This PDF file contains the front matter associated with SPIE Proceedings Volume 9696, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

The onset and progression of cancer introduces changes to the intra-cellular ultrastructural components and to the morphology of the extracellular matrix. While previous work has shown that localized scatter imaging is sensitive to pathology-induced differences in these aspects of tissue microstructure, wide adaptation this knowledge for surgical guidance is limited by two factors. First, the time required to image with confocal-level localization of the remission signal can be substantial. Second, localized (i.e. sub-diffuse) scatter remission intensity is influenced interchangeably by parameters that define scattering frequency and anisotropy. This similarity relationship must be carefully considered in order to obtain unique estimates of biomarkers that define either the scatter density or features that describe the distribution (e.g. shape, size, and orientation) of scatterers. This study presents a novel approach that uses structured light imaging to address both of these limitations. Monte Carlo data were used to model the reflectance intensity over a wide range of spatial frequencies, reduced scattering coefficients, absorption coefficients, and a metric of the scattering phase function that directly maps to the fractal dimension of scatter sizes. The approach is validated in tissue-simulating phantoms constructed with user-tuned scattering phase functions. The validation analysis shows that the phase function can be described in the presence of different scatter densities or background absorptions. Preliminary data from clinical tissue specimens show quantitative images of both the scatter density and the tissue fractal dimension for various tissue types and pathologies. These data represent a novel wide-field quantitative approach to mapping microscopic structural biomarkers that cannot be obtained with standard diffuse imaging. Implications for the use of this approach to assess surgical margins will be discussed.

The extra-cellular space in connective tissue of animals and humans alike is comprised in large part of collagen. Monitoring of collagen arrangement and cross-linking has been utilized to diagnose a variety of medical conditions and guide surgical intervention. For example, collagen monitoring is useful in the assessment and treatment of cervical cancer, skin cancer, myocardial infarction, and non-arteritic anterior ischemic optic neuropathy. We have developed a suite of tools and models based on polarized light transfer for the assessment of collagen presence, cross-linking, and orientation in living tissue. Here we will present some example of such approach applied to the human cervix. We will illustrate a novel Mueller Matrix (MM) imaging system for the study of cervical tissue; furthermore we will show how our model of polarized light transfer through cervical tissue compares to the experimental findings. Finally we will show validation of the methodology through histological results and Second Harmonic imaging microscopy.

With 50% of all interventional procedures in the US being minimally invasive, there is a need for objective tools to help guide surgeons in this challenging environment. Tissue oxygenation is a useful biomarker of tissue viability and suitable for surgical guidance. Here we present our efforts to perform real-time quantitative optical imaging through a rigid endoscope using Single Snapshot of Optical Properties (SSOP) imaging. In particular, in this work we introduce for the first time 3 dimensionally-corrected dual wavelength optical properties imaging using SSOP through an endoscope, allowing accurate oxygenation maps to be obtained on tissue simulating phantoms and in vivo samples. We compared the results with state-of-the-art wide-field spatial frequency domain imaging (SFDI). Overall, results from the novel endoscopic imaging system agreed within 10% in absorption, reduced scattering, and oxygenation. Moreover, we introduce here real-time, video-rate quantitative optical imaging with 3D profile correction through an endoscope. These results demonstrate the potential of endoscopic SSOP as an objective surgical guidance tool for the clinic.

Fluorescence-guided surgery has demonstrated more complete tumor resections in both preclinical models and clinical applications. However, intraoperative fluorescence-based imaging can be challenging due to attenuation of the fluorescence by intrinsic tissue scattering and absorption. Removing attenuation in fluorescence imaging is critical in many applications. We have developed both a model based approach and an experimental approach to retrieve attenuation corrected fluorescence based on spatial frequency domain imaging (SFDI). In the model based approach, we extended an attenuation correction model initially developed for point measurement into wide-field imaging with SFDI. To achieve attenuation correction, tissue optical properties were evaluated at both excitation and emission wavelengths, which were later applied in the model. In an in-vitro phantom study, we achieved a relative flat intensity profile over entire absorption range compared to over 80% drop at the highest absorption level before correction. Similar performance was also observed in an ex-vivo tissue study. However, lengthy image acquisition and image processing make this method ideal for static imaging instead of video-rate imaging. To achieve video-rate correction, we developed an experimental approach to reduce absorption by limiting the imaging depth using a high spatial frequency pattern. The absorption reduced fluorescence image was obtained by performing a simple demodulation. The in-vitro phantom study suggested an approximate 20% intensity drop at the highest absorption level compared to over 70% intensity drop before correction. This approach enabled video-rate attenuation corrected imaging at 19 fps, making this technique viable for clinical image guided surgery.

One of the major complications with conventional imaging-agent-based molecular imaging, particularly for cancer imaging, is variability in agent delivery and nonspecific retention in biological tissue. Such factors can account to “swamp” the signal arising from specifically bound imaging agent, which is presumably indicative of the concentration of targeted biomolecule. In the 1950s, Pressman et al. proposed a method of accounting for these delivery and retention effects by normalizing targeted antibody retention to the retention of a co-administered “untargeted”/control imaging agent [1]. Our group resurrected the approach within the last 5 years, finding ways to utilize this so-called “paired-agent” imaging approach to directly quantify biomolecule concentration in tissue (in vitro, ex vivo, and in vivo) [2]. These novel paired-agent imaging approaches capable of quantifying biomolecule concentration provide enormous potential for being adapted to and optimizing molecular-guided surgery, which has a principle goal of identifying distinct biological tissues (tumor, nerves, etc…) based on their distinct molecular environment. This presentation will cover the principles and nuances of paired-agent imaging, as well as the current status of the field and future applications. [1] D. Pressman, E. D. Day, and M. Blau, “The use of paired labeling in the determination of tumor-localizing antibodies,” Cancer Res, 17(9), 845-50 (1957). [2] K. M. Tichauer, Y. Wang, B. W. Pogue et al., “Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging,” Phys Med Biol, 60(14), R239-69 (2015).

Fluorescence lifetime imaging has been shown to be a robust technique for biochemical and functional characterization of tissues and to present great potential for intraoperative tissue diagnosis and guidance of surgical procedures. We report a technique for real-time mapping of fluorescence parameters (i.e. lifetime values) onto the location from where the fluorescence measurements were taken. This is achieved by merging a 450 nm aiming beam generated by a diode laser with the excitation light in a single delivery/collection fiber and by continuously imaging the region of interest with a color CMOS camera. The interrogated locations are then extracted from the acquired frames via color-based segmentation of the aiming beam. Assuming a Gaussian profile of the imaged aiming beam, the segmentation results are fitted to ellipses that are dynamically scaled at the full width of three automatically estimated thresholds (50%, 75%, 90%) of the Gaussian distribution's maximum value. This enables the dynamic augmentation of the white-light video frames with the corresponding fluorescence decay parameters. A fluorescence phantom and fresh tissue samples were used to evaluate this method with motorized and hand-held scanning measurements. At 640x512 pixels resolution the area of interest augmented with fluorescence decay parameters can be imaged at an average 34 frames per second. The developed method has the potential to become a valuable tool for real-time display of optical spectroscopy data during continuous scanning applications that subsequently can be used for tissue characterization and diagnosis.

Inspired by the visual system of the morpho butterfly, we have designed, fabricated, tested and clinically translated an ultra-sensitive, light weight and compact imaging sensor capable of simultaneously capturing near infrared (NIR) and visible spectrum information. The visual system of the morpho butterfly combines photosensitive cells with spectral filters at the receptor level. The spectral filters are realized by alternating layers of high and low dielectric constant, such as air and cytoplasm. We have successfully mimicked this concept by integrating pixelated spectral filters, realized by alternating silicon dioxide and silicon nitrate layers, with an array of CCD detectors. There are four different types of pixelated spectral filters in the imaging plane: red, green, blue and NIR. The high optical density (OD) of all spectral filters (OD>4) allow for efficient rejections of photons from unwanted bands. The single imaging chip weighs 20 grams with form factor of 5mm by 5mm. The imaging camera is integrated with a goggle display system. A tumor targeted agent, LS301, is used to identify all spontaneous tumors in a transgenic PyMT murine model of breast cancer. The imaging system achieved sensitivity of 98% and selectivity of 95%. We also used our imaging sensor to locate sentinel lymph nodes (SLNs) in patients with breast cancer using indocyanine green tracer. The surgeon was able to identify 100% of SLNs when using our bio-inspired imaging system, compared to 93% when using information from the lymphotropic dye and 96% when using information from the radioactive tracer.

Residual tumor deposits missed by conventional treatments frequently seed local and distal recurrence utilizing a network of molecular signaling mechanisms. Beyond providing contrast for molecular-guided surgery, this talk will highlight new concepts in phototherapy to address residual cancer cells in danger zones of recurrence, including selective treatment of microscopic disease using molecular-targeted, activatable immunoconjugates, and photo-initiated release of multikinase inhibitors that suppress multiple modes of tumor escape using optically active nanoparticles. These new approaches support an expanded role for the use of light in fluorescence-guided surgery—for phototherapy and for focused drug release to maximize tumor debulking with suppression of disease recurrence.

Local disease control is a major problem in the treatment of pancreatic cancer, because curative-intent surgery is only possible in a minority of patients, and radiotherapy cannot be delivered in curative doses. Despite the promise of photothermal therapy (PTT) for ablation of pancreatic tumors, this approach remains under investigated. Using photothermal sensitizers in combination with laser light for PTT can result in more efficient conversion of light energy to heat, and confinement of thermal destruction to the tumor, thus sparing adjacent organs and vasculature. Porphyrins have been previously employed as photosensitizers for PDT and PTT, however their incorporation in to “porphysomes”, lipid-based nanoparticles each containing ~80,000 porphyrins through conjugation of pyropheophorbide to phospholipids, carries two distinct advantages: 1) high-density porphyrin packing imparts the nanoparticles with enhanced photonic properties for imaging and phototherapy; 2) the enhanced permeability and retention effect may be exploited for optimal delivery of porphysomes to the tumor region thus high payload porphyrin delivery. The feasibility of porphysome-enhanced PTT for pancreatic cancer treatment was investigated using a patient-derived orthotopic pancreas xenograft tumor model. Uptake of porphysomes at the orthotopic tumor site was validated using ex vivo fluorescence imaging of intact organs of interest. The accumulation of porphysomes in orthotopic tumor microstructure was also confirmed by fluorescence imaging of excised tissue slices. PTT progress was monitored as changes in tumor surface temperature using IR optical imaging. Histological analyses were conducted to examine microstructure changes in tissue morphology, and the viability of remaining tumor tissues following exposure to heat. These studies may also provide insight as to the contribution of heat sink in application of thermal therapies to highly vascularized pancreatic tumors.

The success of optical surgical navigation depends upon being able to intraoperatively employ a contrast agent and an imaging device to successfully guide surgery. Development of devices and contrast agents typically occur separately even though it is their combined performance that ultimately determines success and clinical adoption. Herein, we review critical issues and summarize our strategies and approaches for validating molecularly-targeted, near-infrared fluorescent contrast agents and the devices sufficiently sensitive enough for their detection in order to guide lymph node dissection.

The use of tissue phantoms as calibrators to transfer SI-referenced scale to an imager offers convenience, compared to other methods of calibration. The tissue phantoms are calibrated separately for radiance at emission wavelength per irradiance at excitation wavelength. This calibration is only performed at a single geometric configuration, typically with the detector normal to the sample. In the clinic however, the imager can be moved around, resulting in a geometric configuration different from the calibration configuration. In this study, radiometric measurements are made at different sample-imager angles to test whether the tissue phantoms are Lambertian and the angular limits to which the calibration values hold true.

Near infrared (NIR) fluorescence imaging technique can provide precise and real-time information about tumor location during a cancer resection surgery. However, many intraoperative fluorescence imaging systems are based on wearable devices or stand-alone displays, leading to distraction of the surgeons and suboptimal outcome. To overcome these limitations, we design a projective fluorescence imaging system for surgical navigation. The system consists of a LED excitation light source, a monochromatic CCD camera, a host computer, a mini projector and a CMOS camera. A software program is written by C++ to call OpenCV functions for calibrating and correcting fluorescence images captured by the CCD camera upon excitation illumination of the LED source. The images are projected back to the surgical field by the mini projector. Imaging performance of this projective navigation system is characterized in a tumor simulating phantom. Image-guided surgical resection is demonstrated in an ex-vivo chicken tissue model. In all the experiments, the projected images by the projector match well with the locations of fluorescence emission. Our experimental results indicate that the proposed projective navigation system can be a powerful tool for pre-operative surgical planning, intraoperative surgical guidance, and postoperative assessment of surgical outcome. We have integrated the optoelectronic elements into a compact and miniaturized system in preparation for further clinical validation.

The use of fluorescence imaging for aiding oncologic surgery is a fast growing field in biomedical imaging, revolutionizing open and minimally invasive surgery practices. We have designed, constructed, and tested a system for fluorescence image acquisition and direct display on the surgical field for fluorescence guided surgery. The system uses a near-infrared sensitive CMOS camera for image acquisition, a near-infra LED light source for excitation, and DLP digital projector for projection of fluorescence image data onto the operating field in real time. Instrument control was implemented in Matlab for image capture, processing of acquired data and alignment of image parameters with the projected pattern. Accuracy of alignment was evaluated statistically to demonstrate sensitivity to small objects and alignment throughout the imaging field. After verification of accurate alignment, feasibility for clinical application was demonstrated in large animal models of sentinel lymph node biopsy. Indocyanine green was injected subcutaneously in Yorkshire pigs at various locations to model sentinel lymph node biopsy in gynecologic cancers, head and neck cancer, and melanoma. Fluorescence was detected by the camera system during operations and projected onto the imaging field, accurately identifying tissues containing the fluorescent tracer at up to 15 frames per second. Fluorescence information was projected as binary green regions after thresholding and denoising raw intensity data. Promising results with this initial clinical scale prototype provided encouraging results for the feasibility of optical projection of acquired luminescence during open oncologic surgeries.

Near-infrared (NIR) contrast agents are becoming more frequently studied in medical imaging due to their advantageous characteristics, most notably the ability to capture near-infrared signal across the tissue and the safety of the technique. This produces a need for imaging technology that can be specific for both the NIR dye and medical application. Indocyanine green (ICG) is currently the primary NIR dye used in neurosurgery. Here we report on using the augmented microscope we described previously for image guidance in a rat glioma resection. Luc-C6 cells were implanted in a rat in the left-frontal lobe and grown for 22 days. Surgical resection was performed by a neurosurgeon using augmented microscopy guidance with ICG contrast. Videos and images were acquired to evaluate image quality and resection margins. ICG accumulated in the tumor tissue due to enhanced permeation and retention from the compromised bloodbrain- barrier. The augmented microscope was capable of guiding the rat glioma resection and intraoperatively highlighted tumor tissue regions via ICG fluorescence under normal illumination of the surgical field.

In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

Negative surgical margins are critical to prevent recurrence in cancer surgery. This is because with current technology in many cases negative margins are impossible due the inability of the surgeon to detect the margin. Our laboratory has developed fluorophore-labeled monoclonal antibodies to aid in cancer visualization in orthotopic nude mouse models of human gastrointestinal (GI) cancer in order to achieve negative margins in fluorescence-guided surgery (FGS). The technologies described herein have the potential to change the paradigm of surgical oncology to engender significantly improved outcomes.

Pre-anastomotic bowel perfusion is a key factor for a successful healing process. Clinical judgment has limited accuracy to evaluate intestinal microperfusion. Fluorescence videography is a promising tool for image-guided intraoperative assessment of the bowel perfusion at the future anastomotic site in the setting of minimally invasive procedures. The standard configuration for fluorescence videography includes a Near-Infrared endoscope able to detect the signal emitted by a fluorescent dye, more frequently Indocyanine Green (ICG), which is administered by intravenous injection. Fluorescence intensity is proportional to the amount of fluorescent dye diffusing in the tissue and consequently is a surrogate marker of tissue perfusion. However, fluorescence intensity alone remains a subjective approach and an integrated computer-based analysis of the over-time evolution of the fluorescence signal is required to obtain quantitative data. We have developed a solution integrating computer-based analysis for intra-operative evaluation of the optimal resection site, based on the bowel perfusion as determined by the dynamic fluorescence intensity. The software can generate a "virtual perfusion cartography", based on the “fluorescence time-to-peak”. The virtual perfusion cartography can be overlapped onto real-time laparoscopic images to obtain the Enhanced Reality effect. We have defined this approach FLuorescence-based Enhanced Reality (FLER). This manuscript describes the stepwise development of the FLER concept.

The key to fluorescence guided surgical oncology is the ability to create specific contrast between normal and glioma tissue. The blood brain barrier that limits the delivery of substances to the normal brain is broken in tumors, allowing accumulation of agents in the tumor interior. However, for a clinical success, imaging agents should be in the infiltrative edges to minimize the resection of normal brain while enable the removal of tumor. The aberrant overexpression and/or activation of EGFR is associated with many types of cancers, including glioblastoma and the injection of a fluorescent molecule targeted to these receptors would improve tumor contrast during fluorescence guided surgery. Affibody molecules have intentional medium affinity and high potential specificity, which are the desirable features of a good surgical imaging agent. The aim of this study was evaluate the brain/glioma uptake of ABY029 labeled with near-infrared dye IRDye800CW after intravenous injection. Rats were either inoculated with orthotopic implantations of U251 human glioma cell line or PBS (shams control) in the brain. The tumors were allowed to grow for 2–3 weeks before carrying out fluorescent tracer experiments. Fluorescent imaging of ex vivo brain slices from rats was acquired at different time points after infection of fluorescently labeled EGFR-specific affibody to verify which time provided maximal contrast tumor to normal brain. Although the tumor was most clearly visualized after 1h of IRDye800CW-labeled ABY029 injection, the tumor location could be identified from the background after 48h. These results suggest that the NIR-labeled affibody examined shows excellent potential to increase surgical visualization for confirmed EGFR positive tumors.

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.

We present a novel technology based on a high sensitivity/specificity cancer targeting agent and of a novel fluorescence-guided microscopy (FGM) scheme for intraoperative assessment of surgical margins in breast cancer patients. Cancer cells are targeted using an optically silent peptide substrate coupled to a near infrared (NIR) fluorochrome that is cleaved by highly mediated breast cancer enzymes, like urokinase-type plasminogen activator (uPA), to become highly fluorescent when excited by a NIR laser beam. A FGM instrument is used to localize cancer-suspect areas on the lumpectomy specimen and visualize tissue morphology at the sub-cellular scale, such that a trained pathologist can read these images in real-time and confirm or rule-out cancer presence. The proposed technology will enable efficient assessment of surgical specimens during surgery, when it is mostly needed, and therefore help the clinician to determine if additional tissue excision is needed or not. The preliminary testing of this technology on breast surgical specimens will be discussed.

Ninety percent of patients with head and neck squamous cell carcinomas (HNSCC) have overexpression of epidermal growth factor receptor (EGFR), which is correlated with poor prognosis. Complete surgical resection of HNSCC tumors has a large impact on patient survival, where detection of tumor at or close to surgical margins increases the risk of death at 5-years by 90%. In addition, large surgical margins can greatly increase the morbidity experienced by the patient due to functional and cosmetic damage of oral and facial structures. Single fluorescence targeting agents are often used for tumor detection in in vivo pre-clinical imaging; however, the arising signal is qualitative at best because it is a complex mixture of vascular perfusion, vascular leakage, inhibited lymphatic clearance, and receptor binding. In vivo ratiometric receptor concentration imaging (RCI) allows quantification of receptor expression (hence identification of cancerous tissue) by utilizing co-administered paired-agents consisting of a targeted agent and non-targeted perfusion agent to reference the plasma delivery and leakage. A panel of HNSCC tumors with varying levels of EGFR expression (SCC-15 >SCC-25 > SCC-09) have been imaged using ABY-029, a clinically relevant anti-EGFR affibody labeled with IRDye 800CW, and affibody control imaging agent labeled with IRDye 680RD. RCI maps of in vivo tissue have been created and are spatially correlated with EGFR and CD31 immunohistochemistry and basic H and E staining. The RCI threshold parameters for distinguishing tumor from normal tissues (skin and muscle) and the accuracy of margin detection in these tumors will be presented. RCI surgical resection will be further developed using a novel multi-channel, gated fluorescence-guided surgery (FGS) imaging system that is capable of performing RCI in normal room light.

Complete initial resection can give cancer patients the best opportunity for long-term survival. There is unmet need in surgical oncology for optical imaging that enables simple and precise visualization of tumors and consistent contrast with surrounding normal tissues. Near-infrared (NIR) contrast agents and camera systems that can detect them represent an area of active research and development. The investigational Tumor Paint agent BLZ-100 is a conjugate of a chlorotoxin peptide and the NIR dye indocyanine green (ICG) that has been shown to specifically bind to a broad range of solid tumors. Clinical efficacy studies with BLZ-100 are in progress, a necessary step in bringing the product into clinical practice. To ensure a product that will be useful for and accepted by surgeons, the early clinical development of BLZ- 100 incorporates multiple tumor types and imaging devices so that surgeon feedback covers the range of anticipated clinical uses. Key contrast agent characteristics include safety, specificity, flexibility in timing between dose and surgery, and breadth of tumor types recognized. Imaging devices should use wavelengths that are optimal for the contrast agent, be sensitive enough that contrast agent dosing can be adjusted for optimal contrast, include real-time video display of fluorescence and white light image, and be simple for surgeons to use with minimal disruption of surgical flow. Rapid entry into clinical studies provides the best opportunity for early surgeon feedback, enabling development of agents and devices that will gain broad acceptance and provide information that helps surgeons achieve more complete and precise resections.

Morbidity and complexity involved in lymph node staging via surgical resection and biopsy calls for staging techniques that are less invasive. While visible blue dyes are commonly used in locating sentinel lymph nodes, since they follow tumor-draining lymphatic vessels, they do not provide a metric to evaluate presence of cancer. An area of active research is to use fluorescent dyes to assess tumor burden of sentinel and secondary lymph nodes. The goal of this work was to successfully perform fluorescence imaging of IRDye®680RD in the lymphatics, in a repeatable manner.

Techniques that provide a non-invasive method for evaluation of intraoperative skin flap perfusion are currently available but underutilized. We hypothesize that intraoperative vascular imaging can be used to reliably assess skin flap perfusion and elucidate areas of future necrosis by means of a standardized critical perfusion threshold. Five animal groups (negative controls, n=4; positive controls, n=5; chemotherapy group, n=5; radiation group, n=5; chemoradiation group, n=5) underwent pre-flap treatments two weeks prior to undergoing random pattern dorsal fasciocutaneous flaps with a length to width ratio of 2:1 (3 x 1.5 cm). Flap perfusion was assessed via laser-assisted indocyanine green dye angiography and compared to standard clinical assessment for predictive accuracy of flap necrosis. For estimating flap-failure, clinical prediction achieved a sensitivity of 79.3% and a specificity of 90.5%. When average flap perfusion was more than three standard deviations below the average flap perfusion for the negative control group at the time of the flap procedure (144.3±17.05 absolute perfusion units), laser-assisted indocyanine green dye angiography achieved a sensitivity of 81.1% and a specificity of 97.3%. When absolute perfusion units were seven standard deviations below the average flap perfusion for the negative control group, specificity of necrosis prediction was 100%. Quantitative absolute perfusion units can improve specificity for intraoperative prediction of viable tissue. Using this strategy, a positive predictive threshold of flap failure can be standardized for clinical use.

The abstract is not available

Fluorescence imaging using non-targeted fluorescent agents has been extensively studied during the last decade. Although proven feasible, tumor-specific imaging can be dramatically enhanced using tumor-specific fluorescent contrast agents. Clinical translation of these agents is challenging and hurdles have to be overcome. In this overview paper we recapitulate the key regulations for first-in-human studies with fluorescent agents, provide insight in different strategies for fast clinical translation and discuss how clinical introduction of the first fluorescent targeted agents was achieved.

During fluorescence-guided surgery, a cancer-specific optical probe is injected and visualized using a compatible device intraoperatively to provide visual contrast between diseased and normal tissues to maximize resection of cancer and minimize the resection of precious adjacent normal tissues. Six patients with squamous cell carcinomas of the head and neck region (oral cavity (n=4) or cutaneous (n=2)) were injected with an EGFR-targeting antibody (Cetuximab) conjugated to a near-infrared (NIR) fluorescent dye (IRDye800) 3, 4, or 7 days prior to surgical resection of the cancer. Each patient’s tumor was then imaged using a commercially available, open-field NIR fluorescence imaging device each day prior to surgery, intraoperatively, and post-operatively. The mean fluorescence intensity (MFI) of the tumor was calculated for each specimen at each imaging time point. Adjacent normal tissue served as an internal anatomic control for each patient to establish a patient-matched “background” fluorescence. Resected tissues were also imaged using a closed-field NIR imaging device. Tumor to background ratios (TBRs) were calculated for each patient using both devices. Fluorescence histology was correlated with traditional pathology assessment to verify the specificity of antibody-dye conjugate binding. Peak TBRs using the open-field device ranged from 2.2 to 11.3, with an average TBR of 4.9. Peak TBRs were achieved between days 1 and 4. This study demonstrated that a commercially available NIR imaging device suited for intraoperative and clinical use can successfully be used with a fluorescently-labeled dye to delineate between diseased and normal tissue in this single cohort human study, illuminated the potential for its use in fluoresence-guided surgery.

Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.