclusionsWe have reproduced the high total and bilateral SLN mapping using cervical ICG injection and NIR fluorescence. Practical application of the MSKCC algorithm allowed high lymph node metastasis detection in combination with a low extent of lymph node removal.

Implementation of indocyanine green and near-infrared fluorescence sentinel lymph node mapping in endometrial and cervical cancer at a community-based academic hospital

Implementation of indocyanine green and near-infrared fluorescence sentinel lymph node mapping in endometrial and cervical cancer at a community-based academic hospital

55. Near-infrared fluorescence sentinel lymph node mapping in patients with muscle-invasive bladder cancer

55. Near-infrared fluorescence sentinel lymph node mapping in patients with muscle-invasive bladder cancer

Near-infrared fluorescence sentinel lymph node mapping in breast cancer: a multicenter experience

Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has the potential to improve the sentinel lymph node (SLN) procedure by facilitating percutaneous and intraoperative identification of lymphatic channels and SLNs. Previous studies suggested that a dose of 0.62mg (1.6mL of 0.5mM) ICG is optimal for SLN mapping in breast cancer. The aim of this study was to evaluate the diagnostic accuracy of NIR fluorescence for SLN mapping in breast cancer patients when used in conjunction with conventional techniques. Study subjects were 95 breast cancer patients planning to undergo SLN procedure at either the Dana-Farber/Harvard Cancer Center (Boston, MA, USA) or the Leiden University Medical Center (Leiden, the Netherlands) between July 2010 and January 2013. Subjects underwent the standard-of-care SLN procedure at each institution using (99)Technetium-colloid in all subjects and patent blue in 27 (28%) of the subjects. NIR fluorescence-guided SLN detection was performed using the Mini-FLARE imaging system. SLN identification was successful in 94 of 95 subjects (99%) using NIR fluorescence imaging or a combination of both NIR fluorescence imaging and radioactive guidance. In 2 of 95 subjects, radioactive guidance was necessary for initial in vivo identification of SLNs. In 1 of 95 subjects, NIR fluorescence was necessary for initial in vivo identification of SLNs. A total of 177 SLNs (mean 1.9, range 1-5) were resected: 100% NIR fluorescent, 88% radioactive, and 78% (of 40 nodes) blue. In 2 of 95 subjects (2.1%), SLNs-containing macrometastases were found only by NIR fluorescence, and in one patient this led to upstaging to N1. This study demonstrates the safe and accurate application of NIR fluorescence imaging for the identification of SLNs in breast cancer patients, but calls into question what technique should be used as the gold standard in future studies.

Effective Low-dose Escalation of Indocyanine Green for Near-infrared Fluorescent Sentinel Lymph Node Mapping in Melanoma

Regional lymph node metastasis is the strongest prognostic factor in patients with melanoma. Published reports that used lymphoscintigraphy with radioactive colloids and blue dye demonstrated accurate sentinel lymph node (SLN) identification in inguinal nodes and axillary nodes, but decreased accuracy in cervical, popliteal, epitrochlear, and parascapular nodes. Near-infrared imaging (NIR) may utilize indocyanine green (ICG) to improve SLN identification. The safety, feasibility and optimal dose of albumin-bound ICG (ICG:HSA) was assessed by NIR to improve SLN mapping in patients with melanoma. Twenty-five consecutive patients with biopsy-proven melanoma underwent standard SLN mapping with preoperatively administered technetium-99 m nanocolloid (Tc-99 m). Intraoperative NIR fluorescence imaging was performed after injection of 1.0 ml of 100, 250 or 500 μM of ICG:HSA in four quadrants around the primary lesion. NIR fluorescent imaging demonstrated accuracy of 98 % when compared with radioactive colloid. A total of 65 lymph nodes were identified (65 with Tc-99 m, 64 with ICG:HSA). Overall, successful mapping that used either technique was 96 % as one patient failed to map with either modality. As the dose of ICG was increased, the signal-to-background ratio increased from a median of 3.1 to 8.4 to 10.9 over the range of 100, 250, and 500 μM, respectively. SLN mapping with ICG:HSA is feasible and accurate in melanoma. ICG has the added advantage of a low cost and an intraoperative technique that does not alter the surgical field, thus allowing for easy identification of SLNs.

46. Near-infrared fluorescence sentinel lymph node mapping of the oral cavity in head and neck cancer patients

46. Near-infrared fluorescence sentinel lymph node mapping of the oral cavity in head and neck cancer patients

121. Dose optimization for near-infrared fluorescence sentinel lymph node mapping in melanoma patients

Background: Regional lymph node involvement is the most important prognostic factor in cutaneous melanoma. Since only 20% of melanoma patients have occult nodal disease and would benefit from a regional lymphadenectomy, the sentinel lymph node (SLN) biopsy was introduced. NIR fluorescence has been hypothesized to improve SLN mapping. The objective was to assess the potential of intraoperative near-infrared (NIR) fluorescence imaging to improve SLN mapping in melanoma patients and to establish the optimal dose of indocyanine green adsorbed to human serum albumin (ICG:HSA).

Near-infrared fluorescence sentinel lymph node mapping of the oral cavity in head and neck cancer patients

Elective neck dissection is frequently performed during surgery in head and neck cancer patients. The sentinel lymph node (SLN) procedure can prevent the morbidity of a neck dissection and improve lymph node staging by fine pathology. Near-infrared (NIR) fluorescence imaging is a promising technique to identify the sentinel lymph node (SLN) intraoperatively. This feasibility study explored the use of indocyanine green adsorbed to human serum albumin (ICG:HSA) for SLN mapping in head and neck cancer patients. A total of 10 consecutive patients with oral cavity or oropharyngeal cancer and a clinical N0 neck were included. After exposure of the neck, 1.6 mL of ICG:HSA (500 μM) was injected at four quadrants around the tumor. During the neck dissection, levels I-IV were measured for fluorescence using the Mini-FLARE imaging system. In all 10 patients, NIR fluorescence imaging enabled visualization of one or more SLNs. A total of 17 SLNs were identified. The mean contrast between the fluorescent signal of the lymph nodes and of the surrounding tissue was 8.7±6.4. In 3 patients, of which 1 was false-negative, lymph node metastases were found. After administration of ICG:HSA, the average number of fluorescent lymph nodes significantly increased over time (P<0.001). This study demonstrated feasibility to detect draining lymph nodes in head and neck cancer patients using NIR fluorescence imaging. However, the fluorescent tracer quickly migrated beyond the SLN to higher tier nodes.

Optimization of Near-Infrared Fluorescent Sentinel Lymph Node Mapping in Cervical Cancer Patients

In early cervical cancer, a total pelvic lymphadenectomy is the standard of care, even though most patients have negative nodes and thus undergo lymphadenectomy unnecessarily. Although the value of sentinel lymph node (SLN) mapping in early-stage cervical cancer has not yet been established, near-infrared (NIR) fluorescence imaging is a promising technique to perform this procedure. Near-infrared fluorescence imaging is based on invisible NIR light and can provide high sensitivity, high-resolution, and real-time image guidance during surgery. Clinical trial subjects were 9 consecutive cervical cancer patients undergoing total pelvic lymphadenectomy. Before surgery, 1.6 mL of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected transvaginally and submucosally in 4 quadrants around the tumor. Patients were allocated to 500-, 750-, or 1000-μM ICG:HSA concentration groups. The Mini-FLARE imaging system was used for NIR fluorescence detection and quantitation. Sentinel lymph nodes were identified in all 9 patients. An average of 3.4 ± 1.2 SLNs was identified per patient. No differences in signal to background of the SLNs between the 500-, 750-, and 1000-μM dose groups were found (P = 0.73). In 2 patients, tumor-positive lymph nodes were found. In both patients, tumor-positive lymph nodes confirmed by pathology were also NIR fluorescent. This study demonstrated preliminary feasibility to successfully detect SLNs in cervical cancer patients using ICG:HSA and the Mini-FLARE imaging system. When considering safety, cost-effectiveness, and pharmacy preferences, an ICG:HSA concentration of 500 μM was optimal for SLN mapping in cervical cancer patients.

Optimization of near-infrared fluorescent sentinel lymph node mapping for vulvar cancer

Near-infrared fluorescence imaging has the potential to improve sentinel lymph node mapping in vulvar cancer, which was assessed in the current study. Furthermore, dose optimization of indocyanine green adsorbed to human serum albumin was performed. Nine vulvar cancer patients underwent the standard sentinel lymph node procedure using (99m)technetium-nancolloid and patent blue. In addition, intraoperative imaging was performed after peritumoral injection of 1.6 mL of 500, 750, or 1000 μM of indocyanine green adsorbed to human serum albumin. Near-infrared fluorescence sentinel lymph node mapping was successful in all patients. A total of 14 sentinel lymph nodes (average, 1.6; range, 1-4) were detected: 14 radioactive (100%), 11 blue (79%), and 14 near-infrared fluorescent (100%). This study demonstrates feasibility and accuracy of sentinel lymph node mapping using indocyanine green adsorbed to human serum albumin. Considering safety, cost, and pharmacy preferences, an indocyanine green adsorbed to human serum albumin concentration of 500 μM appears optimal for sentinel lymph node mapping in vulvar cancer.

Optimization of Coded Aperture Radioscintigraphy for Sentinel Lymph Node Mapping

Radioscintigraphic imaging during sentinel lymph node (SLN) mapping could potentially improve localization; however, parallel-hole collimators have certain limitations. In this study, we explored the use of coded aperture (CA) collimators. Equations were derived for the six major dependent variables of CA collimators (i.e., masks) as a function of the ten major independent variables, and an optimized mask was fabricated. After validation, dual-modality CA and near-infrared (NIR) fluorescence SLN mapping were performed in pigs. Mask optimization required the judicious balance of competing dependent variables, resulting in sensitivity of 0.35%, XY resolution of 2.0 mm, and Z resolution of 4.2 mm at an 11.5-cm field of view. The findings in pigs suggested that NIR fluorescence imaging and CA radioscintigraphy could be complementary, but present difficult technical challenges. This study lays the foundation for using CA collimation for SLN mapping, and also exposes several problems that require further investigation.

Toward Optimization of Imaging System and Lymphatic Tracer for Near-Infrared Fluorescent Sentinel Lymph Node Mapping in Breast Cancer

BackgroundNear-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers.Materials and MethodsA small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using 99mTechnetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 μM.ResultsThe Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1–3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 μM. No adverse reactions were observed.ConclusionsWe describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-011-1566-x) contains supplementary material, which is available to authorized users.

Image-guided near-infrared fluorescent sentinel lymph node mapping in human breast cancer

e11591 Background: Breast cancer surgery is presently performed without real-time image-guidance. We have developed a novel optical imaging system for image-guided surgery that uses invisible near-infrared (NIR) fluorescent light to highlight structures on the surgical field with high sensitivity, specificity, and contrast. We have also performed the first human clinical trial of the imaging system in women undergoing SLN mapping for breast cancer. Methods: We used a portable imaging system with an articulating arm that has 6 degrees of freedom, high power LED light source, custom optics, custom software, and sterile drape. The imaging system provided simultaneous and real-time imaging of color video and NIR fluorescence at up to 15 frames per second. N = 6 women with biopsy- confirmed breast cancer undergoing SLN mapping gave informed consent. All subjects received conventional mapping with Tc-99m sulfur colloid using a handheld gamma probe as well as NIR fluorescence-guided SLN mapping using a mixture of indocyanine green (ICG) diluted to a final concentration of 10 μM in human serum albumin (ICG:HSA). Results: The imaging system was easy to position in the operating room, with the articulating arm providing 50” horizontal reach and 70” vertical reach. Working distance to the patient was 18”. NIR fluorescence excitation was 20 mW/cm2 at 760 nm. NIR-depleted white light was 40,000 lux. A total of 1.6 ml of ICG:HSA was injected intra-tumorally and peri-tumorally and the site massaged for 5 min. 8 of 9 SLNs identified by Tc- 99m sulfur colloid were also identified by NIR fluorescence. However, NIR fluorescence identified an SLN, confirmed to have cancer in it, that was not identified by Tc-99m sulfur colloid. These differences were consistent with asynchrony in the injection techniques. Unlike the gamma-ray probe, NIR fluorescence provided high-resolution, large area optical imaging of the surgical field, and helped guide surgical resection. Conclusions: In this 6-patient pilot study, a novel NIR fluorescence optical imaging system was used for the first time, and provided real-time image-guided surgery for SLN mapping of breast cancer. No significant financial relationships to disclose.

Organic Alternatives to Quantum Dots for Intraoperative Near-Infrared Fluorescent Sentinel Lymph Node Mapping

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.