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This translational study aims to investigate the clinical benefits of indocyanine green (ICG) based near-infrared window II (NIR-II) fluorescence image-guided surgery (FGS) on high-grade glioma (HGG) patients.Patients were randomly assigned to receive FGS or traditional white light image-guided surgery (WLS). The detection rate of NIR-II fluorescence was observed. Complete resection rate, progression-free survival (PFS), overall survival (OS), and neurological status were compared. Tissue samples were obtained from the FGS group, with the diagnosis based on the surgeons and the fluorescence recorded for comparison of diagnostic capability. Patients with WHO grade III gliomas or glioblastomas (GBM) were analyzed separately.15 GBM and 4 WHO grade III glioma patients in the FGS group and 18 GBM and 4 WHO grade III glioma patients in the WLS group were enrolled. The detection rate of NIR-II fluorescence was 100% for GBM. The complete resection rate was significantly increased by the FGS for GBM (FGS, 100% [95% CI 73.41-100] vs. WLS, 50% [95% CI 29.03-70.97], P = 0.0036). The PFS and OS of the FGS group were also significantly prolonged (Median PFS: FGS, 9.0 months vs. WLS, 7.0 months, P0.0001; Median OS: FGS, 19.0 months vs. WLS, 15.5 months, P = 0.0002). No recurrence was observed in WHO grade III glioma patients.NIR-II FGS achieves a much better complete resection rate of GBM than conventional WLS, leading to greatly improved survival of GBM patients.NIR-II FGS is a highly promising technique worthy of exploring more clinical applications.

Over the last decade fluorescence-guided surgery has been primarily focused on the NIR-I window. However, the NIR-I window has constraints, such as limited penetration and scattering. Consequently, exploring the performance of NIR-I dyes at longer wavelengths (i.e., the NIR-II window) is crucial to expanding its application. Two fluorophores were used in three pigs to identify the mean fluorescence intensity (MFI) using two commercially available NIR-I and NIR-II cameras. The near-infrared coating of equipment (NICE) was used to identify endoluminal surgical catheters and indocyanine green (ICG) for common bile duct (CBD) characterization. The NIR-II window evaluation showed an MFI of 0.4 arbitrary units (a.u.) ± 0.106 a.u. in small bowel NICE-coated catheters and an MFI of 0.09 a.u. ± 0.039 a.u. in gastric ones. In CBD characterization, the ICG MFI was 0.12 a.u. ± 0.027 a.u., 0.18 a.u. ± 0.100 a.u., and 0.22 a.u. ± 0.041 a.u. at 5, 35, and 65 min, respectively. This in vivo imaging evaluation of NIR-I dyes confirms its application in the NIR-II domain. To the best of our knowledge, this is the first study assessing the MIF of NICE in the NIR-II window using a commercially available system. Further comparative trials are necessary to determine the superiority of NIR-II imaging systems.

Simple Summary The most frequent post-operative complication in thyroid surgery is hypoparathyroidism leading to temporary or definitive low blood calcium levels. This complication can result from intentional or inadvertent extirpation, trauma, or devascularization of the parathyroid glands. They are located right next to the thyroid, and are responsible for the blood calcium level regulation. Hypoparathyroidism is even more common when a lymph node dissection is needed in addition to thyroidectomy in case of thyroid cancer. The safeguarding of all four parathyroid glands with their vascularization can be extremely challenging, even for experienced surgeons. Fluorescence imaging is a relatively novel intraoperative tool to help identify, visualize, and preserve the parathyroid glands during thyroid surgery. In this review, we summarize the current scientific landscape and the potential benefits of fluorescence imaging to preserve the parathyroid glands and to prevent post-operative hypoparathyroidism in thyroid cancer surgery. Abstract Background: Hypoparathyroidism is one of the most frequent complications of thyroid surgery, especially when associated with lymph node dissection in cases of thyroid cancer. Fluorescence-guided surgery is an emerging tool that appears to help reduce the rate of this complication. The present review aims to highlight the utility of fluorescence imaging in preserving parathyroid glands during thyroid cancer surgery. Methods: We performed a systematic review of the literature according to PRISMA guidelines to identify published studies on fluorescence-guided thyroid surgery with a particular focus on thyroid cancer. Articles were selected and analyzed per indication and type of surgery, autofluorescence or exogenous dye usage, and outcomes. The Methodological Index for Non-Randomized Studies (MINORS) was used to assess the methodological quality of the included articles. Results: Twenty-five studies met the inclusion criteria, with three studies exclusively assessing patients with thyroid cancer. The remaining studies assessed mixed cohorts with thyroid cancer and other thyroid or parathyroid diseases. The majority of the papers support the potential benefit of fluorescence imaging in preserving parathyroid glands in thyroid surgery. Conclusions: Fluorescence-guided surgery is useful in the prevention of post-thyroidectomy hypoparathyroidism via enhanced early identification, visualization, and preservation of the parathyroid glands. These aspects are notably beneficial in cases of associated lymphadenectomy for thyroid cancer.

Hepatocellular carcinoma (HCC) is a common cancer worldwide, and complete surgical resection of diseased tissue is a reliable strategy to cure cancer. Fluorescence image-guided surgery is a promising tool for surgeons to identify and remove malignant lesions. While non-targeted fluorescent dyes have been used for HCC diagnosis and resection, insufficient specificity and false positive uptake from inflammatory tissue result in a high recurrence rate or excessive excision of healthy liver tissue. To circumvent these problems, we focused on developing novel tumor-specific targeting probe to selectively illuminate cancer region during surgery. Given overexpression of histone deacetylases (HDACs) in HCC and many other cancers, HDAC-targeted imaging has been emerged as a promising tool for tumor detection. Recently, high expression of HDACs, in particular HDAC6, has been observed in tumor samples of HCC patient, and a few HDAC inhibitors, including FDA-approved suberoylanilide hydroxamic acid (SAHA), display potent antitumor effect on HCC. Correspondingly, in this study, we utilized a small molecule SAHA with the high HDAC-binding affinity as the HCC-specific targeting ligand to develop HDAC-targeted fluorescence probe for HCC detection and fluorescence image-guided resection. In in vitro imaging, SAHA was labelled with fluorescein isothiocyanate (FITC) to evaluate targeting property, and the imaging results demonstrated that FITC-SAHA was specific uptake by HCC Bel-7402 cells. In in vivo imaging, near infrared fluorescence dye IRDye800CW-labelled SAHA (NIR probe IRDye800CW-SAHA) showed rapid tumor accumulation with high tumor-to-background contrast on both the subcutaneous and orthotopic HCC mouse tumor models. Furthermore, the orthotopic HCC was successfully resected by the IRDye800CW-SAHA fluorescence image-guided surgery. Moreover, IRDye800CW-SAHA showed no toxicity toward healthy tissues. Our results indicate that IRDye800CW-SAHA is a clinical translatable probe for HCC detection and resection.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue–bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with (18)F-labeled trans-cyclooctene ((18)F-TCO). To generate a dual-modality antibody fragment–based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with (18)F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and (18)F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with (18)F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image–guided identification of tumor margins during surgery.

Introduction The c-Met transmembrane protein is vital for cell differentiation and migration and is overexpressed in many gastrointestinal cancers. This study aimed to investigate a novel c-Met targeted peptide coupled to a fluorophore (EMI-137, Edinburgh Molecular Imaging Ltd.) for use in fluorescence image-guided colorectal cancer (CRC) surgery. Method A high c-Met expressing cell-line, HT29, was identified with temporary RNA suppression and used to develop a mouse xenograph CRC model. Tumours were allowed to grow to 10mm. EMI-137 was injected into the tail vein and biodistribution analysed using the IVIS system. Nine patients undergoing elective surgery for colon cancer received a single IV dose EMI-137 1-3 hours before surgery. Tumour and LN fluorescence was assessed with a prototype Karl Storz laparoscope. Intraoperative fluorescence was correlated with radiological and pathological TNM stage and tissue c-Met expression using immunohistochemistry. Result The HT29 xenograph CRC model demonstrated selective EMI-137 uptake and fluorescence 1- 6 hours post administration. Nine participants aged 67-77 years received EMI-137 106 minutes (S.D±17) before surgery. Marked background fluorescence was observed in all patients. 4/9 (44%) patients showed mild increase in tumour fluorescence over background. 5/9 patients had histological LN disease, but no fluorescent nodes were detected intraoperatively. There was no correlation with T-stage. At histopathological assessment 8/9 participants showed moderate or high tumour c-Met expression. 8/8 malignant LNs demonstrated high c-Met expression. Conclusion EMI-137 is specific for human c-Met in 2D and xenograph CRC models. EMI-137 is safe for human use but its utility is limited by insufficient tumour-to-background ratios. Take-home message This first-in-man study of a novel fluorescent peptide targeted to the c-Met receptor, found EMI-137 lacked the sensitivity and specificity to accurately map the tumour margins and lymph node burden in laparoscopic colonic cancer resection surgery.

Complete cytoreductive surgery is the cornerstone of the treatment of epithelial ovarian cancer (EOC). The application of fluorescence image-guided surgery (FIGS) allows for the increased intraoperative visualization and delineation of malignant lesions by using fluorescently labeled targeting biomarkers, thereby improving intraoperative guidance. CD24, a small glycophosphatidylinositol-anchored cell surface receptor, is overexpressed in approximately 70% of solid cancers, and has been proposed as a prognostic and therapeutic tumor-specific biomarker for EOC. Recently, preclinical studies have demonstrated the benefit of CD24-targeted contrast agents for non-invasive fluorescence imaging, as well as improved tumor resection by employing CD24-targeted FIGS in orthotopic patient-derived xenograft models of EOC. The successful detection of miniscule metastases denotes CD24 as a promising biomarker for the application of fluorescence-guided surgery in EOC patients. The aim of this review is to present the clinical and preclinically evaluated biomarkers for ovarian cancer FIGS, highlight the strengths of CD24, and propose a future bimodal approach combining CD24-targeted fluorescence imaging with radionuclide detection and targeted therapy.

PURPOSE: Negative surgical margins (NSMs) have favorable prognostic implications in breast tumor resection surgery. Fluorescence image-guided surgery (FIGS) has the ability to delineate surgical margins in real time, potentially improving the completeness of tumor resection. We have recently developed indocyanine green (ICG)-loaded self-assembled hyaluronic acid (HA) nanoparticles (NanoICG) for solid tumor imaging, which were shown to enhance intraoperative contrast. PROCEDURES: This study sought to assess the efficacy of NanoICG on completeness of breast tumor resection and post-surgical survival. BALB/c mice bearing iRFP(+)/luciferase(+) 4T1 syngeneic breast tumors were administered NanoICG or ICG, underwent FIGS, and were compared to bright light surgery (BLS) and sham controls. RESULTS: NanoICG increased the number of complete resections and improved tumor-free survival. This was a product of superior intraoperative contrast enhancement and the identification of a greater number of small, occult lesions than ICG and BLS. Additionally, NanoICG identified chest wall invasion and predicted recurrence in a model of late-stage breast cancer. CONCLUSIONS: NanoICG is an efficacious intraoperative contrast agent and could potentially improve surgical outcomes in breast cancer.

In the past several decades, a number of novel fluorescence image-guided surgery (FGS) contrast agents have been under development, with many in clinical translation and undergoing clinical trials. In this review, we have identified and summarized the contrast agents currently undergoing clinical translation. In total, 39 novel FGS contrast agents are being studied in 85 clinical trials. Four FGS contrast agents are currently being studied in phase III clinical trials and are poised to reach FDA approval within the next two to three years. Among all novel FGS contrast agents, a wide variety of probe types, targeting mechanisms, and fluorescence properties exists. Clinically available FGS imaging systems have been developed for FDA approved FGS contrast agents, and thus further clinical development is required to yield FGS imaging systems tuned for the variety of contrast agents in the clinical pipeline. Additionally, study of current FGS contrast agents for additional disease types and development of anatomy specific contrast agents is required to provide surgeons FGS tools for all surgical specialties and associated comorbidities. The work reviewed here represents a significant effort from many groups and further development of this promising technology will have an enormous impact on surgical outcomes across all specialties.

Background The completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suitable biomarker for tumour-targeted imaging. Methods CD24 expression was investigated in cell lines and heterogenous patient-derived xenograft (PDX) tumour samples of high-grade serous ovarian carcinoma (HGSOC). After conjugation of the monoclonal antibody CD24 to the NIR dye Alexa Fluor 750 and the evaluation of the optimal pharmacological parameters (OV-90, n = 21), orthotopic HGSOC metastatic xenografts (OV-90, n = 16) underwent cytoreductive surgery with real-time feedback. The impact of intraoperative CD24-targeted fluorescence guidance was compared to white light and palpation alone, and the recurrence of disease was monitored post-operatively (OV-90, n = 12). CD24-AF750 was further evaluated in four clinically annotated orthotopic PDX models of metastatic HGSOC, to validate the translational potential for intraoperative guidance. Findings CD24-targeted intraoperative NIR FIGS significantly (47•3%) improved tumour detection and resection, and reduced the post-operative tumour burden compared to standard white-light surgery in orthotopic HGSOC xenografts. CD24-AF750 allowed identification of minuscule tumour lesions which were undetectable with the naked eye in four HGSOC PDX. Interpretation CD24-targeted FIGS has translational potential as an aid to improve debulking surgery of ovarian cancer. Funding This study was supported by the H2020 program MSCA-ITN [675743], Helse Vest RHF, and Helse Bergen HF [911809, 911852, 912171, 240222, 911974, HV1269], as well as by The Norwegian Cancer Society [182735], and The Research Council of Norway through its Centres of excellence funding scheme [223250, 262652].

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors.

OBJECTIVE The maximum resection of Glioblastoma (GBM) is the standard therapy and is expected to improve prognosis. Image-guided surgery using a neuronavigation system is the standard technique for glioma. However, due to the brain shift during surgery, intraoperative technologies, such as 5-ALA fluorescence and intraoperative MRI (IoMRI), are employed. Radiotracers are used during positron emission tomography (PET) for metabolic imaging and assist the evaluation of glioma metabolism. We compared the effectiveness of these intraoperative technologies. METHODS Between January 2016 and May 2021, 52 patients with gliomas underwent IoMRI. 21 patients were selected for 5-ALA fluorescence-guided resection of GBM and underwent multiple PET studies (MET, FLT, and FMISO). We graded fluorescence level as strong, vague, or none. Following tumor resection, we identified the fluorescence level and evaluated the residual volume of gadolinium-enhanced T1WI (T1-Gd) on IoMRI and at each PET study. After calculating the extent of resection (EOR) for T1-Gd, we compared the residual volume on T1-Gd for IoMRI and each PET study, between EOR ≥ 93% and EOR < 93%. RESULTS We detected strong 5-ALA fluorescence during induction and before tumor resection in all 21 (100%) patients with a newly-diagnosed and histopathologically-confirmed GBM. Following tumor resection, we noted an EOR ≥ 93% for T1-Gd in 12 cases (vague, 4; none, 8) and an EOR < 93% for T1-Gd in 9 cases (vague, 5; none, 4). The compared median residual volume (mL) with no fluorescence between EOR ≥ 93% and EOR < 93% for T1-Gd were T1-Gd (0.22, 0.74), MET (0.29, 3.31), FLT (0.24, 1.77), and FMISO (0.22, 1.02). CONCLUSIONS GBM cells are difficult to distinguish in cases without 5-ALA fluorescence. For cases without 5-ALA fluorescence, we were able to maximize the resection of GBM by extracting the area of MET accumulation.

International audience; PURPOSE:Fluorescence-guided surgery (FGS) provides surgeons with new opportunities to improve real-time cancer nodule detection and tumor margin visualization. Currently, the most important challenge in this field is the development of fluorescent dyes that specifically target tumors. We developed, characterized and evaluated SGM-101, an innovative antibody-dye conjugate in which the fluorochrome BM104, which has an absorbance band centered at 700 nm, is coupled to a chimeric monoclonal antibody (mAb) against carcinoembryonic antigen (CEA).METHODS:The dye to mAb ratio, binding to CEA and photobleaching of SGM-101 were determined. FGS was performed and results analyzed using different mouse models of human digestive tumors.RESULTS:SGM-101 allowed the detection of tumor nodules in three different colon cancer models: LS174T human colorectal adenocarcinoma cell-induced peritoneal carcinomatosis (PC) and liver metastases, and orthotopic grafts of HT29 human colorectal adenocarcinoma cells. In the PC model, submillimeter-sized nodules were detected during SGM-101-based FGS and SGM-101 predictive positive values ranged from 99.04% to 90.24% for tumor nodules >10 mg and nodules

Introduction/Background Intraoperative tumour-specific fluorescence imaging enables visualisation of cancer lesions,1 and benefits staging and debulking of ovarian cancer.2 In patients with advanced epithelial ovarian cancer (EOC), the completeness of cytoreduction significantly impacts prognosis.3 We have demonstrated that EOC cell lines express high levels of the stem cell marker CD24. To improve the degree of cytoreduction in EOC, we aim to develop a near-infrared (NIR) fluorescence image-guided surgery (FIGS) protocol, using CD24-targeting probes in orthotopic xenograft and patient-derived xenograft (PDX) models. Methodology OV90luc+ and patient-derived EOC cells were injected orthotopically into immunodeficient NSG mice, and tumour growth was monitored by bioluminescence imaging and physical examination until metastatic disease had established. The monoclonal antibody CD24 was conjugated to the NIR fluorophore AlexaFluor750, and injected intravenously into the xenograft and PDX models 48hrs prior to surgery. Survival debulking surgery was performed, using a FIGS camera system for real-time NIR fluorescence-guided resection of tumour tissue. To evaluate the effect of FIGS, mice (n=16) were operated under fluorescence guidance, and the other half had standard bright light surgery. Resected tumour volume, post-surgical imaging and post-operative survival were measured. Results CD24-targeted FIGS allows for improved detection of tumour tissue and metastatic spread with high sensitivity and specificity in OV90luc and PDX models, compared to standard bright light surgery, leading to improved resection rates and improved outcome in these preclinical murine models. Conclusion The EOC biomarker CD24 conjugated to AlexaFluor750 demonstrates high sensitivity and specificity of tumour lesion detection in a preclinical murine model. CD24 is a promising agent for clinical implementation of fluorescence image-guided surgery in patients with ovarian cancer. Disclosure Nothing to disclose.

Cancer photodynamic therapy (PDT) represents an attractive local treatment in combination with immunotherapy. Successful cancer PDT relies on image guidance to ensure the treatment accuracy. However, existing nanotechnology for co-delivery of photosensitizers and image contrast agents slows the clearance of PDT agents from the body and causes a disparity between the release profiles of the imaging and PDT agents. We have found that the photosensitizer Chlorin e6 (Ce6) is inherently bound to immunoglobulin G (IgG) in a nanomolarity range of affinity. Ce6 and IgG self-assemble to form the nanocomplexes termed Chloringlobulin (Chlorin e6 + immunoglobulin G). Chloringlobulin enhances the Ce6 concentration in the tumor without changing its elimination half-life in blood. Utilizing the immune checkpoint inhibitor antiprogrammed death ligand 1 (PD-L1) (αPD-L1) to prepare αPD-L1 Chloringlobulin, we have demonstrated a combination of Ce6-based red-light fluorescence image-guided surgery, stereotactic PDT, and PD-L1 blockade therapy of mice bearing orthotopic glioma. In mice bearing an orthotopic colon cancer model, we have prepared another Chloringlobulin that allows intraoperative fluorescence image-guided PDT in combination with PD-L1 and cytotoxic T lymphocyte antigen 4 (CTLA-4) dual checkpoint blockade therapy. The Chloringlobulin technology shows great potential for clinical translation of combinatorial intraoperative fluorescence image-guided PDT and checkpoint blockade therapy.

Breast cancer patients that experience complete removal of the primary tumor, or negative surgical margins (NSMs), benefit from decreased rates of local recurrence and increased survival. However, intraoperative margin detection is limited to visualization, palpation, and experience to identify malignant vs. healthy tissue. As a result, roughly 1/3 of patients treated with breast conserving surgery (BCS) have residual cancer cells left at the resection border, or positive surgical margins (PSMs). Fluorescence image-guided surgery (FIGS) is a promising alternative for intraoperative margin detection, providing surgeons with real-time feedback on tumor location, increasing the likelihood of achieving NSMs. Our past work has demonstrated that the use of self-assembled hyaluronic acid (HA) nanoparticles improves the delivery of indocyanine green (ICG) to breast tumors, enhancing intraoperative tumor signal and contrast. This study built upon these findings by assessing the surgical efficacy of ICG-loaded HA nanoparticles (NanoICG) for the image-guided resection of orthotopic iRFP+/luciferase+ 4T1 breast tumors in BALB/c mice. Tumors were resected with FIGS in mice treated with ICG or NanoICG and compared to bright light surgery (BLS) or sham controls. Tumor growth and recurrence were monitored with bioluminescence imaging. NanoICG improved complete resection and prolonged tumorfree survival. Additionally, NanoICG provided greater intraoperative contrast in malignant tissue than ICG or BLS. Furthermore, NanoICG demonstrated a greater ability to identify small, occult lesions than ICG. Overall, the use of NanoICG for the fluorescence image-guided resection of breast tumors could potentially decrease PSM rates and improve complete tumor removal.

Background Though meningiomas are often benign and well circumscribed in nature, many are associated with recurrences and poor outcome because of their attachment to neurovascular nearby structures or invasion of adjacent venous sinuses or bone, forcing incomplete excision and deployment of further salvage therapy. Recently, ALA-FIGS has been reported as an aid to increase the chances of complete resection and minimizing collateral damage. Methods Critical review and meta-analyses of the literature published to date. All studies reporting ALA-FIGS in meningiomas were critically reviewed. Nineteen studies fulfilled the inclusion criteria with a total of 222 patients, of which 206 were included in the meta-analyses. Results ALA-FIGS was highly specific, and highly sensitive (95%, range 91.8–97.7%). There was no correlation between WHO grading (WHO grade I versus WHO grades II & III) and fluorescence (odds ratio 1.2, p > 0.05). ALA-FIGS altered the surgical plan intraoperatively in 75% of high-grade and 19% of low-grade meningiomas, improving the extent of surgical excision. ALA-FIGS was also highly sensitive and specific in differentiating hypertrophy and tumor invasion of adjacent dura and bone. Conclusions ALA-FIGS in meningiomas is very selective, highly sensitive, and improves the extent of surgical resection in meningiomas. Long-term outcome of these tumors in the future should be categorized into those with and without residual fluorescent tissue. A new surgical resection grading system based on ALA-FIGS is proposed.

In laparotomy surgery guided by near-infrared fluorescence imaging, the access to the field of operation is limited by the illumination and/or the imaging field. The side of cavities or organs such as the liver or the heart cannot be examined with the systems available on the market, which are too large and too heavy. In this article, we describe and evaluate a palm sized probe, whose properties, weight, size and sensitivity are adapted for guiding laparotomy surgery. Different experiments have been performed to determine its main characteristics, both on the illumination and imaging sides. The device has been tested for fluorescent molecular probe imaging in preclinical procedures, to prove its ability to be used in cancer nodule detection during surgery. This system is now CE certified for clinical procedures and Indocyanine Green imaging has been performed during clinical investigations: lymphedema and surgical resection of liver metastases of colorectal cancers.

Nerve damage plagues surgical outcomes, significantly affecting post-surgical quality of life. Surprisingly, no method exists to enhance direct nerve visualization in the operating room, and nerve detection is completed through a combination of palpation and visualization when possible. Fluorescence image-guided surgery offers a potential means of enhanced nerve identification and preservation, however a clinically approved nerve-specific contrast agent does not yet exist. Several classes of nerve-specific fluorophores have recently been demonstrated including the distyrylbenzenes (DSB), select oxazines (oxazine 4 perchlorate), and certain cyanines (3,3’-diethylthiatricarbocyanine iodine), which could provide intraoperative guidance. The nerve-sparing radical prostatectomy is a surgical procedure that could benefit from fluorescence image-guided nerve identification. Although the nerve-sparing surgical technique was developed over 30 years ago, nerve damage following radical prostatectomy is reported in some form in up to 60% of patients one to two years post-surgery. To facilitate clinical translation of fluorescence image guided surgery to the nerve sparing prostatectomy, a direct administration methodology was developed that allows selective nerve highlighting with a significantly lower fluorophore dose than systemic administration, where large animal studies have confirmed the technique’s translatability. Tissue penetration will be critical for clinical feasibility of the direct administration methodology and novel formulation strategies have been explored to enhance tissue penetration for identifying buried nerves. In addition, several biomolecular targets of Oxazine 4, a promising candidate for nerve-specific fluorophore development into a near-infrared (NIR) agent, have been identified, providing insight into the mechanism of nerve-specificity. Fluorophore development has made progress towards the goal of creating a NIR nerve-specific fluorophore and determining the structure-activity relationship responsible for nerve binding.

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

We present a biomedical application of Digital Micro-mirror technologies by adapting the DLP4500 module for quasi real-time intraoperative tumor imaging. Fluorescence image guided surgery has been increasingly popular due to its ability to inform surgeons about tumor boundaries in real-time. We have extended this technique to provide 3D tomographic images of a tumor, by adapting a DLP4500 device to illuminate the surgical field with spatially modulated near-infrared (NIR) light. We combine the digital micro-mirror device (DMD) with two simultaneously triggered CMOS cameras to realize a spatial frequency domain imaging system. Spatial frequency domain imaging utilizes sinusoidally modulated illumination at different spatial frequencies and three different phases; corresponding signals are readily demodulated, and analyzed to derive a 3D fluorescence image. Our DMD device is commercially modified and equipped with high-power (5W) NIR diode laser. We present a brief discussion of data acquisition using DLP4500 module, and corrections for spatial inhomogeneity and gamma adjust in order to create linear/desired sinusoidal illumination of NIR light. We discuss results from a tissue phantom study and in-vivo experiments.

Surgical resection is the most effective treatment strategy for solid tumors, but complete removal of the tumor is critical for post-surgical recovery/long-term survival and is dependent on correct identification of the tumor margin and accurate excision of microscopic residual tumor in the surgical field. Fluorescence image guided surgery is an emerging technique that has shown promise for intraoperative location of tumors and tumor margins. Current versions of such intraoperative fluorescence imaging, however, are generally limited to 2D near-surface images, i.e., without information about tumor depth. Here we present an intraoperative fluorescence imaging system for 3D volumetric imaging of tumors; the system uses spatial frequency domain diffuse optical tomography with an analytic inversion reconstruction method. The new instrument can derive depth-sensitive 3D tumor images at depths up to 1 cm, and it employs compact epi-imaging and illumination suitable for the operating room, with quasi-real-time image reconstruction for surgical visualization. We present experimental results with FDA-approved Indocynanine Green using an extensive array of tissue phantoms and in a pilot in-vivo study.

Segmentectomy using indocyanine green (ICG) has become a primary treatment option to achieve a complete resection and preserve lung function in early-stage lung cancer. However, owing to a lack of appropriate intraoperative imaging systems, it is a huge challenge for surgeons to identify the intersegmental plane during the operation, leading to poor prognosis. Thus, we developed a novel wireless wearable fluorescence image-guided surgery system (LIGHTEN) for fast and accurate identification of intersegmental planes in human patients. The system consists of a handle, light source, Google glass and laptop. Application software is written to capture clear real-time images and Google glass is adopted to display with augmented reality. Twelve in vivo studies of pulmonary segmentectomy in swine by intravenous injection of ICG were conducted to test the performance of the system. A distinct black-and-white transition zone image was observed and displayed simultaneously on the Google glass in all swine. The results demonstrated that surgeons using LIGHTEN can effortlessly and quickly discern intersegmental planes during the operation. Our system has enormous potential in helping surgeons to precisely identify intersegmental planes with mobility and high-sensitivity.

Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low-grade and high-grade glioma patients. However, radiographically complete resections are not often achieved in many cases because of the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins. Here, we provide a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care.

A complete surgical excision with negative tumor margins is the single most important factor in the prediction of long-term survival for most cancer patients with solid tumors. We hypothesized that image-guided surgery using nanoparticle-enhanced photoacoustic and fluorescence imaging could significantly reduce the rate of local recurrence.A murine model of invasive mammary carcinoma was utilized. Three experimental groups were included: (1) control; (2) tumor-bearing mice injected with non-targeted nanoprobe; and (3) tumor-bearing mice injected with targeted nanoprobe. The surgeon removed the primary tumor following the guidance of photoacoustic imaging (PAI), then inspected the surgical wound and removed the suspicious tissue using intraoperative near-infrared (NIR) fluorescence imaging. The mice were followed with bioluminescence imaging weekly to quantify local recurrence.Nanoprobe-enhanced photoacoustic contrast enabled PAI to map the volumetric tumor margins up to a depth of 31 mm. The targeted nanoparticles provided significantly greater enhancement than non-targeted nanoparticles. Seven mice in the group injected with the targeted nanoprobes underwent additional resections based upon NIR fluorescence imaging. Pathological analysis confirmed residual cancer cells in the re-resected specimens in 5/7 mice. Image-guided resection resulted in a significant reduction in local recurrence; 8.7 and 33.3 % of the mice in the targeted and control groups suffered recurrence, respectively.These results suggest that photoacoustic and NIR intraoperative imaging can effectively assist a surgeon to locate primary tumors and to identify residual disease in real-time. This technology has promise to overcome current clinical challenges that result in the need for second surgical procedures.

Nerve tissue can be hard to spot during surgery. Hundreds of thousands of nerve injuries occur annually in the US when surgeons cut nerve tissue by mistake. To help surgeons better avoid nerves, researchers at the Oregon Health & Science University have made the first near-infrared (NIR) fluorophores that specifically bind to nerves. Lei Wang, a postdoctoral fellow working with biomedical engineer Summer Gibbs, reported the findings last week at the American Chemical Society national meeting in Orlando, Florida. Other nerve-specific fluorophores exist, but they get excited by, and emit light in, the visible spectrum. NIR probes are preferable for biological imaging because the light can penetrate deeper into tissue and background fluorescence from the tissue itself is at a minimum. Wang and his colleagues designed and synthesized a library of 66 fluorophores using an oxazine scaffold, starting from a known visible-light, nerve-specific fluorophore. Because what makes a fluorophore nerve

Nerve damage remains a major morbidity following nerve sparing radical prostatectomy, significantly affecting quality of life post-surgery. Nerve-specific fluorescence guided surgery offers a potential solution by enhancing nerve visualization intraoperatively. However, the prostate is highly innervated and only the cavernous nerve structures require preservation to maintain continence and potency. Systemic administration of a nerve-specific fluorophore would lower nerve signal to background ratio (SBR) in vital nerve structures, making them difficult to distinguish from all nervous tissue in the pelvic region. A direct administration methodology to enable selective nerve highlighting for enhanced nerve SBR in a specific nerve structure has been developed herein. The direct administration methodology demonstrated equivalent nerve-specific contrast to systemic administration at optimal exposure times. However, the direct administration methodology provided a brighter fluorescent nerve signal, facilitating nerve-specific fluorescence imaging at video rate, which was not possible following systemic administration. Additionally, the direct administration methodology required a significantly lower fluorophore dose than systemic administration, that when scaled to a human dose falls within the microdosing range. Furthermore, a dual fluorophore tissue staining method was developed that alleviates fluorescence background signal from adipose tissue accumulation using a spectrally distinct adipose tissue specific fluorophore. These results validate the use of the direct administration methodology for specific nerve visualization with fluorescence image-guided surgery, which would improve vital nerve structure identification and visualization during nerve sparing radical prostatectomy.

Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

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.

125I-labeled anti-TAG-72 antibodies were applied in radioimmunoguided surgery (RIGS) to remove gross and occult tumors. It is challenging to handle 125I-labeled materials. PET/CT image-guided surgery utilizes 18FDG to monitor the biochemical activity of the tumor and to integrate pre- and postoperative imaging for complete tumor removal. PET/CT image-guided surgery only detects later stage disease. Fluorescence image-guided surgery using anti-TAG-72 antibodies may provide opportunities for intraoperative cancer detection of both gross and occult tumors. J. Surg. Oncol. 2007;96:297–308. © 2007 Wiley-Liss, Inc.

A wearable all-plastic imaging system for real-time fluorescence image-guided surgery is presented. The compact size of the system is especially suitable for applications in the operating room. The system consists of a dual-mode imaging system, see-through goggle, autofocusing, and auto-contrast tuning modules. The paper will discuss the system design and demonstrate the system performance.