Search

Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively.

Considerable advances in cancer-specific optical imaging have improved the precision of tumor resection. In comparison to traditional imaging modalities, this technology is unique in its ability to provide real-time feedback to the operating surgeon. Given the significant clinical implications of optical imaging, there is an urgent need to standardize surgical navigation tools and contrast agents to facilitate swift regulatory approval. Because fluorescence-enhanced surgery requires a combination of both device and drug, each may be developed in conjunction, or separately, which are important considerations in the approval process. This report is the result of a one-day meeting held on May 4, 2016 with officials from the National Cancer Institute, the FDA, members of the American Society of Image-Guided Surgery, and members of the World Molecular Imaging Society, which discussed consensus methods for FDA-directed human testing and approval of investigational optical imaging devices as well as contrast agents for surgical applications. The goal of this workshop was to discuss FDA approval requirements and the expectations for approval of these novel drugs and devices, packaged separately or in combination, within the context of optical surgical navigation. In addition, the workshop acted to provide clarity to the research community on data collection and trial design. Reported here are the specific discussion items and recommendations from this critical and timely meeting. Cancer Res; 77(9); 2197–206. ©2017 AACR.

The ability to predict the future viability of tissue while still in the operating room and able to intervene would have a major impact on patient outcome. Although several objective methods to evaluate tissue perfusion have been reported, none to date has sufficient accuracy. In eight Sprague-Dawley rats, reverse McFarlane dorsal skin flaps were created. Continuous near-infrared fluorescence angiography using indocyanine green was performed immediately after surgery, for a total of 30 minutes. These dynamic measurements were used to quantify indocyanine green biodistribution and clearance, and to develop a simple metric that accurately predicted tissue viability at postoperative day 7. The new metric was compared to previously described metrics. Reproducible patterns of indocyanine green biodistribution and clearance from the flap permitted quantitative metrics to be developed for predicting flap viability at postoperative day 7. Previously described metrics, which set the boundary between healthy and necrotic tissue as either 17 or 25 percent of peak near-infrared fluorescence at 2 minutes after indocyanine green injection, underestimated the area of necrosis by 75 and 48 percent, respectively. Our data suggest that both the shape and area of clinical necrosis occurring at postoperative day 7 can be predicted intraoperatively, with the boundary defined as near-infrared fluorescence intensities of 40 to 55 percent of peak fluorescence measured at 5 minutes. Two 750-msec intraoperative near-infrared fluorescence images obtained at time 0 and at 5 minutes after injection of indocyanine green accurately predicted skin flap viability 7 days after surgery.

Iatrogenic injury of the ureters is a feared complication of abdominal surgery. Zwitterionic near-infrared fluorophores are molecules with geometrically-balanced, electrically-neutral surface charge, which leads to renal-exclusive clearance and ultralow non-specific background binding. Such molecules could solve the ureter mapping problem by providing real-time anatomic and functional imaging, even through intact peritoneum. Here we present the first-in-human experience of this chemical class, as well as the efficacy study in patients undergoing laparoscopic abdominopelvic surgery. The zwitterionic near-infrared fluorophore ZW800-1 is safe, has pharmacokinetic properties consistent with an ideal blood pool agent, and rapid elimination into urine after a single low-dose intravenous injection. Visualization of structure and function of the ureters starts within minutes after ZW800-1 injection and lasts several hours. Zwitterionic near-infrared fluorophores add value during laparoscopic abdominopelvic surgeries and could potentially decrease iatrogenic urethral injury. Moreover, ZW800-1 is engineered for one-step covalent conjugatability, creating possibilities for developing novel targeted ligands., Iatrogenic injury of the ureters is a feared complication of laparoscopic abdominal surgery. Here the authors present the NIR fluorophore ZW800-1 as an intraoperative imaging agent for ureter mapping, showing its safety, pharmacokinetic properties, and efficacy in healthy volunteers and patients undergoing abdominopelvic surgery.

BACKGROUND Vascularized composite allotransplantation represents an important advancement in the field of reconstructive microsurgery and has continued to increase in popularity. The significant clinical morbidity associated with flap failure represents an important barrier to even more widespread use of these techniques. Early identification of vascular compromise has been associated with a higher salvage rate, yet most surgeons rely only on clinical assessment intraoperatively. Spatial frequency domain imaging (SFDI) presents a noncontact, objective measurement of tissue oxygenation over a large field of view. This study aims to evaluate the use of SFDI technology in hemifacial composite flap compromise as could occur during facial transplant. METHODS Six composite hemifacial flaps were created in three 35-kg Yorkshire pigs and continuously imaged using SFDI before, during, and after 15-minute selective vascular pedicle occlusion. Arterial and venous clamping trials were performed for each flap. Changes in oxyhemoglobin concentration, deoxyhemoglobin concentration, and total hemoglobin were quantified over time. RESULTS The SFDI successfully measured changes in oxygenation parameters in all 6 composite tissue flaps. Significant changes in oxyhemoglobin, deoxyhemoglobin, and total hemoglobin were seen relative to controls. Early and distinct patterns of alteration were noted in arterial and in venous compromise relative to one another. CONCLUSIONS The need for noninvasive, reliable assessment of composite tissue graft viability is apparent, given the morbidity associated with flap failure. The results of this study suggest that SFDI technology shows promise in providing intraoperative guidance with regard to pedicle vessel integrity during reconstructive microsurgery.

The adrenal glands (AGs) are relatively small yet require definitive identification during their resection, or more commonly their avoidance. To enable image-guided surgery involving the AGs, we have developed novel near-infrared (NIR) fluorophores that target the AGs after a single intravenous injection, which provided dual-NIR image-guided resection or avoidance of the AGs during both open and minimally-invasive surgery.

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA-targeted near-infrared fluorescent (NIRF) tracer, based on a disulfide-stabilized single-chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell-based plate assays and orthotopic colorectal (HT-29, well differentiated) and pancreatic (BXPC-3, poorly differentiated) xenogeneic human-mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor-to-background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent-guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.

Pre- and intraoperative diagnostic techniques facilitating tumor staging are of paramount importance in colorectal cancer surgery. The urokinase receptor (uPAR) plays an important role in the development of cancer, tumor invasion, angiogenesis, and metastasis and over-expression is found in the majority of carcinomas. This study aims to develop the first clinically relevant anti-uPAR antibody-based imaging agent that combines nuclear (111In) and real-time near-infrared (NIR) fluorescent imaging (ZW800-1). Conjugation and binding capacities were investigated and validated in vitro using spectrophotometry and cell-based assays. In vivo, three human colorectal xenograft models were used including an orthotopic peritoneal carcinomatosis model to image small tumors. Nuclear and NIR fluorescent signals showed clear tumor delineation between 24h and 72h post-injection, with highest tumor-to-background ratios of 5.0 ± 1.3 at 72h using fluorescence and 4.2 ± 0.1 at 24h with radioactivity. 1-2 mm sized tumors could be clearly recognized by their fluorescent rim. This study showed the feasibility of an uPAR-recognizing multimodal agent to visualize tumors during image-guided resections using NIR fluorescence, whereas its nuclear component assisted in the pre-operative non-invasive recognition of tumors using SPECT imaging. This strategy can assist in surgical planning and subsequent precision surgery to reduce the number of incomplete resections.

Background Advances in microsurgical techniques have increased the use of free tissue transfer. Methods of intraoperative flap perfusion assessment, however, still rely primarily on subjective evaluation of traditional clinical parameters. Anastomotic thrombosis, if not expeditiously identified and revised, can result in flap loss with significant associated morbidity. This study aims to evaluate the use of near-infrared (NIR) fluorescence imaging in the assessment of microsurgical anastomotic patency, thrombosis, and vascular revision. Materials and Methods A model of pedicle thrombosis was created using bilateral abdominal flaps isolated on deep superior epigastric vascular pedicles in four Yorkshire pigs. Following flap elevation, microvascular arterial and venous anastomoses were performed unilaterally, preserving an intact contralateral control flap. Thrombosis was induced at the arterial anastomosis site using ferric chloride, and both flaps imaged using NIR fluorescence angiography. The thrombosed vascular segments were subsequently excised and new anastomoses performed to restore flow. Follow-up imaging of both flaps was then obtained to confirm patency using fluorescence imaging technology. Results Pedicled abdominal flaps were created and successful anastomotic thrombosis was induced unilaterally in each pig. Fluorescence imaging technology identified large decreases in tissue perfusion of the thrombosed flap within 2 minutes. After successful revision anastomosis, NIR imaging demonstrated dramatic increase in flow to the reconstructed flap, but intensity did not return to pre-thrombosis levels. Conclusions Early identification of anastomotic thrombosis is important in successful free tissue transfer. Real-time, intraoperative evaluation of flap perfusion, anastomotic thrombosis, and successful revision can be performed using NIR fluorescence imaging. © 2015 Wiley Periodicals, Inc. Microsurgery 35:309–314, 2015.

Objective: Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. Design: After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. Results: T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR ≥ 2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Conclusion: Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.

Background Several studies demonstrated that intraoperative near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) identifies (sub)capsular colorectal liver metastases (CRLM) missed by other techniques. It is unclear if this results in any survival benefit. This study evaluates long-term follow-up after NIRF-guided resection of CRLM using ICG. Methods First, patients undergoing resection of CRLM with or without NIRF imaging were analyzed retrospectively. Perioperative details, liver-specific recurrence-free interval and overall survival were compared. Second, the prognosis of patients in whom additional metastases were identified solely by NIRF was studied. Results Eighty-six patients underwent resection with NIRF imaging and 87 without. In significantly more patients of the NIRF imaging cohort additional metastases were identified during surgery (25% vs. 13%, p = 0.04). Tumors identified solely by NIRF imaging were significantly smaller compared to additional metastases identified also by inspection, palpation or intraoperative ultrasound (3.2 ± 1.8 mm vs. 7.4 ± 2.6 mm, p < 0.001). Liver-specific recurrence-free survival at 4 years was 47% with NIRF imaging and 39% without (hazard ratio at multivariate analysis 0.73, 95% CI 0.42–1.28, p = 0.28). Overall survival at 4 years was 62% and 59%, respectively (p = 0.79). No liver recurrences occurred within 3 years follow-up in 52% of patients in whom additional metastases were resected based on only NIRF imaging. Conclusions This study suggests that NIRF imaging identifies significantly more and smaller tumors during resection of CRLM, preventing recurrences in a subset of patients. Given its safety profile and low expense, routine use can be considered until tumor targeting fluorescent tracers are clinically available.

Background Tumor recurrence after radical resection of hepatic tumors is not uncommon, suggesting that malignant lesions are missed during surgery. Intraoperative navigation using fluorescence guidance is an innovative technique enabling real-time identification of (sub)capsular liver tumors. The objective of the current study was to compare fluorescence imaging (FI) and conventional imaging modalities for laparoscopic detection of both primary and metastatic tumors in the liver. Methods Patients undergoing laparoscopic resection of a malignant hepatic tumor were eligible for inclusion. Patients received standard of care, including preoperative CT and/or MRI. In addition, 10 mg indocyanine green was intravenously administered 1 day prior to surgery. After introduction of the laparoscope, inspection, FI, and laparoscopic ultrasonography (LUS) were performed. Histopathological examination of resected suspect tissue was considered the gold standard. Results Twenty-two patients suspected of having hepatocellular carcinoma (n = 4), cholangiocarcinoma (n = 2) or liver metastases from colorectal carcinoma (n = 12), uveal melanoma (n = 2), and breast cancer (n = 2) were included. Two patients were excluded because their surgery was unexpectedly postponed several days. Twenty-six malignancies were resected in the remaining 20 patients. Sensitivity for various modalities was 80 % (CT), 84 % (MRI), 62 % (inspection), 86 % (LUS), and 92 % (FI), respectively. Three metastases (12 %) were identified solely by FI. All 26 malignancies could be detected by combining LUS and FI (100 % sensitivity). Conclusion This study demonstrates added value of FI during laparoscopic resections of several hepatic tumors. Although larger series will be needed to confirm long-term patient outcome, the technology already aids the surgeon by providing real-time fluorescence guidance. Electronic supplementary material The online version of this article (doi:10.1007/s00464-016-5007-6) contains supplementary material, which is available to authorized users.

Background and objectives Unlike other cancers, the Sentinel Lymph Node (SLN) procedure in bladder cancer requires special attention to the injection technique. The aim of this study was to assess feasibility and to optimize tracer injection technique for SLN mapping in bladder cancer patients using NIR fluorescence imaging. Methods Twenty patients with invasive bladder cancer scheduled for radical cystectomy were prospectively enrolled. Indocyanine green (ICG) bound to human serum albumin (complex ICG:HSA; 500 µM) was injected peritumourally to permit SLN mapping. ICG:HSA was first administrated serosally (n = 5), and subsequently mucosally by cystoscopic injection (n = 15). In the last cohort of 12 patients treated with cystoscopic injection, the bladder was kept filled with saline for at least 15 min. Results Fluorescent lymph nodes were observed only in the patient group with cystoscopic injection of ICG:HSA. Filling of the bladder post-injection was of added value to promote drainage of ICG:HSA to the lymph nodes, and in 11 of these 12 patients (92%) one or more NIR fluorescent lymph nodes were identified. Conclusions The current study demonstrates proof-of-principle of using NIR fluorescence imaging for SLN identification in bladder cancer. Cystoscopic injection with distension of the bladder appears optimal for SLN mapping. J. Surg. Oncol. 2014 110:845–850. © 2014 Wiley Periodicals, Inc.

The resection of regional lymph nodes in the basin of a primary tumor is of paramount importance in surgical oncology. Although sentinel lymph node mapping is now the standard of care in breast cancer and melanoma, over 20% of patients require a completion lymphadenectomy. Yet, there is currently no technology available that can image all lymph nodes in the body in real time, or assess both the sentinel node and all nodes simultaneously. In this study, we report an optical fluorescence technology that is capable of simultaneous mapping of pan lymph nodes (PLNs) and sentinel lymph nodes (SLNs) in the same subject. We developed near-infrared fluorophores, which have fluorescence emission maxima either at 700 nm or at 800 nm. One was injected intravenously for identification of all regional lymph nodes in a basin, and the other was injected locally for identification of the SLN. Using the dual-channel FLARE intraoperative imaging system, we could identify and resect all PLNs and SLNs simultaneously. The technology we describe enables simultaneous, real-time visualization of both PLNs and SLNs in the same subject.

Background: During laparoscopic cholecystectomy, common bile duct (CBD) injury is a rare but severe complication. To reduce the risk of injury, near-infrared (NIR) fluorescent cholangiography using indocyanine green (ICG) has recently been introduced as a novel method of visualizing the biliary system during surgery. To date, several studies have shown feasibility of this technique; however, liver background fluorescence remains a major problem during fluorescent cholangiography. The aim of the current study was to optimize ICG dose and timing for NIR cholangiography using a quantitative intraoperative camera system during open hepatopancreatobiliary (HPB) surgery. Subsequently, these results were validated during laparoscopic cholecystectomy using a laparoscopic fluorescence imaging system. Methods: Twenty-seven patients who underwent NIR imaging using the Mini-FLARE image-guided surgery system during open HPB surgery were analyzed to assess optimal dosage and timing of ICG administration. ICG was intravenously injected preoperatively at doses of 5, 10, and 20 mg, and imaged at either 30 min (early) or 24 h (delayed) post-injection. Next, the optimal doses found for early and delayed imaging were applied to two groups of seven patients (n = 14) undergoing laparoscopic NIR fluorescent cholangiography during laparoscopic cholecystectomy. Results: Median liver-to-background contrast was 23.5 (range 22.1-35.0), 16.8 (range 11.3-25.1), 1.3 (range 0.7-7.8), and 2.5 (range 1.3-3.6) for 5 mg/30 min, 10 mg/30 min, 10 mg/24 h, and 20 mg/24 h, respectively. Fluorescence intensity of the liver was significantly lower in the 10 mg delayed-imaging dose group compared with the early imaging 5 and 10 mg dose groups (p = 0.001), which resulted in a significant increase in CBD-to-liver contrast ratio compared with the early administration groups (p < 0.002). These findings were qualitatively confirmed during laparoscopic cholecystectomy. Conclusion: This study shows that a prolonged interval between ICG administration and surgery permits optimal NIR cholangiography with minimal liver background fluorescence. © 2013 Springer Science+Business Media.

Nerve preservation is an important issue during most surgery because accidental transection or injury results in significant morbidity, including numbness, pain, weakness, or paralysis. Currently, nerves are still identified only by gross appearance and anatomical location during surgery, without intraoperative image guidance. Near-infrared (NIR) fluorescent light, in the wavelength range of 650-900 nm, has the potential to provide high-resolution, high-sensitivity, and real-time avoidance of nerve damage, but only if nerve-specific NIR fluorophores can be developed. In this study, we evaluated a series of Oxazine derivatives to highlight various peripheral nerve structures in small and large animals. Among the targeted fluorophores, Oxazine 4 has peak emission near into the NIR, which provided nerve-targeted signal in the brachial plexus and sciatic nerve for up to 12 h after a single intravenous injection. In addition, recurrent laryngeal nerves were successfully identified and highlighted in real time in swine, which could be preserved during the course of thyroid resection. Although optical properties of these agents are not yet optimal, chemical structure analysis provides a basis for improving these prototype nerve-specific NIR fluorophores even further.

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Background. Intraoperative identification of parathyroid adenomas can be challenging. We hypothesized that low-doses methyl- ene blue (MB) and near-infrared fluorescence (NIRF) imaging could be used to identify parathyroid adenomas intraoperatively. Methods. MB was injected intravenously after exploration at a dose of 0.5 mg/kg into 12 patients who underwent parathyroid surgery. NIRF imaging was performed using the Mini-FLARE imaging system. Results. In 10 of 12 patients, histology confirmed a parathyroid ade- noma. In 9 of these patients, NIRF could clearly identify the parathyroid adenoma during surgery. Seven of these 9 patients had a positive preoperative 99m Tc-sestamibi single photon emission CT (SPECT) scan. Importantly, in 2 patients, parathyroid adenomas could be identified only using NIRF. Conclusion. This is the first study to show that low-dose MB can be used as NIRF tracer for identification of parathyroid adenomas, and suggests a correlation with preoperative 99m Tc-sestamibi SPECT scan- ning. V C 2013 Wiley Periodicals, Inc. Head Neck 36: 853-858, 2014

Facial transplantation is a complex procedure that corrects severe facial defects due to traumas, burns, and congenital disorders. Although face transplantation has been successfully performed clinically, potential risks include tissue ischemia and necrosis. The vascular supply is typically based on the bilateral neck vessels. As it remains unclear whether perfusion can be based off a single pedicle, this study was designed to assess perfusion patterns of facial transplant allografts using near-infrared (NIR) fluorescence imaging.Upper facial composite tissue allotransplants were created using both carotid artery and external jugular vein pedicles in Yorkshire pigs. A flap validation model was created in n = 2 pigs and a clamp occlusion model was performed in n = 3 pigs. In the clamp occlusion models, sequential clamping of the vessels was performed to assess perfusion. Animals were injected with indocyanine green and imaged with NIR fluorescence. Quantitative metrics were assessed based on fluorescence intensity.With NIR imaging, arterial perforators emitted fluorescence indicating perfusion along the surface of the skin. Isolated clamping of one vascular pedicle showed successful perfusion across the midline based on NIR fluorescence imaging. This perfusion extended into the facial allograft within 60 s and perfused the entire contralateral side within 5 min.Determination of vascular perfusion is important in microsurgical constructs as complications can lead to flap loss. It is still unclear if facial transplants require both pedicles. This initial pilot study using intraoperative NIR fluorescence imaging suggests that facial flap models can be adequately perfused from a single pedicle.

INTRODUCTION: Although various methods exist for monitoring flaps during reconstructive surgery, surgeons primarily rely on assessment of clinical judgment. Early detection of vascular complications improves rate of flap salvage. Spatial frequency domain imaging (SFDI) is a promising new technology that provides oxygenation images over a large field of view. The goal of this clinical pilot study is to use SFDI in perforator flap breast reconstruction. METHODS: Three women undergoing unilateral breast reconstruction after mastectomy were enrolled for our study. The SFDI system was deployed in the operating room, and images acquired over the course of the operation. Time points included images of each hemiabdominal skin flap before elevation, the selected flap after perforator dissection, and after microsurgical transfer. RESULTS: Spatial frequency domain imaging was able to measure tissue oxyhemoglobin concentration (ctO2Hb), tissue deoxyhemoglobin concentration, and tissue oxygen saturation (stO2). Images were created for each metric to monitor flap status and the results quantified throughout the various time points of the procedure. For 2 of 3 patients, the chosen flap had a higher ctO2Hb and stO2. For 1 patient, the chosen flap had lower ctO2Hb and stO2. There were no perfusion deficits observed based on SFDI and clinical follow-up. CONCLUSIONS: The results of our initial human pilot study suggest that SFDI has the potential to provide intraoperative oxygenation images in real-time during surgery. With the use of this technology, surgeons can obtain tissue oxygenation and hemoglobin concentration maps to assist in intraoperative planning; this can potentially prevent complications and improve clinical outcome. Copyright © 2013 by Lippincott Williams & Wilkins.

Objectives Early-stage non–small cell lung cancer (NSCLC) has a high recurrence rate and poor 5-year survival, particularly if lymph nodes are involved. Our objective was to perform a dose-escalation study to assess safety and feasibility of intraoperative near-infrared (NIR) fluorescence imaging to identify the first tumor-draining lymph nodes (ie, sentinel lymph nodes [SLNs] in patients with NSCLC). Methods A-dose escalation phase 1 clinical trial assessing real-time NIR imaging after peritumoral injection of 3.8 to 2500 μg indocyanine green (ICG) was initiated in patients with suspected stage I/II NSCLC. Visualization of lymphatic migration, SLN identification, and adverse events were recorded. Results Thirty-eight patients underwent ICG injection and NIR imaging via thoracotomy (n = 18) or thoracoscopic imaging (n = 20). SLN identification increased with ICG dose, with fewer than 25% SLNs detected in dose cohorts of 600 μg or less versus 89% success at 1000 μg or greater. Twenty-six NIR + SLNs were identified in 15 patients, with 7 NIR + SLNs (6 patients) harboring metastatic disease on histologic analysis. Metastatic nodal disease was never identified in patients with a histologically negative NIR + SLN. No adverse reactions were noted. Conclusions NIR-guided SLN identification with ICG was safe and feasible in this initial dose-escalation trial. ICG doses greater than 1000 μg yielded nearly 90% intrathoracic SLN visualization, with the presence or absence of metastatic disease in the SLN directly correlating with final nodal status of the lymphadenectomy specimen. Further studies are needed to optimize imaging parameters and confirm sensitivity and specificity of SLN mapping in NSCLC using this promising imaging technique.

BACKGROUND The fundamental principle of oncologic surgery is the complete resection of malignant cells. However, small tumors are often difficult to find during surgery using conventional techniques. The objectives of this study were to determine if optical imaging, using a contrast agent already approved for other indications, could improve hepatic metastasectomy with curative intent, to optimize dose and timing, and to determine the mechanism of contrast agent accumulation. METHODS The high tissue penetration of near-infrared (NIR) light was exploited by use of the FLARE (Fluorescence-Assisted Resection and Exploration) image-guided surgery system and the NIR fluorophore indocyanine green in a clinical trial of 40 patients undergoing hepatic resection for colorectal cancer metastases. RESULTS A total of 71 superficially located (

Methylene blue (MB) is a near-infrared fluorophore that provides a stable visual map of skin perfusion after intravenous injection. We explored the capability of MB to predict submental flap postoperative outcome using a single intraoperative measurement. Submental flaps were created in N = 15 pigs and imaged using the FLARE imaging system immediately after surgery and at 72 hours. Using the first 3 pigs, optimal MB dosing was found to be 2.0 mg/kg. Training and validation sets of 6 pigs each were then used for receiver operating characteristic analysis. In the training set, a contrast-to-background ratio (CBR) threshold of 1.24 provided the highest sensitivity and specificity to predict tissue necrosis at 72 hours. In the validation set, this threshold provided a prediction sensitivity of 95.3% and a specificity of 98.0%. We demonstrate that a single intraoperative near-infrared measurement can predict submental flap outcome at 72 hours.

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.

Preserving the nipple-areolar complex with a nipple-sparing mastectomy improves cosmesis compared with skin-sparing mastectomy. However, complications such as necrosis of the nipple-areolar complex significantly affect cosmetic outcome. Many factors influence nipple-areolar perfusion, and no consensus currently exists on optimal incisional choice. This study evaluates 2 nipple-sparing mastectomy incision models using near-infrared fluorescence to assess perfusion quantitatively. The periareolar and radial incisions were compared with 2 control models in Yorkshire pigs (N = 6). Methylene blue and indocyanine green were injected intravenously, and near-infrared fluorescence images were recorded at 3 time points: before surgery, immediately after (0 hour), and 3 days postoperatively. Contrast-to-background ratio was used to assess perfusion. At 72 hours, radial incisions showed a statistically significantly higher perfusion compared with periareolar incisions (P < 0.05). Based on our findings, radial incisions for nipple-sparing mastectomy may be preferable due to higher perfusion; however, clinical trials are necessary for further assessment.

// Henricus J.M. Handgraaf 1, * , Martin C. Boonstra 1, * , Hendrica A.J.M. Prevoo 1 , Joeri Kuil 2 , Mark W. Bordo 3 , Leonora S.F. Boogerd 1 , Babs G. Sibinga Mulder 1 , Cornelis F.M. Sier 1 , Maaike L. Vinkenburg-van Slooten 1 , A. Rob P.M. Valentijn 2 , Jacobus Burggraaf 4, 5 , Cornelis J.H. van de Velde 1 , John V. Frangioni 3, 6, 7 , Alexander L. Vahrmeijer 1 1 Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands 3 Curadel, LLC, Marlborough, MA, U.S.A 4 Centre for Human Drug Research, Leiden, The Netherlands 5 Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands 6 Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, U.S.A 7 Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA, U.S.A * These authors contributed equally and share first authorship Correspondence to: Alexander L. Vahrmeijer, email: a.l.vahrmeijer@lumc.nl Keywords: integrins, RGD, fluorescence-guided surgery, preclinical validation, in vivo diagnosis Received: December 15, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT Incomplete resections and damage to critical structures increase morbidity and mortality of patients with cancer. Targeted intraoperative fluorescence imaging aids surgeons by providing real-time visualization of tumors and vital structures. This study evaluated the tumor-targeted zwitterionic near-infrared fluorescent peptide cRGD-ZW800-1 as tracer for intraoperative imaging of multiple cancer types. cRGD-ZW800-1 was validated in vitro on glioblastoma (U-87 MG) and colorectal (HT-29) cell lines. Subsequently, the tracer was tested in orthotopic mouse models with HT-29, breast (MCF-7), pancreatic (BxPC-3), and oral (OSC-19) tumors. Dose-ranging studies, including doses of 0.25, 1.0, 10, and 30 nmol, in xenograft tumor models suggest an optimal dose of 10 nmol, corresponding to a human equivalent dose of 63 μg/kg, and an optimal imaging window between 2 and 24 h post-injection. The mean half-life of cRGD-ZW800-1 in blood was 25 min. Biodistribution at 4 h showed the highest fluorescence signals in tumors and kidneys. In vitro and in vivo competition experiments showed significantly lower fluorescence signals when U-87 MG cells (minus 36%, p = 0.02) or HT-29 tumor bearing mice (TBR at 4 h 3.2 ± 0.5 vs 1.8 ± 0.4, p = 0.03) were simultaneously treated with unlabeled cRGD. cRGD-ZW800-1 visualized in vivo all colorectal, breast, pancreatic, and oral tumor xenografts in mice. Screening for off-target interactions, cRGD-ZW800-1 showed only inhibition of COX-2, likely due to binding of cRGD-ZW800-1 to integrin α V β 3 . Due to its recognition of various integrins, which are expressed on malignant and neoangiogenic cells, it is expected that cRGD-ZW800-1 will provide a sensitive and generic tool to visualize cancer during surgery.

Background There are currently no thymus-specific contrast agents for biomedical imaging. Thus, finding ectopic thymic tissue during certain operations is extremely difficult. The purpose of the present study was to determine if near-infrared (NIR) fluorescence imaging could provide high sensitivity, real-time identification of thymic tissue during the operation. Methods After initial in vivo screening of a 315-compound NIR fluorophore library for thymic uptake, methylene blue and five different 700-nm emitting candidate molecules were injected into CD-1 mice for quantitation of the signal-to-background ratio as a function of kinetics and dosing. Results were confirmed in 35-kg Yorkshire pigs. Dual-channel NIR imaging was also performed using a variety of 800-nm emitting NIR fluorophores targeted to various tissues in the mediastinum and neck. Results The compound Oxazine 170 demonstrated the highest signal-to-background ratio (≥3) for thymic tissue relative to mediastinal fat, heart, lung, muscle, thyroid gland, and parathyroid gland, with peak signal-to-background ratio occurring 4 h after 1 intravenous injection of a human equivalent dose of approximately 7 mg. Simultaneous dual-channel NIR imaging permitted unambiguous identification of the thymus from surrounding tissues, such as endocrine glands and lymph nodes. Conclusions In mouse and pig, NIR fluorescence imaging using Oxazine 170 permits high sensitivity, real-time identification of thymic tissue for surgical procedures requiring its resection or avoidance. The performance of Oxazine 170 for imaging human thymic tissue is currently not known.

To investigate feasibility and accuracy of near-infrared fluorescence imaging using indocyanine green: nanocolloid for sentinel lymph node (SLN) detection in gastric cancer.A prospective, single-institution, phase I feasibility trial was conducted. Patients suffering from gastric cancer and planned for gastrectomy were included. During surgery, a subserosal injection of 1.6 mL ICG:Nanocoll was administered around the tumor. NIR fluorescence imaging of the abdominal cavity was performed using the Mini-FLARE™ NIR fluorescence imaging system. Lymphatic pathways and SLNs were visualized. Of every detected SLN, the corresponding lymph node station, signal-to-background ratio and histopathological diagnosis was determined. Patients underwent standard-of-care gastrectomy. Detected SLNs outside the standard dissection planes were also resected and evaluated.Twenty-six patients were enrolled. Four patients were excluded because distant metastases were found during surgery or due to technical failure of the injection. In 21 of the remaining 22 patients, at least 1 SLN was detected by NIR Fluorescence imaging (mean 3.1 SLNs; range 1-6). In 8 of the 21 patients, tumor-positive LNs were found. Overall accuracy of the technique was 90% (70%-99%; 95%CI), which decreased by higher pT-stage (100%, 100%, 100%, 90%, 0% for respectively Tx, T1, T2, T3, T4 tumors). All NIR-negative SLNs were completely effaced by tumor. Mean fluorescence signal-to-background ratio of SLNs was 4.4 (range 1.4-19.8). In 8 of the 21 patients, SLNs outside the standard resection plane were identified, that contained malignant cells in 2 patients.This study shows successful use of ICG:Nanocoll as lymphatic tracer for SLN detection in gastric cancer. Moreover, tumor-containing LNs outside the standard dissection planes were identified.

A multi-wavelength fluorescence camera add on for existing cryoslicing instruments is described. Ex-vivo fluorescently-labeled tracers are recovered in 3D with high resolution, including 2-Deoxy-D-glucose, ZW800-1, and an antisense oligonucleotide.

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.

For academic discoveries eschewed by traditional venture capital business models, there is now an alternative path.

ObjectiveIntraoperative imaging of intravascular thrombi is limited by the inability of visible light to penetrate thick-walled vessels. Near-infrared (NIR) light has relatively high tissue penetration and low autofluorescence and scatter, offering significant advantages. We hypothesized that the development of 700-nm NIR fluorophores for platelet labeling, in conjunction with existing 800-nm NIR fluorophores, would permit simultaneous and separable quantitation of intravascular thrombi and measurement of the antiplatelet effect of drugs.MethodsWe synthesized a series of lipophilic, cationic, polymethine indocyanine dyes (MHI-86, 94, 106, and 114) that emit at approximately 700 nm. Platelet uptake was optimized in vitro and the bioactivity and blood half-life of labeled platelets was characterized in vitro and in vivo. FeCl3-induced injury of the femoral arteries and intravascular thrombus formation was performed in 35-kg Yorkshire pigs. A combination of 700-nm and 800-nm NIR fluorophore-labeled platelets was used in conjunction with the fluorescence-assisted resection and exploration imaging system to image and quantify the antiplatelet effect of cilostazol and acetylsalicylic acid.ResultsMHI-114 was incorporated at nearly 4.1 × 106 molecules per platelet without affecting platelet function. When infused into pigs, the signal-to-background ratio of MHI-114-labeled platelets exhibited a blood half-life of 16.4 ± 2.2 (mean ± SEM; n = 3) minute and generated a signal-to-background ratio of 2.5 ± 0.5 (mean ± SEM; n = 3) at the site of thrombi. Using dual-NIR-labeled platelet populations, cilostazol and acetylsalicylic acid were found to cause a reduction in platelet incorporation into thrombi of 51 ± 2% and 10 ± 1% (mean ± SEM; n = 3), respectively, relative to vehicle-only treated control thrombi.ConclusionsNew platelet-avid 700-nm NIR fluorophores permit simultaneous two-wavelength NIR fluorescence imaging and quantitation of intravascular thrombi in intact vessels approaching the size of humans and can be used to study the antiplatelet effect of drugs.Clinical RelevanceOccult thrombus formation during surgery leads to significant morbidity and mortality. We describe the synthesis of a novel family of 700-nm near-infrared fluorophores that concentrate into platelets and render them highly fluorescent while maintaining bioactivity. Using these platelets and a near-infrared imaging system, we can detect thrombi in thick-walled vessels intraoperatively with high signal-to-background ratio ≈ 2.5. The optical properties and blood half-life of these 700-nm fluorophores complement a previously described 800-nm platelet-avid fluorophore and enable the assessment of antiplatelet interventions intraoperatively.

A panel of five experts discuss the implications of cancer heterogeneity for diagnostics and therapy.

Background The aim of this study was to demonstrate feasibility of migration and in situ chemotherapy delivery to regional lymph nodes (LN) in a large animal model using an expansile polymer nanoparticle (eNP) delivery system. Study Design Dual-labeled 50-nm and 100-nm eNP were prepared by encapsulating an IR-813 near-infrared (NIR) fluorescent dye within coumarin-conjugated expansile polymer nanoparticles (NIR-C-eNP). NIR imaging and fluorescent microscopy were used to identify intralymphatic migration of NIR-nanoparticles to draining inguinal or mesenteric LN after injection in swine hind legs or intestine. Nanoparticle-mediated intranodal delivery of chemotherapy was subsequently assessed with Oregon Green paclitaxel-loaded NIR-eNP (NIR-OGpax-eNP). Results NIR imaging demonstrated direct lymphatic migration of 50-nm, but not 100-nm, NIR-C-eNP and NIR-OGpax-eNP to the draining regional LNs after intradermal injection in the hind leg or subserosal injection in intestine. Fluorescent microscopy demonstrated that IR-813 used for NIR real-time trafficking colocalized with both the coumarin-labeled polymer and paclitaxel chemotherapy and was identified within the subcapsular spaces of the draining LNs. These studies verify nodal migration of both nanoparticle and encapsulated payload, and confirm the feasibility of focusing chemotherapy delivery directly to regional nodes. Conclusions Regionally-targeted intranodal chemotherapy can be delivered to draining LNs for both skin and solid organs using 50-nm paclitaxel-loaded eNP.

Background Predicting the long-term viability of ischaemic bowel during surgery is challenging. The aim was to determine whether intraoperative near-infrared angiography (NIR-AG) of ischaemic bowel might provide metrics that were predictive of long-term outcome. Methods NIR-AG using indocyanine green was performed on 24 pigs before, and after inducing bowel ischaemia to determine the feasibility of NIR-AG for detecting compromised perfusion. Contrast-to-background ratio (CBR) over time was measured in regions of interest throughout the bowel, and various metrics of the CBR–time curve were developed. Sixty rat small bowels, with or without strangulation, were imaged during surgery and on day 3 after operation. CBR metrics and clinical findings were assessed quantitatively for their ability to predict animal survival, histological grade of ischaemic injury and visible necrosis on day 3. Results In ischaemic pig small bowel, various qualitative and quantitative CBR metrics appeared to correlate with bowel injury as a function of distance from normal bowel. In rats, intraoperative clinical assessment showed high specificity but low sensitivity for predicting outcome on day 3 after operation. Qualitative patterns of the CBR–time curve, such as absence of an arterial inflow peak and presence of a NIR filling defect, resulted in better prediction of survival (90 per cent), histological grade (85 per cent) and visible necrosis on day 3 (92 per cent). Conclusion Survival of ischaemic bowel was predicted by intraoperative NIR-AG with greater accuracy than clinical evaluation alone.

International audience; Near-infrared (NIR) fluorescence imaging has the potential to improve sentinel lymph node (SLN) mapping in breast cancer. Indocyanine green (ICG) is currently the only clinically available fluorophore that can be used for SLN mapping. Preclinically, ICG adsorbed to human serum albumin (ICG:HSA) improves its performance as a lymphatic tracer in some anatomical sites. The benefit of ICG:HSA for SLN mapping of breast cancer has not yet been assessed in a clinical trial. We performed a double-blind, randomized study to determine if ICG:HSA has advantages over ICG alone. The primary endpoint was the fluorescence brightness, defined as the signal-to-background ratio (SBR), of identified SLNs. Clinical trial subjects were 18 consecutive breast cancer patients scheduled to undergo SLN biopsy. All patients received standard of care using Technetium-nanocolloid and patent blue. Patients were randomly assigned to receive 1.6 ml of 500 μM ICG:HSA or ICG that was injected periareolarly directly after patent blue. The Mini-Fluorescence-Assisted Resection and Exploration (Mini-FLARE) imaging system was used for NIR fluorescence detection and quantitation. SLN mapping was successful in all patients. Patient, tumor, and treatment characteristics were equally distributed over the treatment groups. No significant difference was found in SBR between the ICG:HSA group and the ICG alone group (8.4 vs. 11.3, respectively, = 0.18). In both groups, the average number of detected SLNs was 1.4 ± 0.5 SLNs per patient ( = 0.74). This study shows that there is no direct benefit of premixing ICG with HSA prior to injection for SLN mapping in breast cancer patients, thereby reducing the cost and complexity of the procedure. With these optimized parameters that eliminate the necessity of HSA, larger trials can now be performed to determine patient benefit.

Background: Perforator flaps have become increasingly popular in reconstructive surgery, as patients experience less donor-site morbidity than with conventional musculocutaneous flaps. Previously, the authors' laboratory described the intraoperative use of near-infrared fluorescence angiography for patient-specific perforator flap design. This study evaluates the predictive capability of near-infrared fluorescence angiography for flap survival in submental flap reconstruction. Methods: Near-infrared angiography was performed using indocyanine green at 0, 0.5, 24, 48, and 72 hours after surgery for flap creation in 12 pigs. A single perforator artery was preserved during flap creation based on location (central or noncentral) and dominance (dominant or nondominant). Venous drainage, arterial perfusion, and perfused area as a percentage of total flap area were analyzed. Clinical assessments of perfusion were compared with those made using near-infrared imaging and histology. Results: Use of near-infrared fluorescence angiography immediately after flap creation accurately predicted areas of perfusion at 72 hours (p = 0.0013), compared with the initial clinical assessment (p = 0.3085). Identification of necrosis by histology at 72 hours correlated with near-infrared findings of insufficient arterial perfusion immediately after flap creation. No statistically significant differences in perfusion metrics were detected based on location or dominance of the preserved perforator; however, flaps containing central perforators had a higher percentage perfused area than those with noncentral perforators. Conclusions: The use of near-infrared angiography immediately after flap creation can predict areas of perfusion at 72 hours. This predictive capability may permit intraoperative revision of compromised flaps that have a high likelihood of failure.

The ability to determine flap perfusion in reconstructive surgery is still primarily based on clinical examination. In this study, the authors demonstrate the use of an intraoperative, near-infrared fluorescence imaging system for evaluation of perforator location and flap perfusion.Indocyanine green was injected intravenously in six breast cancer patients undergoing a deep inferior epigastric perforator flap breast reconstruction after mastectomy. Three dose levels of indocyanine green were assessed using the fluorescence-assisted resection and exploration (FLARE) imaging system. This system uses light-emitting diodes for fluorescence excitation, which is different from current commercially available systems. In this pilot study, the operating surgeons were blinded to the imaging results.Use of the FLARE system was successful in all six study subjects, with no complications or sequelae. Among the three dose levels, 4 mg per injection resulted in the highest observed contrast-to-background ratio, signal-to-background ratio, and signal-to-noise ratio. However, because of small sample size, the authors did not have sufficient power to detect statistical significance for these pairwise comparisons at the multiple-comparison adjusted type I error of 0.017. Six milligrams per injection provided a similar contrast-to-background ratio but also a higher residual background signal.Based on this pilot study, the authors conclude that near-infrared assessment of perforator flap breast reconstruction is feasible with a light-emitting diode-based system, and that a dose of 4 mg of indocyanine green per injection yields the best observed contrast-to-background ratio compared with a dose of 2 or 6 mg for assessment of flap perfusion.

Background Iatrogenic bile duct injuries are serious complications with patient morbidity. We hypothesized that the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB) and indocyanine green (ICG) could be exploited for real-time, intraoperative imaging of the extrahepatic bile ducts during open and laparoscopic surgeries. Methods In all, 2.0 mg/kg of MB and 0.05 mg/kg of ICG were injected intravenously into 35-kg female Yorkshire pigs and the extrahepatic bile ducts were imaged over time using either the Fluorescence-Assisted Resection and Exploration (FLARE) image-guided surgery system (open surgery) or a custom NIR fluorescence laparoscopy system. Surgical anatomy was confirmed using x-ray cholangiography. The contrast-to-background ratio (CBR), contrast-to-liver ratio (CLR), and chemical concentrations in the cystic duct (CD) and common bile duct (CBD) were measured, and the performance of each agent was quantified. Results Using NIR fluorescence of MB, the CD and CBD could be identified with good sensitivity (CBR and CLR ≥4), during both open and laparoscopic surgeries, from 10 to 120 min postinjection. Functional impairment of the ducts, including constriction and injury were immediately identifiable. Using NIR fluorescence of ICG, extrahepatic bile ducts did not become visible until 90 min postinjection because of strong residual liver retention; however, between 90 and 240 min, ICG provided exquisitely high sensitivity for both CD and CBD, with CBR ≥8 and CLR ≥4. Conclusion We demonstrate that 2 clinically available NIR fluorophores, MB fluorescing at 700 nm and ICG fluorescing at 800 nm, provide sensitive, prolonged identification of the extrahepatic bile ducts and assessment of their functional status.

Despite recent advances in perforator flap reconstruction, there can be significant variability in vessel size and location. Although pre-operative evaluation may provide valuable information, real-time intraoperative methods have the potential to provide the greatest benefit. Our laboratory has developed the Fluorescence-Assisted Resection and Exploration (FLARE™) near-infrared (NIR) fluorescence imaging system for visualizing, intraoperatively, details of the underlying vasculature. The FLARE™ system uses indocyanine green, a safe and reliable NIR fluorophore already FDA-approved for other indications. The system has been optimized in large animal models for the identification of perforator size, location, and perfusion, and has also been translated to the clinic for use during breast reconstruction after mastectomy. In this paper, we review our pre-clinical and clinical data, as well as literature describing the use of similar NIR fluorescence imaging systems in plastic and reconstructive surgery.

Current methods for sentinel lymph node (SLN) mapping and nodal treatment in lung cancer remain inadequate for routine clinical use. Here we discuss the potential for using the combination of invisible near-infrared (NIR) fluorescent light and nanotechnology for these applications. NIR fluorescence imaging has recently received significant attention for in vivo imaging applications because of its low tissue autofluorescence, high photon penetration into living tissue, and high signal-to-background ratio. Our large animal in vivo studies have been able to successfully identify sentinel lymph nodes in lung tissue and several clinical studies have examined the use of NIR fluorescence imaging systems for SLN mapping in breast and gastric cancer. Promising new nanoparticle technologies, when combined with NIR fluorescence imaging, offer the potential for image-guided treatment of lymph nodes at high risk for tumor recurrence. This review provides a theoretical and empirical framework for developing the next-generation of diagnostic and therapeutic agents for lung cancer.

Techniques currently used to determine flap perfusion are mainly subjective, with the majority of reconstructive surgeons still relying on clinical examination. In this study, the authors demonstrate the use of near-infrared fluorescence angiography to directly quantify normal and abnormal perfusion in perforator flaps.Indocyanine green was injected intravenously into anesthetized adult pigs (n = 38). A custom near-infrared fluorescence imaging system was used for image acquisition and quantitation. Thirty-nine flaps were designed based on identified perforators, and postoperative imaging was performed for comparison. In select flaps, isolated occlusion of the arterial and venous pedicle was performed. In select flaps, vascular spasm was induced by local irrigation of the vessels with epinephrine. The fluorescence intensities of select regions of interest were quantified. From these data, the authors defined two indices for abnormal perfusion: the Tmax ratio and the drainage ratio.The authors identified a normal pattern of perfusion before flap elevation, composed of a distinct fluorescence intensity peak at maximal arterial inflow followed by a smooth drop representing venous drainage. Delay of this peak after flap elevation, as indicated by the Tmax ratio, identified vascular spasm and arterial occlusion (p0.0001). Abnormal fall of fluorescence intensities after this peak, as indicated by the drainage ratio, identified venous occlusion (p0.0001).Quantitation of fluorescence intensities by near-infrared angiography accurately characterizes arterial and venous compromise. The authors' technique can assess perfusion characteristics during the intraoperative and postoperative periods and therefore complements clinically based subjective criteria now used for flap assessment.

Invisible NIR fluorescent light can provide high sensitivity, high-resolution, and real-time image-guidance during oncologic surgery, but imaging systems that are presently available do not display this invisible light in the context of surgical anatomy. The FLARE imaging system overcomes this major obstacle.Color video was acquired simultaneously, and in real-time, along with two independent channels of NIR fluorescence. Grayscale NIR fluorescence images were converted to visible "pseudo-colors" and overlaid onto the color video image. Yorkshire pigs weighing 35 kg (n = 5) were used for final preclinical validation of the imaging system. A six-patient pilot study was conducted in women undergoing sentinel lymph node (SLN) mapping for breast cancer. Subjects received (99m)Tc-sulfur colloid lymphoscintigraphy. In addition, 12.5 microg of indocyanine green (ICG) diluted in human serum albumin (HSA) was used as an NIR fluorescent lymphatic tracer.The FLARE system permitted facile positioning in the operating room. NIR light did not change the look of the surgical field. Simultaneous pan-lymphatic and SLN mapping was demonstrated in swine using clinically available NIR fluorophores and the dual NIR capabilities of the system. In the pilot clinical trial, a total of nine SLNs were identified by (99m)Tc- lymphoscintigraphy and nine SLNs were identified by NIR fluorescence, although results differed in two patients. No adverse events were encountered.We describe the successful clinical translation of a new NIR fluorescence imaging system for image-guided oncologic surgery.

Objectives We have developed an image-guided surgical system based on invisible near-infrared fluorescent light. Presently, the only clinically available near-infrared fluorophore is indocyanine green, which fluoresces at approximately 800 nm and is used for coronary angiography. Our objective was to determine whether methylene blue, already US Food and Drug Administration approved for other indications, has useful near-infrared fluorescence properties for image-guided cardiac surgery. Methods The optical properties of methylene blue were measured after dissolution in 100% serum. Biodistribution and clearance were quantified in organs and tissue after intravenous bolus injection of 2 mg/kg methylene blue in 3 rats. Coronary arteriography and cardiac perfusion were imaged in real time after intravenous bolus injection of 1 mg/kg methylene blue in 5 pigs with coronary obstructions. Coronary angiography and acute thrombi were assessed by using 800-nm fluorophores, indocyanine green, and IR-786–labeled platelets, respectively. Results The peak absorbance and emission of methylene blue as a near-infrared fluorophore occur at 667 nm and 686 nm, respectively. After intravenous injection, methylene blue provides highly sensitive coronary angiography. A lipophilic cation, methylene blue is extracted rapidly into tissue, with myocardium displaying unusually high uptake. Methylene blue permits real-time visualization and quantitative assessment of myocardial perfusion. Because of absent spectral overlap, use of 2 independent fluorophores in our imaging system permits simultaneous quantification of perfusion, venous drainage, and/or intravascular thrombi. Conclusions Methylene blue is an effective near-infrared fluorophore that provides direct visualization of coronary arteriography and cardiac perfusion. In conjunction with approximately 800-nm near-infrared fluorophores, important functional assessments during cardiac surgery are also possible.

Presently, signal generation in MRI depends on the concentration and relaxivity of protons or other MR-active nuclei, and contrast depends on local differences in signal. In this proof-of-principle study, we explore the use of nonchemical, solid-state devices for generating detectable signal and/or contrast in vitro and in vivo. We introduce the concept of microresonant devices (MRDs), which are micron-sized resonators fabricated using microelectromechanical systems (MEMS) technology. Fifteen-micrometer (15-μm)-thick, coil MRDs were designed to resonate at the 3T Larmor frequency of protons (127.7 MHz) and were fabricated using tantalum (Ta) oxide thin-film capacitors and copper-plated spiral inductors. The performance of MRDs having final diameters of 300, 500, and 1000 μm were characterized in saline using a radio frequency (RF) scanning microscope and a clinical 3T MR scanner. The measured B1 fields of 300 μm to 1000 μm MRDs ranged from 3.25 μT to 3.98 μT, and their quality factors (Q) ranged from 3.9 to 7.2. When implanted subcutaneously in the flank of a mouse, only MRDs tuned to the resonant frequency of protons generated a measurable in vivo B1 field. This study lays the foundation for a new class of solid-state contrast agents for MRI. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc.

Magnetic resonance imaging (MRI) and MR spectroscopy can probe a variety of physiological (e.g. blood vessel permeability) and metabolic characteristics of prostate cancer. However, little is known about the changes in gene expression that underlie the spectral and imaging features observed in prostate cancer. Tumor induced changes in vascular permeability and angiogenesis are thought to contribute to patterns of dynamic contrast enhanced (DCE) MRI images of prostate cancer even though the genetic basis of tumor vasculogenesis is complex and the specific mechanisms underlying these DCEMRI features have not yet been determined. In order to identify the changes in gene expression that correspond to MRS and DCEMRI patterns in human prostate cancers, we have utilized tissue print micropeel techniques to generate “whole mount” molecular maps of radical prostatectomy specimens that correspond to pre-surgical MRI/MRS studies. These molecular maps include RNA expression profiles from both Affymetrix GeneChip microarrays and quantitative reverse transcriptase PCR (qrt-PCR) analysis, as well as immunohistochemical studies. Using these methods on patients with prostate cancer, we found robust over-expression of choline kinase a in the majority of primary tumors. We also observed overexpression of neuropeptide Y (NPY), a newly identified angiogenic factor, in a subset of DCEMRI positive prostate cancers. These studies set the stage for establishing MRI/MRS parameters as validated biomarkers for human prostate cancer.

Clinical trials in cardiac cell-based therapy (CBT) have demonstrated the immense potential of stem progenitor cells (SPCs) to repair the injured myocardium. The bulk of evidence so far has shown that CBT can lead to structural and functional improvements. Unresolved issues remain, however, including gaps in the understanding of mechanisms and mixed results from CBT trials. To try to provide answers for these issues, assessment of the biological fate of SPCs once delivered to the injured heart has been called for. Advances in contrast agents and imaging modalities have made feasible the objective assessment of the in vivo molecular and cellular evolution of transplanted SPCs. In vivo imaging can target fundamental processes related to SPCs to gain information on their biological activities and outcomes within specific authentic microenvironments. Advantages and inherent drawbacks of imaging techniques, such as reporter-gene systems, optical imaging, radionuclide imaging, and MRI, are discussed in this Review. More than ever, it has become clear to scientists and clinicians that parallel developments in cell-based therapies and in vivo imaging modalities will strengthen this blossoming field.

Cell-based therapy has been heralded as a promising, novel therapeutic strategy for cardiovascular diseases. Despite a rapid transition from animal studies to clinical trials, there remain numerous unresolved, and at times, controversial issues with respect to underlying molecular mechanisms. In parallel, recent advances in the field of molecular imaging has provided a means to bridge the gap in knowledge through in vivo stem cells tracking. Herein, we review current in vivo imaging techniques and future directions for tracking the effects of cell-based therapy.

Although the sentinel lymph node (SLN) hypothesis has been applied to many tissues and organs, liver has remained unstudied. Currently, it is unclear whether hepatic SLNs even exist. If so, they could alter the management of intrahepatic cholangiocarcinoma and other hepatic malignancies by minimizing the extent of surgery while still providing precise nodal staging. This study investigated whether invisible yet tissue-penetrating near-infrared (NIR) fluorescent light can provide simultaneous identification of both the SLN and all other regional lymph nodes (RLNs) in the liver. In 25 Yorkshire pigs, this study determined whether SLNs exist in liver and compared the effectiveness of two clinically available NIR fluorophores [methylene blue and indocyanine green (ICG)], and two novel NIR fluorophores previously described by our group (ESNF14 and ZW800-3C) for SLN and RLN mapping. In this study, ESNF14 showed the highest signal-to-background ratio and the longest retention time in SLNs without leakage to second-tier lymph nodes. The findings showed that ICG had apparent leakage to second-tier nodes, and ZW800-3C had poor migration after intraparenchymal injection. However, when injected intravenously, ZW800-3C was able to highlight all RLNs in liver during a 4- to 6-h period. Simultaneous dual-channel imaging of SLN (ESNF14) and RLN (ZW800-3C) permitted unambiguous identification and image-guided resection of SLNs and RLNs in liver. The NIR imaging technology enables real-time intraoperative identification of SLNs and RLNs in the liver of swine. If these results are confirmed in patients, new strategies for the surgical management of intrahepatic malignancies should be possible.