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)

<p>Cancer is now the leading cause of death in developed countries. Despite advances in strategies aimed at the prevention and treatment of the disease, early detection of precancerous growths remains the most effective method of reducing associated morbidity and mortality. Pathological examination of physical tissues that are collected via systematic biopsy is the current "gold standard" in this pursuit. Despite widespread acceptance of this methodology and high confidence in its performance, it is not without limitations. Recently, much attention has been given to the development of optical biopsy techniques that can be used clinically and are able to overcome these limitations. This dissertation describes one such optical biopsy technique, angle-resolved low coherence interferometry (a/LCI), its adaptation to a clinical technology, and its evaluation in clinical studies.</p><p> The dissertation presents the theory that underlies the operation of the a/LCI technique, the design and validation of the clinical instrument, and its evaluation by means of two clinical trials. First, an account of the manner in which the depth-resolved angular scattering profiles that are collected by a/LCI can be used to determine nuclear characteristics of the investigated tissues is given. The design of the clinical system that is able to collect these scattering profiles through an optical fiber probe that can be passed through the accessory channel of an endoscope for <italic>in vivo</italic> use is presented. To demonstrate the ability of this system to accurately determine the size of cell nuclei, a set of validation experiments are described.</p><p> In order to evaluate the clinical utility of this a/LCI system, two clinical trials intended to assess the ability of a/LCI to detect the presence of early, pre-cancerous dysplasias in human tissues are presented. The first of these, an <italic>in vivo</italic> study of Barrett's esophagus (BE) patients undergoing routine surveillance for the early signs of esophageal adenocarcinoma, is described. This study represents the first use of the a/LCI technique in vivo, and confirms its ability to provide clinically useful information regarding the disease state of the tissue that it examines, with performance that compares favorably to other optical biopsy techniques. Next, an <italics>ex vivo</italics> study of resected intestinal tissue is presented. The results of this study demonstrate the ability of a/LCI to provide information that can be used to detect dysplasia in the lower gastrointestinal tract with high accuracy. This study will enable future development of the technology to allow conduction of <italic>in vivo</italic> trials of intestinal tissue. The results of these two clinical studies demonstrate the clinical utility a/LCI, illustrating its potential as an optical biopsy technique that has great potential to provide diagnostically relevant information during surveillance procedures. This is particularly relevant in the case of BE, where its successful use has been demonstrated <italic>in vivo</italic>.</p> / Dissertation

Complete resection is correlated to better patient outcome in aggressive cancers such as glioblastoma. Optical biopsy refers to a family of techniques utilizing optical properties of living targets to make diagnoses where a biopsy would conventionally be used. Such a technology can potentially guide neurosurgeons in removing glioblastomas. Diffuse reflectance (DR) and Time-resolved fluorescence (TRF) have previously been investigated for their ability to measure biomarkers indicative of cancer. One of the difficulties faced in using TRF as a diagnostic tool is that multiple endogenous fluorophores will simultaneously contribute to the signal. This makes it difficult to attribute fluorescence lifetimes or spectral changes to one type of molecule in the tissue. This thesis focuses on the challenge of separating the components in a TRF measurement and their fractional contributions. A DR-TRF instrument was designed and built and characterized using fluorescent dyes. An orthonormal basis deconvolution method combined with a Fourier-domain method were tested for their ability to unmix fluorescent components in a hyperspectral TRF measurement. This method was tested on dye mixtures and retrieved fluorescence lifetimes of 4.6±0.4 ns and 2.7±0.2 ns in a mixture of Fluorescein and Coumarin-6 at concentrations of 5 μM each. It was also tested on an ex-vivo brain tissue where the fluorescence was approximated as a sum of 2 components. / Thesis / Master of Applied Science (MASc)

Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (~1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography data. However, the multi-mode characteristic of the fibers in the fiber-bundle affects the depth sensitivity of the imaging system. A description of light interference in a multi-mode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.

The first real-time uorescence confocal microlaparoscope has been de- veloped that provides instant in vivo cellular images, comparable to those provided by histology, through a nondestructive procedure. The device in- cludes an integrated contrast agent delivery mechanism and a computerized depth scan system. The instrument uses a fiber bundle to relay the image plane of a slit-scan confocal microlaparoscope into tissue. The confocal laparoscope was used to image the ovaries of twenty-one patients in vivo using uorescein sodium and acridine orange as the uorescent contrast agents. The results indicate that the device is safe and functions as designed. A Monte Carlo model was developed to characterize the system performance in a scattering media representative of human tissues. The results indicate that a slit aperture has limited ability to image below the surface of tissue. In contrast, the results show that multi-pinhole apertures such as a Nipkow disk or a linear pinhole array can achieve nearly the same depth performance as a single pinhole aperture. The model was used to determine the optimal aperture spacing for the multi-pinhole apertures. The confocal microlaparoscope represents a new type of in vivo imaging device. With its ability to image cellular details in real time, it has the potential to aid in the early diagnosis of cancer. Initially, the device may be used to locate unusual regions for guided biopsies. In the long term, the device may be able to supplant traditional biopsies and allow the surgeon to identify early stage cancer in vivo.

One in eight women in America will develop breast cancer at some point in their lives. Breast cancer is the second deadliest form of cancer for women in the United States. When a suspicious region of the breast is detected, the tissue is diagnosed by removing a sample, preparing an H&E section, and performing histopathology. This procedure is expensive, invasive, and can take days to return a diagnosis. An alternative to excision biopsies is to instead perform an optical biopsy. This work details endomicroscopes intended to perform optical biopsies in breast tissue. The work address two issues limiting current optical biopsy systems: insufficient resolution and inability to reject out of focus light. To improve the resolution of current endomicroscopes, ultra-slim objectives are developed using optical plastics and zero alignment fabrication techniques. These objectives can outperform current alternative endomicroscope objectives in terms of performance across the field of view and chromatic aberration correction, while remaining as narrow as a biopsy needle. Next, an endomicroscope which utilizes structured illumination to perform optical section is designed, tested, and evaluated on ex vivo breast biopsies. The new endomicroscope provides high contrast images by reducing out of focus background light. Finally, an achromatic, ultra-slim objective and the structured illumination endomicroscope are integrated to form an optical biopsy system with improved lateral resolution and axial response. This integrated system is a step forward for in vivo microscopy and cancer diagnoses.

Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-04-23T17:39:54Z No. of bitstreams: 1 Tese - Gilberth Acioli.pdf: 3536665 bytes, checksum: a37b496b111b3eb77680d5e81b8fac30 (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-05-08T12:01:41Z (GMT) No. of bitstreams: 1 Tese - Gilberth Acioli.pdf: 3536665 bytes, checksum: a37b496b111b3eb77680d5e81b8fac30 (MD5) / Made available in DSpace on 2013-05-08T12:01:42Z (GMT). No. of bitstreams: 1 Tese - Gilberth Acioli.pdf: 3536665 bytes, checksum: a37b496b111b3eb77680d5e81b8fac30 (MD5) Previous issue date: 2010 / O objetivo deste estudo foi avaliar o efeito da fotobiomodulação laser (780nm, 50mW, 4x4J/cm2 = 16J/cm2, ϕ 0,5cm2, CW) associada ou não a implante de Enxerto Ósseo Cerâmico Bifásico e Reparação Óssea Guiada através da técnica de histologia, histomorfometria, espectroscopia Raman e Fluorescência laser no reparo de fraturas cirúrgicas fixadas com o sistema de fixação rígida (miniplacas) ou semi-rígida (fio de aço) em tíbias de coelhos. Foram utilizados 27 coelhos Oryctolagus que foram divididos em nove grupos e mantidos em gaiolas individuais em temperatura média de 22°C, ambientação dia/noite, alimentação sólida e água ad libidum. As fraturas foram produzidas sob anestesia geral (Ketamina 0,4ml/Kg IP e Xilazina 0,2ml/Kg IP). No período pós operatório os mesmos receberam em dose única, como terapia antimicrobiana (Pentabiótico 0,2ml/Kg IM) e como terapia antiinflamatória e analgésica (Banamine 0,1ml/Kg IM). Nos grupos II, III, IV e V foram realizadas as fraturas e os cotos ósseos fixados com sistema rígido (FIR). Nos grupos VI, VII, VIII e IX a fratura foi realizada e logo depois fixada com sistema semi-rígido (FISR). Em seguida, foi feita a colocação do enxerto e da membrana nos grupos III, V, VII e IX. Os animais dos grupos IV, V, VIII e IX foram irradiados durante 14 (catorze) dias, a cada 48 horas com uma dose de 16J/cm2, de forma pontual em 4 (quatro) regiões adjacentes a área da fratura óssea (4 x 4J/cm2). Os animais foram sacrificados no 30° dia pós-operatório através de overdose de anestesia geral (Ketamina e Xilazina IP) e administração de Cloreto de Potássio (5ml/Kg, IV). Em seguida os espécimes foram removidos, sendo metade encaminhado para análises histológica e histomorfométrica e a outra metade para análise por espectroscopia Raman. Antes da cirurgia e da morte animal a fluorescência laser foi medida. Histologicamente, observou-se um preenchimento das fraturas por um trabeculado ósseo maduro nos grupos onde houve o uso da associação laser, HATCP e ROG nos grupos tratados com FIR e FISR. Histomorfometricamente verificou-se maior neoformação óssea e maior deposição de colágeno, menor quantidade reabsorção óssea e de infiltrado inflamatório nos grupos nos quais o laser foi associado a HATCP. As análises por fluorescência laser (DIAGNOdent®) e por espectroscopia Raman, observaram-se diferenças significantes entre os grupos (p<0.001) entre os grupos tratados com FIR e FISR. A correlação de Pearson evidenciou uma correlação negativa entre as medidas de fluorescência e deslocamento Raman. Concluiu-se que a fotobiomodulação Laser infravermelho acelerou o reparo de fraturas ósseas e que quando o laser foi associado a HATCP e ROG esta causou aumento da deposição da HAC. Adicionalmente, o uso do DIAGNOdent® como instrumento de biópsia óptica pode ser útil. / Salvador

<p>The annual incidence rate of tumours in the brain and central nervous system (CNS) was 19.89 per 100,000 persons between 2004 and 2008 in the United States. Surgery is a common treatment option for brain and CNS tumours. Typically, biopsy followed by histological analysis is used to confirm tumour types and margin during neurosurgery as an intraoperative diagnostic tool. However, this biopsy method is invasive, sampling number limited and not in real-time. To overcome these problems, many minimally invasive optical techniques, called optical biopsies, have been developed towards intraoperative diagnosis.</p> <p>The research work carried out in this dissertation focuses on combining the time-resolved fluorescence (TRF) and diffuse reflectance (DR) spectroscopy towards intraoperative tumour margin detection in neurosurgery. Combining these two modalities allows us to obtain additional contrast features, thus potentially improving the diagnostic accuracy. To achieve this goal, first, a clinically compatible integrated TRF-DR spectroscopy instrument was developed for <em>in vivo</em> brain tumour study. An acousto-optical-tunable-filter-based spectrometer was designed to acquire the time-resolved fluorescence signal. A dual-modality fibre optic probe was used to collect the TRF and DR signals in a small volume. The system’s capabilities of resolving fluorescence spectrum and lifetime, and optical properties were characterized and validated using tissue phantoms. Second, in order to retrieve the fluorescence impulse response function accurately from measured fluorescence signals, a robust Laguerre-based deconvolution method was optimized by using the constrained linear least squares fitting and high order Laguerre function basis. This optimized Laguerre-based deconvolution method overcomes the over-fitting problem introduced by low signal-to-noise ratio and complex fitting model. Third, an <em>ex vivo</em> clinical study of brain tumours was carried out using the TRF and DR spectroscopy. Fluorescence spectra and lifetime features were selected to classify various tumour types. The sensitivity and specificity of meningioma grade I differentiated from meningioma grade II are both 100%. Finally, in order to increase the measurement tissue volume and obtain imaging contrast features, a scanning-based hyperspectral fluorescence lifetime imaging system was developed. This setup can provide time-, space-, spectrum- resolved multi-dimensional images for tumour margin detection.</p> / Doctor of Philosophy (PhD)

Orientador: Prof. Dr. Herculano da Silva Martinho / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017.

Les avancées technologiques ont ouvert la voie à des approches de biopsie optique innovantes. Elles permettent à l'inverse des méthodes physiques de profiter des avantages d'une procédure mini-invasive, temps réels et répétitive. Le système de tomographie par cohérence optique (OCT) (la technique de biopsie optique utilisée dans cette thèse) propose des approches qui naviguent dans le corps humain grâce à des sondes endoscopiques robotisées. Toutefois, leur contrôle une fois à l’intérieur du corps devient difficile, surtout si l’objectif est de suivre l’évolution d'une zone cible, en faisant un travail de repositionnement dans le temps. L'asservissement visuel est un outil de choix pour le contrôle et le positionnement directement par l'image. Néanmoins, la richesse des informations présentes dans les images autorisent l'utilisation de plusieurs types d'information visuelle. Dans ce contexte, nous proposons l'utilisation de primitives visuelles innovantes fondées sur les ondelettes. Ainsi, deux approches d'asservissements visuels fondées sur les ondelettes ont été développées. La première approche est un asservissement visuel 2D pose fondé sur les ondelettes spectrales continues qui assure une convergence sur un espace plus important avec une bonne robustesse au bruit et une commande découplée. La deuxième est un asservissement visuel 2D direct fondé sur les ondelettes multirésolution, principalement pour faire du positionnement aux petits déplacements. Par ailleurs, la deuxième méthode couvre les 6 DDL quand la première se limite aux 3 DDL dans les images CCD. De plus, ces deux approches ont prouvé leurs aptitudes à faire du positionnement des coupes OCT. Mais encore, nous avons proposé une méthode de positionnement partitionnée que nous pouvons qualifier d'hybride, car elle exploite deux modalités d'images (OCT - CCD) pour assurer un positionnement sur SE(3) d'un échantillon. De même, nous avons proposé une méthode d'étalonnage des images de coupe et de volume OCT, liée aux distorsions générées par le chemin optique parcouru par le faisceau laser OCT. Finalement, ces travaux ouvrent la voie vers des applications dans le positionnement des volumes OCT, la compensation de mouvement physiologique et le suivi d'outils par des images OCT. / The technological advances have facilitated the optical biopsy approaches, unlike physical methods to take advantage of a minimally invasive, real time and repetitive procedure. The optical coherence tomography system is one of the optical biopsy techniques used in this thesis to prospect in the human body with robotized OCT endoscopic probes. Nevertheless, their control once inside the body becomes difficult, especially if the goal is following changes in the target area. The visual servoing is an ideal tool for the control and positioning of the robot. However, the amount of information present in the images allows the use of several types of visual features. In this thesis, we propose to use an innovative visual servoing feature based on wavelets. This representation developed as the evolution of the Fourier transform for non-stationary signals provides a time-frequency representation of the signal with a better extraction of the relevant information. Indeed, two visual servoing approaches based on wavelets were developed. The first approach is a 2D pose visual servoing based on spectral continuous wavelets, which ensures convergence over a larger area and decoupled control. The second is a direct 2D visual servoing based on multiresolution wavelets, mainly for small displacements positioning. However, the latter covers the 6 DOF when the previous one is limited to 3 DDL with a CCD camera. Both approaches have proven their ability to make the positioning of B-Scan OCT images. After that, we have proposed a method of partitioned positioning, that we can qualify by hybrid because it uses two image modalities to ensure SE(3) positioning of a sample. On the other side, we proposed a calibration method of B-Scan and 3D-Scan OCT images, due to the distortions generated by the optical path of the laser beam in OCT. Finally, these thesis is a beginning work for applications in positioning of 3D-Scan OCT, physiological motion compensation and monitoring tools by OCT images.