Real-time tissue viability assessment using near-infrared light

Despite significant advances in medical imaging technologies, there currently exist no tools to effectively assist healthcare professionals during surgical procedures. In turn, procedures remain subjective and dependent on experience, resulting in avoidable failure and significant quality of care disparities across hospitals. Optical techniques are gaining popularity in clinical research because they are low cost, non-invasive, portable, and can retrieve both fluorescence and endogenous contrast information, providing physiological information relative to perfusion, oxygenation, metabolism, hydration, and sub-cellular content. Near-infrared (NIR) light is especially well suited for biological tissue and does not cause tissue damage from ionizing radiation or heat. My dissertation has been focused on developing rapid imaging techniques for mapping endogenous tissue constituents to aid surgical guidance. These techniques allow, for the first time, video-rate quantitative acquisition over a large field of view (> 100 cm2) in widefield and endoscopic implementations. The optical system analysis has been focused on the spatial-frequency domain for its ease of quantitative measurements over large fields of view and for its recent development in real-time acquisition, single snapshot of optical properties (SSOP) imaging. Using these methods, this dissertation provides novel improvements and implementations to SSOP, including both widefield and endoscopic instrumentations capable of video-rate acquisition of optical properties and sample surface profile maps. In turn, these measures generate profile-corrected maps of hemoglobin concentration that are highly beneficial for perfusion and overall tissue viability. Also utilizing optical property maps, a novel technique for quantitative fluorescence imaging was also demonstrated, showing large improvement over standard and ratiometric methods. To enable real-time feedback, rapid processing algorithms were designed using lookup tables that provide a 100x improvement in processing speed. Finally, these techniques were demonstrated in vivo to investigate their ability for early detection of tissue failure due to ischemia. Both pre-clinical studies show endogenous contrast imaging can provide early measures of future tissue viability. The goal of this work has been to provide the foundation for real-time imaging systems that provide tissue constituent quantification for tissue viability assessments.2018-01-09T00:00:00

Rapid wide-field imaging of soft-tissue microstructure

Tissue microstructure is pivotal in determining the function, behavior, and disease state of biological tissues. Histology and advanced optical techniques are commonly used to examine the cellular, extracellular, and subcellular constituents that define tissue microstructure. However, these techniques frequently require tedious and destructive tissue preparations and lengthy imaging times, or have limited fields of view. Therefore, it is challenging to study soft-tissue microstructure within the macroscopic spatial and temporal context of tissue- and organ-level function. Wide-field imaging techniques provide a non-destructive alternative to rapidly assess tissue microstructure across macroscopic fields of view. Rather than resolving microstructure directly, these techniques are sensitive to light-scattering characteristics of tissue that indicate the underlying microstructure. This dissertation develops light-scattering models to interpret tissue microstructure from light-scattering across macroscopic fields of view rapidly and non-destructively. The first half of the dissertation uses spatial frequency domain imaging (SFDI) to quantify the sub-diffuse light-scattering characteristics of tissues that are intrinsically linked to microstructure. It then introduces a novel empirical model which allows rapid fitting of SFDI data and is sensitive to changes in microparticle size. This technique is then demonstrated as a potential surgical guidance tool for Mohs Micrographic Surgery by rapidly and non-destructively demarcating tumor boundaries in skin biopsies. The imaging and processing speeds achieved with this technique can improve clinical workflows, particularly tissue-conserving surgical procedures, which are currently hindered by the time necessary to determine tumor boundaries using histopathology. Improvements to this technique by use of higher spatial frequencies are also considered. The second section investigates polarization-dependent scattering in tissues that is a result of collagen fiber microstructure. An experimentally-validated computational model is developed to allow direct conversion of polarized-light measurements into absolute measures of collagen fiber alignment in tissues. Furthermore, a combined polarized light SFDI system (pSFDI) is demonstrated to measure distinct fiber alignments in multi-layered tissue samples. The increased speed and versatility of this system is employed to map wide-field microfiber kinematics during mechanical tissue deformation. This technique enables direct examination of the contributions of local fiber kinematics to tissue- and organ-level scales of growth and remodeling.Biomedical Engineerin

Design, Implementation, and Evaluation of a Fluorescence Laminar Optical Tomography Scanner for Brain Imaging

Implementation of new instrumentation and techniques for neuroscience research in recent years has opened new avenues in the study of the dynamics of large-scale neural networks such as the brain. In many of these techniques, including fluorescence recordings and optogenetic stimulation, a combination of photonics and molecular genetic methods are exploited to manipulate and monitor neural activities. Such techniques have been proven to be highly efficient in unraveling the mysteries of data processing in the micro circuits of the brain and as a result these techniques are widely used nowadays in most neuroscience labs. In optogenetics, cell-types of interest are genetically modified by expressing light-sensitive proteins adapted from microbial opsin. Once these proteins are expressed, we are able to use light of appropriate wavelengths to manipulate, increase or suppress neural activity of specific neurons on command. With a high temporal resolution (in the order of milliseconds) and cell-type-specific precision, optogenetics is able to probe how the nervous system functions in real-time, even in freely-moving animals. Currently, whenever genetic modifications are employed in the study of nervous systems, fluorescence proteins are also co-expressed in the same cells as biological markers to visualize the induced changes in the targeted cells. Despite its importance to trace the signal of such markers in-vivo, capabilities of the developed fluorescence tomography instrumentation are still limited and researchers mostly document the fluorescence distribution and expression of proteins of interest after euthanizing the animal and dissection of the tissue. In this project, we present our effort in implementing a fluorescence laminar optical tomography (FLOT) system which is specifically designed for non-invasive three dimensional imaging of fluorescence proteins within the brain of rodents. The application of the developed technology is not limited to optogenetics, but it can be used as a powerful tool to help improving the precision and accuracy of neuroscience and optogenetic experiments. In this design, a set of galvanometer mirrors are employed for realization of a fast and flexible scanner while a highly sensitive camera records the produced fluorescence signals. Fluorescence laminar optical tomography (FLOT) scanner has shown promising results in imaging superficial areas up to 2mm deep from the surface, with the resolution of ~200µm. Details of the design of the hardware and reconstruction algorithms are discussed and samples of experimental results are presented

Multimodal optical systems for clinical oncology

This thesis presents three multimodal optical (light-based) systems designed to improve the capabilities of existing optical modalities for cancer diagnostics and theranostics. Optical diagnostic and therapeutic modalities have seen tremendous success in improving the detection, monitoring, and treatment of cancer. For example, optical spectroscopies can accurately distinguish between healthy and diseased tissues, fluorescence imaging can light up tumours for surgical guidance, and laser systems can treat many epithelial cancers. However, despite these advances, prognoses for many cancers remain poor, positive margin rates following resection remain high, and visual inspection and palpation remain crucial for tumour detection. The synergistic combination of multiple optical modalities, as presented here, offers a promising solution. The first multimodal optical system (Chapter 3) combines Raman spectroscopic diagnostics with photodynamic therapy using a custom-built multimodal optical probe. Crucially, this system demonstrates the feasibility of nanoparticle-free theranostics, which could simplify the clinical translation of cancer theranostic systems without sacrificing diagnostic or therapeutic benefit. The second system (Chapter 4) applies computer vision to Raman spectroscopic diagnostics to achieve spatial spectroscopic diagnostics. It provides an augmented reality display of the surgical field-of-view, overlaying spatially co-registered spectroscopic diagnoses onto imaging data. This enables the translation of Raman spectroscopy from a 1D technique to a 2D diagnostic modality and overcomes the trade-off between diagnostic accuracy and field-of-view that has limited optical systems to date. The final system (Chapter 5) integrates fluorescence imaging and Raman spectroscopy for fluorescence-guided spatial spectroscopic diagnostics. This facilitates macroscopic tumour identification to guide accurate spectroscopic margin delineation, enabling the spectroscopic examination of suspicious lesions across large tissue areas. Together, these multimodal optical systems demonstrate that the integration of multiple optical modalities has potential to improve patient outcomes through enhanced tumour detection and precision-targeted therapies.Open Acces

Multimodal and multiscale imaging of the human placental vasculature

Minimally invasive fetal interventions, such as those used for therapy of twin-to- twin transfusion syndrome (TTTS), require accurate image guidance to optimise patient outcomes. Photoacoustic imaging can provide molecular contrast based on the optical absorption of the haemoglobin, and in this dissertation, it was proposed as a novel technique to image the human placental vasculature. Normal term and in utero TTTS treated placentas were imaged post-partum using two novel photoacoustic imaging systems. With PA imaging, vasculature was resolved to a depth of approximately 7 mm from the chorionic placental surface; the photocoagulated tissue provided a negative contrast and the ablation depth of the scar was visualised. Complementary imaging of the placental vasculature in a microscopic size scale was performed with a handheld incident dark field illumination video microscope in fresh and formalin-fixed term placentas. Real time visualisation of the villus tree down to the terminal villi level was achieved without any contrast injection or extensive tissue preparation. Additionally, the novel application of photoacoustic imaging to guide minimally invasive fetal interventions motivated the development of tissue-mimicking placental phantoms for bench-top system validation and for clinical training. Ideally, phantoms for this modality comprise materials with optical and acoustic properties that can be precisely and independently controlled, which are stable over time, and which are non-toxic and low-cost. Gel wax was proposed as a novel tissue-mimicking material (TMM) that satisfies these criteria, and that it can be used to represent various soft tissues and fabricate heterogeneous phantoms with structures based on patient-specific anatomy. This dissertation sets the stage for the development of miniaturised photoacoustic imaging probes for intraoperative guidance, and new methods of understanding the placental vascular anatomy in health and disease. Gel wax has strong potential to become a next generation TMM for evaluation, and standardisation of imaging systems, and for clinical training

Diffuse Reflectance Spectroscopy to Quantify In Vivo Tissue Optical Properties: Applications in Human Epithelium and Subcutaneous Murine Colon Cancer

Colorectal cancer is the 4th most common and 2nd deadliest cancer. Problems exist with predicting which patients will respond best to certain therapy regimens. Diffuse reflectance spectroscopy has been suggested as a candidate to optically monitor a patient’s early response to therapy and has been received favorably in experimentally managing other cancers such as breast and skin. In this dissertation, two diffuse reflectance spectroscopy probes were designed: one with a combined high-resolution microendoscopy modality, and one that was optimized for acquiring data from subcutaneous murine tumors. For both probes, percent errors for estimating tissue optical properties (reduced scattering coefficient and absorption coefficient) were less than 5% and 10%, respectively. Then, studies on tissue-simulating phantoms were performed to test probe sensitivity and to serve as testing platforms for investigators in biomedical optics. Next, the diffuse reflectance spectroscopy probe was applied to subcutaneous murine colon tumors (n=61) undergoing either antibody immunotherapy or standard 5-fluorouracil chemotherapy. Mice treated with a combination of these therapies showed reduced tumor growth compared to saline control, isotype control, immunotherapy, and chemotherapy groups (p\u3c0.001, \u3c0.001, \u3c0.001, and 0.046, respectively) 7 days post-treatment. Additionally, at 7 days post-treatment, oxyhemoglobin, a marker currently being explored as a functional prognostic cancer marker, trended to increase in immunotherapy, chemotherapy, and combination therapy groups compared to controls (p=0.315, 0.149, and 0.190). Also of interest, an oxyhemoglobin flare (averageincrease of 1.44x from baseline, p=0.03 compared to controls) was shown in tumors treated with chemotherapy, indicating that diffuse reflectance spectroscopy may be useful as a complimentary tool to monitor early tumor therapeutic response in colon cancer. However, subject-to-subject variability was high and studies correlating survival to early oxyhemoglobin flares are suggested

Multimodal Multispectral Optical Endoscopic Imaging for Biomedical Applications

Optical imaging is an emerging field of clinical diagnostics that can address the growing medical need for early cancer detection and diagnosis. Various human cancers are amenable to better prognosis and patient survival if found and treated during early disease onset. Besides providing wide-field, macroscopic diagnostic information similar to existing clinical imaging techniques, optical imaging modalities have the added advantage of microscopic, high resolution cellular-level imaging from in vivo tissues in real time. This comprehensive imaging approach to cancer detection and the possibility of performing an ‘optical biopsy’ without tissue removal has led to growing interest in the field with numerous techniques under investigation. Three optical techniques are discussed in this thesis, namely multispectral fluorescence imaging (MFI), hyperspectral reflectance imaging (HRI) and fluorescence confocal endomicroscopy (FCE). MFI and HRI are novel endoscopic imaging-based extensions of single point detection techniques, such as laser induced fluorescence spectroscopy and diffuse reflectance spectroscopy. This results in the acquisition of spectral data in an intuitive imaging format that allows for quantitative evaluation of tissue disease states. We demonstrate MFI and HRI on fluorophores, tissue phantoms and ex vivo tissues and present the results as an RGB colour image for more intuitive assessment. This follows dimensionality reduction of the acquired spectral data with a fixed-reference isomap diagnostic algorithm to extract only the most meaningful data parameters. FCE is a probe-based point imaging technique offering confocal detection in vivo with almost histology-grade images. We perform FCE imaging on chemotherapy-treated in vitro human ovarian cancer cells, ex vivo human cancer tissues and photosensitiser-treated in vivo murine tumours to show the enhanced detection capabilities of the technique. Finally, the three modalities are applied in combination to demonstrate an optical viewfinder approach as a possible minimally-invasive imaging method for early cancer detection and diagnosis

Next generation near infrared (NIR) and shortwave infrared (SWIR) wearables for breast cancer imaging

Neoadjuvant chemotherapy (NAC) is a common breast cancer treatment that involves administering chemotherapy for 3-6 months prior to surgery. This treatment enables more breast-conserving surgeries and even allows for the omission of surgery in some cases. However, about 31% of patients receiving NAC do not respond to the treatment. Therefore, there is a need for real-time methods to predict treatment response and improve patient outcomes. Over the last two decades, diffuse optical imaging has been investigated as a potential solution to this problem. This noninvasive and inexpensive technology uses near or shortwave infrared (NIR or SWIR) light to illuminate tissue, and detects multiply-scattered photons. However, bulky instrumentation and complicated imaging procedures have limited the clinical adoption of this technology. Furthermore, measured biomarkers including oxy- and deoxy-hemoglobin (HbO2 and HHb, respectively), water, and lipid, have had mixed results in terms of prognostic capability. To address these limitations, a new wearable optical probe technology was developed and validated in this project, including a high-optode density NIR probe for monitoring hemodynamics and a first-generation SWIR probe for quantifying water and lipid. Measurements on tissue-mimicking channel flow phantoms confirmed the ability of the NIR probe to quantify absorption contrast in vitro, and a cuff occlusion measurement demonstrated sensitivity to HbO2 and HHb in vivo. Hemodynamic oscillations at the respiratory rate were also explored in healthy volunteers and breast cancer patients as a potential new biomarker. It was demonstrated that traditional and novel breathing-related hemodynamic metrics provide tumor contrast and can potentially track treatment response. A deep-learning algorithm was developed to extract water and lipid concentrations from multi-distance SWIR measurements. The SWIR probe was validated by comparing measured water and lipid concentrations against ground truth values in emulsion phantoms. This work represents a significant step toward the development of technologies for frequent breast cancer treatment monitoring in the clinic and potentially at home

Advanced cranial navigation

Neurosurgery is performed with extremely low margins of error. Surgical inaccuracy may have disastrous consequences. The overall aim of this thesis was to improve accuracy in cranial neurosurgical procedures by the application of new technical aids. Two technical methods were evaluated: augmented reality (AR) for surgical navigation (Papers I-II) and the optical technique of diffuse reflectance spectroscopy (DRS) for real-time tissue identification (Papers III-V). Minimally invasive skull-base endoscopy has several potential benefits compared to traditional craniotomy, but approaching the skull base through this route implies that at-risk organs and surgical targets are covered by bone and out of the surgeon’s direct line of sight. In Paper I, a new application for AR-navigated endoscopic skull-base surgery, based on an augmented-reality surgical navigation (ARSN) system, was developed. The accuracy of the system, defined by mean target registration error (TRE), was evaluated and found to be 0.55±0.24 mm, the lowest value reported error in the literature. As a first step toward the development of a cranial application for AR navigation, in Paper II this ARSN system was used to enable insertions of biopsy needles and external ventricular drainages (EVDs). The technical accuracy (i.e., deviation from the target or intended path) and efficacy (i.e., insertion time) were assessed on a 3D-printed realistic, anthropomorphic skull and brain phantom; Thirty cranial biopsies and 10 EVD insertions were performed. Accuracy for biopsy was 0.8±0.43 mm with a median insertion time of 149 (87-233) seconds, and for EVD accuracy was 2.9±0.8 mm at the tip with a median angular deviation of 0.7±0.5° and a median insertion time of 188 (135-400) seconds. Glial tumors grow diffusely in the brain, and patient survival is correlated with the extent of tumor removal. Tumor borders are often invisible. Resection beyond borders as defined by conventional methods may further improve a patient’s prognosis. In Paper III, DRS was evaluated for discrimination between glioma and normal brain tissue ex vivo. DRS spectra and histology were acquired from 22 tumor samples and 9 brain tissue samples retrieved from 30 patients. Sensitivity and specificity for the detection of low-grade gliomas were 82.0% and 82.7%, respectively, with an AUC of 0.91. Acute ischemic stroke caused by large vessel occlusion is treated with endovascular thrombectomy, but treatment failure can occur when clot composition and thrombectomy technique are mismatched. Intra-procedural knowledge of clot composition could guide the choice of treatment modality. In Paper IV, DRS, in vivo, was evaluated for intravascular clot characterization. Three types of clot analogs, red blood cell (RBC)-rich, fibrin-rich and mixed clots, were injected into the external carotids of a domestic pig. An intravascular DRS probe was used for in-situ measurements of clots, blood, and vessel walls, and the spectral data were analyzed. DRS could differentiate clot types, vessel walls, and blood in vivo (p<0,001). The sensitivity and specificity for detection were 73.8% and 98.8% for RBC clots, 100% and 100% for mixed clots, and 80.6% and 97.8% for fibrin clots, respectively. Paper V evaluated DRS for characterization of human clot composition ex vivo: 45 clot units were retrieved from 29 stroke patients and examined with DRS and histopathological evaluation. DRS parameters correlated with clot RBC fraction (R=81, p<0.001) and could be used for the classification of clot type with sensitivity and specificity rates for the detection of RBC-rich clots of 0.722 and 0.846, respectively. Applied in an intravascular probe, DRS may provide intra-procedural information on clot composition to improve endovascular thrombectomy efficiency