Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

In vivo microvascular oximetry using multispectral imaging

This thesis describes the application of multispectral imaging to several novel oximetry applications. Chapter 1 motivates optical microvascular oximetry, outlines oxygen transport in the body, describes the theory of oximetry, and describes the challenges associated with in vivo oximetry, in particular imaging through tissue. Chapter 2 reviews various imaging techniques for quantitative in vivo oximetry of the microvasculature, including multispectral and hyperspectral imaging, photoacoustic imaging, optical coherence tomography, and laser speckle techniques. Chapter 3 describes a two-wavelength oximetry study of two microvascular beds in the anterior segment of the eye: the bulbar conjunctival and episcleral microvasculature. This study reveals previously unseen oxygen diffusion from ambient air into the bulbar conjunctival microvasculature, altering the oxygen saturation of the bulbar conjunctiva. The response of the bulbar conjunctival and episcleral microvascular beds to acute mild hypoxia is quantified and the rate at which oxygen diffuses into bulbar conjunctival vessels is measured. Chapter 4 describes the development and application of a highly novel non-invasive retinal angiography technique: Oximetric Ratio Contrast Angiography (ORCA). ORCA requires only multispectral imaging and a small perturbation of blood oxygen saturation to produce angiographic sequences. A pilot study of ORCA in human subjects was conducted. This study demonstrates that ORCA can produce angiographic sequences with features such as sequential vessel filling and laminar flow. The application and challenges of ORCA are discussed, with emphasis on comparison with other angiography techniques, such as fluorescein angiography. Chapter 5 describes the development of a multispectral microscope for oximetry in the spinal cord dorsal vein of rats. Measurements of blood oxygen saturation are made in the dorsal vein of both healthy rats, and in rats with the Experimental autoimmune encephalomyelitis (EAE) disease model of multiple sclerosis. The venous blood oxygen saturation of EAE disease model rats was found to be significantly lower than that of healthy controls, indicating increased oxygen uptake from blood in the EAE disease model of multiple sclerosis. Chapter 6 describes the development of video-rate red eye oximetry; a technique which could enable stand-off oximetry of the blood-supply of the eye with high temporal resolution. The various challenges associated with video-rate red eye oximetry are investigated and their influence quantified. The eventual aim of this research is to track circulating deoxygenation perturbations as they arrive in both eyes, which could provide a screening method for carotid artery stenosis, which is major risk-factor for stroke. However, due to time constraints, it was not possible to thoroughly investigate if video-rate red eye can detect such perturbations. Directions and recommendations for future research are outlined

Using Fluorescence – Polarization Endoscopy in Detection of Precancerous and Cancerous Lesions in Colon and Pancreatic Cancer

Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real time detection of these lesions. Utilizing a new thermoresponsive sol-gel formulation with targeted molecular probe allowed topical application and detection of precancerous and cancerous lesions during endoscopy. Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy served as a validation strategy prior to obtaining biopsies. In order to reduce repeat surgeries arising from incomplete tumor resection, we demonstrated the efficacy of the targeted molecular probe towards margins of sporadic colorectal cancer (SCC). Fluorescence-polarization microscopy using circular polarized (CP) light served as a rapid, supplementary tool for assessment and validation of excised tissue to ensure complete tumor resection for examining tumor margins prior to H&E-based pathological diagnosis. We extended our platform towards non-invasive directed detection of pancreatic cancer utilizing fluorescence molecular tomography (FMT) and NIR laparoscopy using identified targeted molecular probe. We were able to non-invasively distinguished between pancreatitis and pancreatic cancer and guide pancreatic tumor resection using NIR laparoscopy

Validation de modalités d’imagerie innovantes de l’athérosclérose par cathéter intravasculaire bimodal combinant fluorescence (NIRF) et ultrasons (IVUS) couplé à l’injection locale de sondes moléculaires in vivo ciblant ICAM-1 et le collagène

Les maladies cardiovasculaires sont la principale cause de mortalité à l’échelle mondiale, dont la survenue de l’infarctus du myocarde et de la mort subite découle majoritairement de la rupture d’une plaque d’athérosclérose coronarienne vulnérable. Les modalités d’imagerie invasive contemporaines permettent d’analyser les caractéristiques anatomiques et morphologiques de la plaque d’athérome, mais ne révèlent aucune information sur la biologie de l’athérogenèse. Ainsi, l’identification des déterminant moléculaires et cellulaires définissant la plaque à haut risque de rupture est l’approche de choix afin d’améliorer nos connaissances sur la maladie coronarienne et pour l’optimisation des traitements actuels. L’objectif principal de ce projet de recherche visait à démontrer la faisabilité d’une technique d’imagerie moléculaire innovante par cathéter bimodal IVUS-NIRF couplé à l’utilisation de sondes d’imagerie ciblant spécifiquement l’inflammation (ICAM-1) et la composition de la plaque d’athérosclérose (fibres de collagène type-I non polymérisées) dans deux modèles animaux distincts de l’athérosclérose. Les résultats préliminaires contenus dans cette thèse décrivent une technique d’imagerie intravasculaire originale et novatrice permettant la détection in vivo par cathéter de biomarqueurs de l’inflammation et de la composition de la plaque d’athérosclérose à l’aide de traceurs moléculaires fluorescents spécifiques injectés en faible concentration au sein de plaques visualisées par IVUS. Permettant de discriminer avec précision le signal NIRF, le système d’imagerie par cathéter IVUS-NIRF exploité dans ce projet de recherche permet un multiplexage précis du signal NIRF suivant l’injection simultanée de sondes couplées à des fluorophores distincts, offrant la possibilité de cibler simultanément un panel de biomarqueurs à haut risque au sein d’une même plaque d’athérosclérose. Ainsi, cette application d’imagerie moléculaire in vivo est une stratégie prometteuse dont la translation en clinique permettrait d’identifier les processus biologiques clés impliqués dans l’instabilité de la plaque d’athérosclérose coronarienne humaine, un besoin à combler dans la pratique clinique.Cardiovascular disease is the leading cause of mortality worldwide, with the onset of myocardial infarction and sudden death resulting mainly from the rupture of a vulnerable coronary atherosclerotic plaque. Contemporary invasive imaging modalities enable analysis of anatomical and morphological characteristics of the atheroma plaque without providing information on the biology of atherosclerosis. Thus, the identification of molecular and cellular determinants defining high-risk plaques is a promising approach to improve our current knowledge of coronary heart disease and to optimize patient treatment. This research project aimed to demonstrate the feasibility of a novel molecular imaging technique using a custom bimodal intravascular ultrasound (IVUS) – near-infrared fluorescence (NIRF) catheter imaging system combined to local injection of labeled-specific imaging probes targeting both inflammation (ICAM-1) and plaque composition (unpolymerized type I collagen fibers) in two distinct atherosclerotic animal models. Preliminary results presented in this thesis describe an original and novel intravascular imaging technique for in vivo detection of inflammation and plaque morphology using specific fluorescent molecular tracers injected in low concentration in the vicinity of plaques visualized by IVUS. The bimodal IVUS-NIRF imaging catheter system used in this project enables accurate NIRF signal multiplexing following dual-injection of molecular probes coupled to distinct fluorophores, which might offer the possibility of targeting a panel of high-risk biomarkers simultaneously within in a single atherosclerotic plaque. Thus, this in vivo molecular imaging application is a promising strategy that could translate to future human studies with the purpose to identify key biological processes involved in human coronary atherosclerotic plaque instability, an unmet need in clinical practice

Diagnosis, Treatment and Prevention of Age-Related Macular Degeneration

In this reprint, we hope to review the basics and highlight the latest developments in AMD. This demonstrates the benefits of the international scientific community working on this disease, to limit its negative impacts, the most vital of which is the loss of visual function, leading to a loss of autonomy and a decrease in patients’ quality of life

Diffuse Optical Imaging for Monitoring Peripheral Arterial Disease Revascularizations

Peripheral arterial disease (PAD) affects approximately 200 million individuals worldwide. It is characterized by a reduction in blood flow to the lower extremities due to atherosclerosis. This can result in leg pain, tissue loss, and ultimately amputation. Revascularization procedures aim to restore blood flow, but up to 50% of patients require another intervention within a year. Revascularization monitoring and early detection of failure are crucial in preventing limb loss and adverse cardiovascular events. However, current evaluation methods do not directly measure perfusion and are limited in a significant segment of PAD patients, such as those with diabetes and renal insufficiency. Diffuse optical imaging (DOI) techniques are promising tools to overcome these limitations. Employing near-infrared light, they are non-invasive, non-ionizing, contrast-free, and cost-effective methods that are sensitive to hemodynamic parameters such as changes in oxy-, deoxy-, and total hemoglobin concentration, making DOI ideal for revascularization monitoring. In this dissertation, I investigate and develop DOI systems for the purpose of monitoring lower extremity revascularization procedures in PAD patients. We utilize a contact-based diffuse optical spectroscopy (DOS) system to monitor localized foot perfusion in an ongoing clinical study of 100 patients undergoing lower extremity angiography. I demonstrate the utility of DOS measurements to provide valuable insights into revascularization related hemodynamic remodeling and to predict revascularization success. Furthermore, I also develop a clinic friendly contact-free diffuse optical tomography (DOT) system that is better-suited for PAD patients with ulcers. I show that this system can provide spatial maps of perfusion within the foot. Collectively, this work establishes diffuse optical imaging as a valuable imaging modality for the evaluation of lower extremity perfusion

Added value of acute multimodal CT-based imaging (MCTI) : a comprehensive analysis

Introduction: MCTI is used to assess acute ischemic stroke (AIS) patients.We postulated that use of MCTI improves patient outcome regardingindependence and mortality.Methods: From the ASTRAL registry, all patients with an AIS and a non-contrast-CT (NCCT), angio-CT (CTA) or perfusion-CT (CTP) within24 h from onset were included. Demographic, clinical, biological, radio-logical, and follow-up caracteristics were collected. Significant predictorsof MCTI use were fitted in a multivariate analysis. Patients undergoingCTA or CTA&CTP were compared with NCCT patients with regards tofavourable outcome (mRS ≤ 2) at 3 months, 12 months mortality, strokemechanism, short-term renal function, use of ancillary diagnostic tests,duration of hospitalization and 12 months stroke recurrence