Fluorescence background quenching as a means to increase Signal to Background ratio - a proof of concept during Nerve Imaging

Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up.Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin. functionalized with an azide-containing Cy5 dye (N-3-Cy5-WGA). A Cy7 quencher dye (Cy7-DBCO) was subsequently used to yield Cy7-Cy5-WGA, a compound wherein the Cy5 emission is quenched by Forster resonance energy transfer to Cy7. The photophysical properties of N-3-Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture), ex vivo (muscle tissue from mice; used for dose optimization), and in vivo (nervus ischiadicus in THY-1 YFP mice).Results: In situ, conjugation of Cy7-DBCO to N-3-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N-3-Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N-3-Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo, Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site.Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging

Multispectral-Fluorescence Imaging as a Tool to Separate Healthy from Disease-Related Lymphatic Anatomy During Robot-Assisted Laparoscopy

Prostatic carcinom

The best of both worlds: a hybrid approach for optimal pre- and intraoperative identification of sentinel lymph nodes

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Technologic (r)evolution leads to detection of more sentinel nodes in patients with melanoma in the head and neck region

Sentinel lymph node (SN) biopsy (SNB) has proven to be a valuable tool for staging melanoma patients. Since its introduction in the early 1990s, this procedure has undergone several technologic refinements, including the introduction of SPECT/CT, as well as radioguidance and fluorescence guidance. The purpose of the current study was to evaluate the effect of this technologic evolution on SNB in the head and neck region. The primary endpoint was the false-negative (FN) rate. Secondary endpoints were number of harvested SNs, overall operation time, operation time per harvested SN, and postoperative complications. Methods: A retrospective database was queried for cutaneous head and neck melanoma patients who underwent SNB at The Netherlands Cancer Institute between 1993 and 2016. The implementation of new detection techniques was divided into 4 groups: 1993-2005, with preoperative lymphoscintigraphy and intraoperative use of both a y-ray detection probe and patent blue (n = 30); 2006-2007, with addition of preoperative road maps based on SPECT/CT (n = 15); 2008-2009, with intraoperative use of a portable y-camera (n = 40); and 2010-2016, with addition of near-infrared fluorescence guidance (n = 192). Results: In total, 277 patients were included. At least 1 SN was identified in all patients. A tumor-positive SN was found in 59 patients (21.3%): 10 in group 1 (33.3%), 3 in group 2 (20.0%), 6 in group 3 (15.0%), and 40 in group 4 (20.8%). Regional recurrences in patients with tumor negative SNs resulted in an overall FN rate of 11.9% (group 1, 16.7%; group 2, 0%; group 3, 14.3%; group 4, 11.1%). The number of harvested nodes increased with advancing technologies (P = 0.003), whereas Breslow thickness and operation time per harvested SN decreased (P = 0.003 and P = 0.017, respectively). There was no significant difference in percentage of tumor-positive SNs, overall operation time, and complication rate between the different groups. Conclusion: The use of advanced detection technologies led to a higher number of identified SNs without an increase in overall operation time, possibly indicating an improved surgical efficiency. Operation time per harvested SN decreased; the average FN rate remained 11.9% and was unchanged over 23 y. There was no significant change in postoperative complication rate.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Image guided surgery: clinical validation of lesion identification technologies and exploration of nerve sparing approaches

Surgery is considered the golden standard to treat the primary tumour and regional spread of many different cancer types. In addition to the pathological evaluation of surgical margins, the evaluation of lymph nodes is needed to stage the metastatic dissemination of the disease [1,2]. Both these aspects are critical in accomplishing radical excision and are, as such, providing true prognosis. However, the quest for complete cure should be in balance with the desire for minimally invasive surgery [3–5]. Hereby it can be assumed that surgical side effects may negatively influence the patient’s quality of life. Advanced image guided surgery technologies can be used to improve the surgical resection and to minimize the invasive nature of the procedure [6]. In this thesis both these technologies are discussed. Hereby we focussed on the locoregional assessment of the lymphatic tumour spread via sentinel lymph node procedures [7–11]. In addition technologies that support nerve-sparing surgery have been pursued. To realize these ambitious goals, a combination of imaging modalities has been used, ranging from fluorescence to nuclear imaging and hybrid combinations of the same.</p

Image guided surgery: clinical validation of lesion identification technologies and exploration of nerve sparing approaches

Surgery is considered the golden standard to treat the primary tumour and regional spread of many different cancer types. In addition to the pathological evaluation of surgical margins, the evaluation of lymph nodes is needed to stage the metastatic dissemination of the disease [1,2]. Both these aspects are critical in accomplishing radical excision and are, as such, providing true prognosis. However, the quest for complete cure should be in balance with the desire for minimally invasive surgery [3–5]. Hereby it can be assumed that surgical side effects may negatively influence the patient’s quality of life. Advanced image guided surgery technologies can be used to improve the surgical resection and to minimize the invasive nature of the procedure [6]. In this thesis both these technologies are discussed. Hereby we focussed on the locoregional assessment of the lymphatic tumour spread via sentinel lymph node procedures [7–11]. In addition technologies that support nerve-sparing surgery have been pursued. To realize these ambitious goals, a combination of imaging modalities has been used, ranging from fluorescence to nuclear imaging and hybrid combinations of the same.Hamamatsu Photonics K.K., SurgicEye GmbH, KARL STORZ Endoscopie Nederland B.V., ZEISS, EuroTec B.V. and Astellas Pharma B.V.LUMC / Geneeskunde Repositoriu

Fluorescent lectins for peripheral nerve visualization during surgery

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Reply from Authors re: Francesco Montorsi, Giorgio Gandaglia. Sentinel Node Biopsy for Prostate Cancer: A Useless Surgical Exercise? Eur Urol 2014;66:999-1000

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Multispectral Fluorescence Imaging During Robot-assisted Laparoscopic Sentinel Node Biopsy: A First Step Towards a Fluorescence-based Anatomic Roadmap

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Feasibility study illustrating the use of the Crystal Cam/DeclipseSPECT system for image-guided sentinel node navigation in head and neck cancer

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas