Optical biopsy is a medical technique that uses light to perform non-invasive analysis of tissue in-situ. This technology has many applications in the medical profession, opening up exciting new possibilities for surgical guidance and diagnosis of malignancies and other conditions. Optical biopsy allows a medical professional to perform near instantaneous, real time analysis of tissue composition without the need to physically remove tissue from the body, as required in traditional biopsy. A technique frequently used for this purpose is diffuse reflectance spectroscopy (DRS): collection and analysis of the spectrum of light reflected from a material. Another technique frequently used for optical biopsy is laser induced fluorescence spectroscopy (LIFS): analysis of the fluorescence spectrum returned by a material when illuminated at a specific wavelength. This thesis discusses the design and construction of a spatially resolved DRS system intended for use in a dual modality DRS and time resolved LIFS optical biopsy instrument for clinical analysis of brain tissue. This instrument is specifically intended for use in the surgical removal of malignant gliomas: infiltrating tumours associated with a poor patient prognosis. Theoretical simulation based studies were used to optimize the design of a compact, dual modality fibre optic probe for use in the system and a novel algorithm was developed to allow recovery of the optical properties of tissue from reflectance spectra obtained with this probe. This probe was manufactured and a corresponding spectrometer based system was created for the acquisition of diffuse reflectance spectra. Components were designed to allow sterilization and thus clinical use in an operating room environment. A laboratory trial of this system demonstrated its range and ability to recover the optical properties of lipid emulsion optical phantoms. / Thesis / Doctor of Philosophy (PhD)

This work presents an investigation into the optical scattering of heterogeneous cells with an application to two-photon imaging, optical scattering measurements and STED imaging. Using the finite difference time-domain (FDTD) method, the full-wave scattering by many cells containing multiple organelles with varying indices of refraction is computed. These simulations were previously limited to single cells for reasons of computational cost. A superposition approximation that uses the coherent linear superposition of FDTD-determined farfield scattering patterns of small numbers of cells to estimate the scattering from a larger tissue was developed and investigated. It was found that for the approximation to be accurate, the scattering sub-problems must at minimum extend along the incident field propagation axis for the full depth of the tissue, preserving the scattering that takes place in the direction of propagation. The FDTD method was used to study the scattering effects of multiple inhomogeneous cells on the propagation of a focused Gaussian beam with an application to two-photon imaging. It was found that scattering is mostly responsible for the reduction in two-photon fluorescence signal as depth is increased. It was also determined that for the chosen beam parameters and the cell and organelle configurations used, the nuclei are the dominant scatterers. FDTD was also utilized in an investigation of cellular scattering effects on the propagation of a common depletion beam used in STED microscopy and how scattering impacts the image obtained with a STED microscope. An axial doughnut beam was formulated and implemented in FDTD simulations, along with a corresponding focused Gaussian beam to simulate a fluorescence excitation beam. It was determined that the depletion beam will maintain a well-defined axial null in spite of scattering, although scattering will reduce the resulting fluorescence signal with focal depth. / text

Abstract Determination of blood glucose level is a frequently occurring procedure in diabetes care. As the most common method involves collecting blood drops for chemical analysis, it is invasive and liable to afflict a degree of pain and cause a skin injury. To eliminate these disadvantages, this thesis focuses on pulsed photoacoustic techniques, which have potential ability in non-invasive blood glucose measurement. The fundamental theory of photoacoustics in liquid and soft tissue was studied systematically. The distributions of photoacoustic sources in a near-infrared optical skin model were simulated by the Monte Carlo method. Expansion coefficient and specific heat of glucose solution were measured by thermodynamic method, while the sound velocity in it was determined by photoacoustic approach. The effect of glucose on blood optical scattering was studied by a picosecond pulsed laser together with a streak camera. A photoacoustic apparatus comprising a pulsed laser diode and a piezoelectric transducer was built and applied to measure glucose concentration in water and scattering media. Moreover, this apparatus was also used to non-invasive experiment on human fingers. The measurements showed that the expansion coefficient, specific heat and acoustic velocity change by 1.2%, -0.6% and 0.28%, respectively, in response to a 1% change in glucose concentration. The sum effect of these parameters to photoacoustic signal was much larger than that of optical absorption of glucose in near infrared wavelengths, which provided photoacoustic technique a higher degree of sensitivity than offered by the optical absorption method. At the wavelength of 905 nm, the measured glucose detection sensitivity in a 3% milk solution, a tissue sample and whole human blood was 5.4%, 2.5% and 14%, respectively. Each figure is higher than that of glucose in water, about 2%, for a one percent change in glucose concentration. This was supported by the temporal dispersion curves of glucose in blood samples, which demonstrated that glucose decreased the optical scattering of tissues. The currently photoacoustic apparatus could detect the minimal glucose concentration of 100 mg/dl in whole blood samples. It is sensitive to physiological changes in non-invasive measurement, but insufficient for evaluating change in the physiological glucose concentration. Current photoacoustic techniques have apparently advantages in study of scattering media and made great progress in tissue imaging and diagnosis. However, in non-invasive blood glucose measurement they met similar problems as optical approaches based on scattering effect.

L’imagerie moléculaire occupe une place majeure dans le domaine de la recherche préclinique. Parmi les modalités existantes, les techniques optiques fondées sur la détection d’un rayonnement visible ou proche infrarouge sont les plus récentes et sont principalement représentées par les méthodes de tomographie optique de luminescence. Ces méthodes permettent une caractérisation 3D d’un milieu biologique par la reconstruction de cartes de concentration ou la localisation de marqueurs luminescents sensibles à des processus biologiques et chimiques se déroulant à l’échelle de la cellule ou de la molécule. La tomographie de luminescence se fonde sur un modèle de propagation de la lumière dans les tissus, un protocole d’acquisition du signal en surface du milieu et une procédure numérique d’inversion de ces mesures afin de reconstruire les paramètres d’intérêts. Ce travail de thèse s’articule donc autour de ces trois axes et apporte un élément de réponse à chacun des problèmes. L’objectif principal de cette étude est d’introduire et de présenter des outils d’évaluation des performances théoriques d’une méthode de tomographie optique. L’un des aboutissements majeurs est la réalisation de reconstructions tomographiques expérimentales à partir d’images acquises par un imageur optique conçu pour l’imagerie planaire 2D et développé par la société Quidd. Dans un premier temps nous abordons la théorie du transport en milieu diffusant afin de poser les concepts et outils sur lesquels vont s’appuyer l’ensemble des travaux. Nous présentons particulièrement deux modèles de propagation différents ainsi que les méthodes de résolution et les difficultés théoriques qui leur sont liées. Dans une deuxième partie nous introduisons les outils statistiques utilisés pour caractériser les systèmes tomographiques et leur résolution potentielle. Nous définissons une procédure et nous l’appliquons à l’étude de quelques situations simples en tomographie de luminescence. La dernière partie de ce travail présente la mise au point d’une procédure d’inversion. Après avoir présenté le cadre théorique dans lequel cette procédure s’inscrit nous la validons à partir de données numériques avant de l’appliquer avec succès à des mesures expérimentales. / Molecular imaging is a major modality in the field of preclinical research. Among the existing methods, techniques based on optical detection of visible or near infrared radiation are the most recent and are mainly represented by luminescence optical tomography techniques. These methods allow for 3D characterization of a biological medium by reconstructing maps of concentration or localisation of luminescent beacons sensitive to biological and chemical processes at the molecular or cellular scale. Luminescence optical tomography is based on a model of light propagation in tissues, a protocol for acquiring surface signal and a numerical inversion procedure used to reconstruct the parameters of interest. This thesis is structured around these three axes and provides an answer to each problem. The main objective of this study is to introduce and present the tools to evaluate the theoretical performances of optical tomography methods. One of its major outcomes is the realisation of experimental tomographic reconstructions from images acquired by an optical imager designed for 2D planar imaging and developed by the company Quidd. In a first step we develop the theory of transport in scattering medium to establish the concept on which our work will rely. We present two different propagation models as well as resolution methods and theoretical difficulties associated with them. In a second part we introduce the statistical tools used to characterise tomographic systems. We define and apply a procedure to simple situations in luminescence optical tomography. The last part of this work presents the development of an inversion procedure. After introducing the theoretical frameworkwe validate the procedure fromnumerical data before successfully applying it to experimentalmeasurements.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

The clinical management of glioma remains a challenge. The prognosis is poor—for glioblastoma multiforme, the most virulent of these brain cancers, survival is only ~1 year. Surgical resection of the tumor is the first line of defense. Several studies demonstrate a survival advantage in patients who undergo near-complete tumor resection; however, achieving complete resection is limited by the difficulty of visualizing residual tumor after de-bulking. Intraoperative fluorescence guidance is a promising candidate to better visualize residual tumor. The most clinically developed form uses protoporphyrin IX fluorescence, the precursor to heme in its biosynthesis which preferentially accumulates in tumor cells after the administration of 5-aminolevulinic acid. Challenges remain in quantitatively assessing the fluorescence to reduce variability of outcome and improve tumor detection specificity, and in observing sub-surface tumor fluorescence. To these ends, this work outlines the development of intraoperative techniques to 1) quantify tissue fluorescence using a handheld fiberoptic probe and 2) improve detection by reconstructing the depth-resolved fluorescence topography of sub-surface tumor. As a critical component to achieve these objectives, a technique to measure the tissue optical properties was developed. This technique used diffuse reflectance measurements mediated by a handheld fiberoptic probe to derive the tissue optical properties. The handheld fiberoptic probe was further developed to include fluorescence spectroscopy. A novel algorithm to combine the fluorescence measurement and the tissue optical properties was derived in order to extract the quantitative fluorescence spectrum, i.e. fluorescence without confounding effects of tissue optical properties. The concentration of fluorescent tumor biomarker can then be extracted. The quantitative fluorescence work culminated in deployment of the fiberoptic probe in clinical trials for the resection of intracranial tumors. The quantitative fluorescence probe out-performed a state-of-the-art fluorescence surgical microscope for a broad range of brain tumor pathologies. A novel technique for depth-resolved fluorescence detection was developed utilizing multi-excitation fluorescence imaging. An algorithm to extract depth information from the multi-excitation images was derived, with validation in phantoms and a rat brain tumor model. This demonstrates the potential for depth-resolved fluorescence imaging, which there is a clear need for in tumor resection guidance.

Photodynamic therapy (PDT) derives its tumour selectivity from preferential photosensitizer accumulation and short light penetration in tissue. However, additional strategies are needed to improve the therapeutic index of PDT in oncological applications where light is delivered interstitially to large volumes (e.g. prostate), or when adjacent normal tissue is extremely sensitive (e.g. brain). Much research to improve PDT's selectivity is directed towards developing targeted photosensitizers. Here, I present two alternative strategies to improve PDT's selectivity, without compromising its efficacy. For interstitial delivery, I investigated whether customizable cylindrical diffusers can be used to deliver light doses that conform better to target geometries, specifically the prostate. Additionally, I examined whether the neuroprotectant erythropoietin, used as an adjuvant to PDT for brain tumours, can reduce the sensitivity of normal tissue, thereby improving treatment selectivity. To determine if tailored diffusers constitute an improvement over conventional ones, I introduce a novel optimization algorithm for treatment planning. I also analyze the sensitivity of the resulting plans to changes in the optical properties and diffuser placement. These results are contextualized by a mathematical formalism to characterize the light dose distributions arising from tailored diffusers. In parallel, I investigate the neuroprotective effects of erythropoietin in PDT of primary cortical neurons in culture and normal rat brain in vivo. I show that the most important parameter determining prostate coverage is the number of diffusers employed. Moreover, while tailored diffusers do offer an improvement over conventional ones, the improvement is likely masked by perturbations introduced by the uncertainties of light delivery. Although these results largely discard the use of tailored diffusers in prostate PDT, significant insight has been gained into PDT treatment planning, and tailored diffusers may still be advantageous in more complicated geometries. Additionally, I show that erythropoietin does not improve survival of PDT-treated neurons PDT, nor reduces the volume of necrosis in vivo, for the ranges of conditions and doses studied. To our knowledge, this is the first time this strategy has been tested in brain PDT and deserves to be investigated further, by using later time-points, functional outcomes, and other neuroprotectants.

The clinical management of glioma remains a challenge. The prognosis is poor—for glioblastoma multiforme, the most virulent of these brain cancers, survival is only ~1 year. Surgical resection of the tumor is the first line of defense. Several studies demonstrate a survival advantage in patients who undergo near-complete tumor resection; however, achieving complete resection is limited by the difficulty of visualizing residual tumor after de-bulking. Intraoperative fluorescence guidance is a promising candidate to better visualize residual tumor. The most clinically developed form uses protoporphyrin IX fluorescence, the precursor to heme in its biosynthesis which preferentially accumulates in tumor cells after the administration of 5-aminolevulinic acid. Challenges remain in quantitatively assessing the fluorescence to reduce variability of outcome and improve tumor detection specificity, and in observing sub-surface tumor fluorescence. To these ends, this work outlines the development of intraoperative techniques to 1) quantify tissue fluorescence using a handheld fiberoptic probe and 2) improve detection by reconstructing the depth-resolved fluorescence topography of sub-surface tumor. As a critical component to achieve these objectives, a technique to measure the tissue optical properties was developed. This technique used diffuse reflectance measurements mediated by a handheld fiberoptic probe to derive the tissue optical properties. The handheld fiberoptic probe was further developed to include fluorescence spectroscopy. A novel algorithm to combine the fluorescence measurement and the tissue optical properties was derived in order to extract the quantitative fluorescence spectrum, i.e. fluorescence without confounding effects of tissue optical properties. The concentration of fluorescent tumor biomarker can then be extracted. The quantitative fluorescence work culminated in deployment of the fiberoptic probe in clinical trials for the resection of intracranial tumors. The quantitative fluorescence probe out-performed a state-of-the-art fluorescence surgical microscope for a broad range of brain tumor pathologies. A novel technique for depth-resolved fluorescence detection was developed utilizing multi-excitation fluorescence imaging. An algorithm to extract depth information from the multi-excitation images was derived, with validation in phantoms and a rat brain tumor model. This demonstrates the potential for depth-resolved fluorescence imaging, which there is a clear need for in tumor resection guidance.

<p>Breast conserving surgery (BCS) is a common treatment option for breast cancer patients. The goal of BCS is to remove the entire tumor from the breast while preserving as much normal tissue as possible for a better cosmetic outcome after surgery. Specifically, the excised specimen must have at least 2 mm of normal tissue surrounding the diseased mass. Unfortunately, a staggering 20-70% of patients undergoing BCS require repeated surgeries due to the incomplete removal of the tumor diagnosed post-operatively. Due to these high re-excision rates as well as limited post-operative histopathological sampling of the tumor specimen, there is an unmet clinical need for margin assessment. Quantitative diffuse reflectance spectral imaging has previously been explored as a promising, method for providing real-time visual maps of tissue composition to help surgeons determine breast tumor margins to ensure the complete removal of the disease during breast conserving surgery. We have leveraged the underlying sources of contrast in breast tissue, specifically total hemoglobin content, beta-carotene content, and tissue scattering, and developed various fiber optics based spectral imaging systems for this clinical application. Combined with a fast inverse Monte Carlo model of reflectance, previous studies have shown that this technology may be able to decrease re-excision rates for BCS. However, these systems, which all consist of a broadband source, fiber optics probes, an imaging spectrograph and a CCD, have severe limitations in system footprint, tumor area coverage, and speed for acquisition and analysis. The fiber based spectral imaging systems are not scalable to smaller designs that cover a large surveillance area at a very fast speed, which ultimately makes them impractical for use in the clinical environment. The objective of this dissertation was to design, develop, test, and show clinical feasibility of a novel wide field spectral imaging system that utilizes the same scientific principles of previously developed fiber optics based imaging systems, but improves upon the technical issues, such as size, complexity, and speed,to meet the demands of the intra-operative setting. </p><p>First, our simple re-design of the system completely eliminated the need for an imaging spectrograph and CCD by replacing them with an array of custom annular photodiodes. The geometry of the photodiodes were designed with the goal of minimizing optical crosstalk, maximizing SNR, and achieving the appropriate tissue sensing depth of up to 2 mm for tumor margin assessment. Without the imaging spectrograph and CCD, the system requires discrete wavelengths of light to launch into the tissue sample. A wavelength selection method that combines an inverse Monte Carlo model and a genetic algorithm was developed in order to optimize the wavelength choices specifically for the underlying breast tissue optical contrast. The final system design consisted of a broadband source with an 8-slot filter wheel containing the optimized set of wavelength choices, an optical light guide and quartz light delivery tube to send the 8 wavelengths of light in free space through the back apertures of each annular photodiode in the imaging array, an 8-channel integrating transimpedance amplifier circuit with a switch box and data acquisition card to collect the reflectance signal, and a laptop computer that controls all the components and analyzes the data.</p><p>This newly designed wide field spectral imaging system was tested in tissue-mimicking liquid phantoms and achieved comparable performance to previous clinically-validated fiber optics based systems in its ability to extract optical properties with high accuracy. The system was also tested in various biological samples, including a murine tumor model, porcine tissue, and human breast tissue, for the direct comparison with its fiber optics based counterparts. The photodiode based imaging system achieved comparable or better SNR, comparable extractions of optical properties extractions for all tissue types, and feasible improvements in speed and coverage for future iterations. We show proof of concept in performing fast, wide field spectral imaging with a simple, inexpensive design. With a reduction in size, cost, number of wavelengths used, and overall complexity, the system described by this dissertation allows for a more seamless scaling to higher pixel number and density in future iterations of the technology, which will help make this a clinically translatable tool for breast tumor margin assessment.</p> / Dissertation

This dissertation focuses on the development of algorithms for analyzing and modeling of the signals from optical spectroscopy. This dissertation is motivated by the detection of oral cancer, but some of the methods developed can be generalized to epithelial cancers of other sites. Two main topics are covered in this dissertation: Analysis and Modeling. For analysis, the focus is on developing algorithms to make diagnostic predictions. The analysis methods are empirically tested using an oral cancer dataset. Statistical analyses show that polarized reflectance spectroscopy has the potential to aid screening and diagnosis of oral cancer. Also, a novel adaptive windowing technique is developed to extract spectral features with fewer windows that retain the diagnostic information. For modeling, a Monte Carlo model simulating light-tissue interactions is presented to aid in the design of diagnostic instrumentation. / text