Dentre as diversas aplicações da luz em nosso dia-a-dia, as ligadas à área biomédica merecem destaque e são frequentemente objetos de pesquisa tanto para o desenvolvimento quanto para o aprimoramento de técnicas para o diagnóstico e terapias. Os tecidos biológicos são, em sua maioria, estruturas complexas, não-homogêneas e opticamente muito espalhadoras. Apesar das centenas de estudos existentes acerca da propagação da luz em tecidos biológicos, sua complexidade exige que novos estudos sejam conduzidos a fim de aprimorar o conhecimento já existente, que ainda apresenta muitas lacunas. A presença de heterogeneidades nos tecidos (vasos sanguíneos, hematomas, cistos, tumores e outras alterações macroscópicas) mudam a propagação da luz e dificultam a previsibilidade do seu comportamento por modelos matemáticos. O presente trabalho teve por objetivo estabelecer um método empírico que utiliza imagens de refletância difusa obtidas através de uma instrumentação simples, baseada em uma fonte de luz contínua no visível (laser de diodo em 660 nm) e uma câmera CMOS monocromática, para verificar a possibilidade de localização de inclusões absorvedoras embebidas em phantoms altamente espalhadores. Foi avaliada a capacidade de detectar inclusões de dois diferentes tipos de materiais em diversas geometrias e tamanhos, posicionadas em diferentes profundidades. O ângulo de incidência do feixe laser também foi variado, bem como a distância entre a fonte e o objeto, a fim de avaliar quais as melhores condições experimentais. Os resultados obtidos mostraram que os objetos puderam ser detectados, e suas formas puderam ser satisfatoriamente recuperadas através de um algoritmo desenvolvido para o processamento das imagens. Em algumas situações, mesmo para a maior profundidade utilizada, que foi de 20 mm, a inclusão pôde ser detectada nas imagens de refletância difusa processadas. Apesar da capacidade de detecção das formas geométricas representar um avanço com relação às possibilidades de identificação de estruturas em meios túrbidos, a determinação da profundidade ainda é um desafio a ser superado. / Among the various applications of light in our daily life, those connected with biomedicine should be highlighted and are frequently subject of researches aiming for the development and for the enhancement of techniques for diagnosis and therapy. Biological tissues are mostly complex, non-homogeneous and optically highly scattering structures. Despite the hundreds of existent studies on the propagation of light in biological tissues, its complexity requires new studies to be conducted in order to improve the existing knowledge, which still has many gaps. The presence of heterogeneities in tissue (blood vessels, bruises, cysts, tumors and other macroscopic alteration) changes the light propagation and impedes the predictability of its behavior by mathematical models. This work aimed to establish an empirical method using diffuse reflectance images acquired with simple instrumentation, based on a source of continuous light in the visible (diode laser at 660 nm) and a monochromatic CMOS camera, to check the possibility of the location of absorbing inclusions embedded in highly scattering phantoms. The ability to detect inclusions of two different kinds of materials in different sizes and geometries, positioned at different depths were evaluated. The laser beam angle of incidence was also varied, as well as the distance between the source and the object, in order to evaluate the best experimental conditions. The results showed that the objects could be detected, and their shapes might be satisfactorily recovered by an algorithm developed for image processing. In some situations, even at the greatest depth used, which was 20 mm, the inclusion could be detected in diffuse reflectance processed images. Although the detection capability of geometric shapes represents an improvement over the structures of identification possibilities in turbid media, the determination of depth is still a challenge to be overcome.

L'imagerie à travers les milieux turbides comme le brouillard, les tissus, les colloïdes, etc. répond à plusieurs besoins de la vie courante. L'imagerie à travers de tels milieux diffusants est un défi auquel peuvent répondre les nouveaux systèmes d'imagerie, la théorie de l'information et l'étude des lois de transport de la lumière dans les milieux aléatoires. La thèse est divisée en deux parties adressant deux modalités d'imagerie différentes, à savoir : l'imagerie de contraste polarimétrique et l'imagerie modulée en intensité. Dans les deux cas, des systèmes d'imagerie en temps réel sont proposés et mis au point. Leurs performances sont évaluées à la fois théoriquement et expérimentalement. Dans la première partie de la thèse, une caméra polarimétrique à deux canaux instantanés conçue autour d'un prisme de Wollaston est utilisée pour imager de manière optimale une source de lumière polarisée noyée dans un brouillard. Une expérience en situation réelle a été mise en place à proximité du campus de Beaulieu à Rennes. La source est placée sur une tour de télécommunication située à plus d'un kilomètre du système imageant. Les données acquises dans diverses conditions météorologiques montrent que l'efficacité de cette caméra polarimétrique dépend de la corrélation du bruit de fond dans les deux images initiales. Ceci a été confirmé grâce à une analyse fondée sur la théorie de l'information qui montre que le contraste polarimétriques maximal est obtenu par une combinaison linéaire des deux canaux polarimétriques dont la pondération dépendant de la corrélation du bruit de fond dans les deux canaux. Un système de détection, intégrant cette représentation polarimétrique optimale, a été développé pour explorer de bout en bout les capacités offertes par l'imagerie polarimétrique à deux canaux à travers le brouillard. Ces études trouvent des applications directes dans le transport par temps dégradé, y compris pour l'aide à l'atterrissage d'aéronefs. Dans la même logique, la deuxième partie de la thèse porte sur l'apport de la modulation d'intensité plein champ pour imager les photons balistiques dans les milieux diffusants. En utilisant de concert la théorie de la diffusion et la théorie de l'information, nous avons pu montrer que, pour un budget de photons donné, il existait une fréquence de modulation minimale pour laquelle le filtrage de photons balistique devient efficace. Cette fréquence dépend des propriétés de diffusion du milieu intermédiaire et se trouve être dans la gamme du MHz en situation réelle. L'imagerie en temps réel à de telles fréquences étant un vrai défi, nous avons proposé un système de démodulation plein champ inédit basé sur l'utilisation d'un cristal électro-optique. Ce système d'imagerie, dont nous avons breveté le principe, est en mesure de démoduler avec une caméra standard une scène en temps réel et en plein champ à des fréquences de plusieurs MHz (voire GHz) sans synchronisation de phase. Un prototype de ce système a été développé permettant de confirmer qu'il était robuste, portable et rentable. Le travail présenté dans cette thèse ouvre la voie à la mise en œuvre de systèmes d'imagerie de pointe fonctionnant dans des situations réelles, allant de l'imagerie biomédicale, à la sécurité. / Imaging through turbid media like fog, tissues, colloids etc. has various applications in real-life situations. The problem of imaging through such scattering media presents a challenge that can be addressed by using novel imaging schemes, information theory and laws of light transport through random scattering media. The thesis is divided into two parts corresponding to two different imaging modalities, namely, polarimetric contrast imaging and intensity modulated light imaging. In both the cases, advanced imaging systems, capable of imaging in real-time are used and their performances are evaluated both theoretically and experimentally. In the first part of the thesis, a two-channel, snapshot polarimetric camera, based on a Wollaston prism is used to attain optimal imaging of polarized light source through fog. An original outdoor experiment is setup in the vicinity of the campus Beaulieu in Rennes, France, where a source is placed on a telecommunication tower more than a kilometer away from the imaging system. Data acquired in various weather conditions show that the efficiency of the two-channel polarimetric camera depends on the background noise correlation in the two images. Further, this was confirmed using an information theoretical analysis, which showed that a polarimetric contrast maximizing image representation is a linear combination of the two polarimetric images whose weights depend on the background noise correlation. Based on the derived optimal polarimetric representation, a detection scheme was presented, leading to an end-to-end study of two-channel polarimetric imaging through fog that may be useful in transport applications like aircraft landing/taxiing in degraded weather. The second part of the thesis deals with intensity modulated light and its potential for ballistic photon imaging through scattering media. First, using the diffusion theory of photon transport and information theory, it was shown that for a given photon budget, ballistic imaging can be achieved for a minimum modulation frequency that depends on the scattering properties of the intervening medium. In real-life situation, the minimum frequency can be in the range of MHz. Real-time imaging at these frequencies is a challenge. Hence, a novel demodulation camera system based on electro-optics was proposed and patented. The imaging system is capable of real-time, full-field demodulation at frequencies of several MHz (potentially, in GHz as well), without requiring a phase synchronized source. A prototype of the imaging system was developed and shown that a demodulation camera based on the proposed design is robust, portable and cost-effective. Finally, the work presented in this thesis pave way for implementation of advanced imaging systems in real-life situations, varying from biomedical imaging to transport safety.

Diplomová práca sa zaoberá kvantitatívnym fázovým zobrazovaním objektov umiestnených za rozptyľujúcim prostredím v koherenciou riadenom holografickom mikroskope. Tento mikroskop umožnuje zobrazovať s úplne nekoherentným osvetlením vzorky, čo vyvoláva efekt koherenčnej brány. Koherenčná brána je veľmi dôležitá vlastnosť zobrazovacieho systému umožňujúca separáciu balistických a rozptýlených fotónov, jej dôkladnému vysvetleniu je venovaná značná časť práce. Ďalej sú prezentované základy teórie zobrazenia v koherenciou riadenom holografickom mikroskope. Tie sú využité v závere práce pri interpretácii experimentálnych výsledkov. Cieľom práce je navrhnúť metódu pre pozorovanie fázových objektov v rozptyľujúcich prostrediach a experimentálne túto metódu overiť. Na základe analytických výsledkov a predchádzajúceho výskumu je navrhnutá nová metóda, ktorá je ďalej overovaná pomocou rôzne komplexných vzoriek. Je založená na zázname viacerých obrazov s rôznym posunutím referenčného poľa. Každý posun korešponduje so zobrazovaním pomocou inej skupiny fotónov. Je možné vytvoriť syntetický obraz so zlepšenou kvalitou sčítaním jednotlivých obrazov získaných z interferencie balistických alebo rozptýlených fotónov. Experimenty s rôzne komplexnými vzorkami poskytujú náhľad na obmedzenia prezentovanej metódy.

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Les techniques de microscopie non linéaire connaissent un essor considérable ensciences du vivant, du fait de leur capacité à imager les tissus biologiques en profondeur et àexploiter différents contrastes dont les plus connus sont la fluorescence excitée à deux photons(2PEF) et la génération de second harmonique (SHG).Ce travail de thèse ’articule autour de la métrologie de milieux diffusants et d’applicationsbiomédicales de la microscopie non linéaire. Après une présentation générale de la technique, nousdécrivons une méthode originale de mesure du coefficient de diffusion μs et du facteur d’anisotropieg de milieux turbides épais basée sur la comparaison des intensités de fluorescence épi-collectéesselon trois modalités de notre microscope non-linéaire. Notre méthode est alors appliquée à lacaractérisation de gels biomimétiques et d’échantillons d’intérêt biologique. Le manuscrit abordeensuite le problème de l’imagerie en profondeur d’explants de peau humaine ré-innervée par desneurones sensoriels de rats nouveau-nés. Le choix du marqueur neuronal fluorescent fait l’objetd’une mesure in situ de la section efficace d’absorption à deux photons de différents fluorophores.La faisabilité d’une imagerie bimodale exploitant la fluorescence de ce marqueur et la réponse SHGdu collagène fibrillaire du derme est démontrée. Le manuscrit s’achève par une étude faisant suiteà des travaux de thèse antérieurs relatifs à la quantification de la fibrose hépatique par microscopieSHG/2PEF couplée. Nous appliquons la méthode de scoring SHG développée précédemment àune cohorte de patients infectés par le virus de l’hépatite C et comparons nos résultats aux testsMETAVIR et Ishak. / Nonlinear microscopy techniques are experiencing a considerable growth in lifescience, thanks to their ability to image biological tissues at high depth with different contrastssuch as two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG).This manuscript focuses on metrology of scattering media and on biomedical applications ofnonlinear microscopy. After an overview of the technique, we describe a novel method for measuringthe scattering coefficient μs and anisotropy factor g of thick turbid media based on the comparisonof fluorescence intensities epi-collected through the three modalities of our nonlinear microscope.Our method is then applied to biomimetic gels and to biological samples. The manuscript thentackles the problem of imaging deeply human skin explants re-innervated by sensory neurons fromneonatal rats. The choice of the fluorescence probe is the subject of an in situ measurement ofthe two-photon action cross-sections of various fluorophores. The feasibility of bimodal nonlinearimaging of re-innervated skin explants based on the 2PEF signal of the molecular probe and onthe SHG response of fibrillar collagen of the dermis is demonstrated. The manuscript ends witha study subsequent to the work of previous thesis regarding the quantification of liver fibrosisby SHG/2PEF microscopy. We apply the method of fibrillar collagen scoring by SHG previouslydeveloped to a new cohort of patients infected with hepatitis C virus and we analyze our resultsin terms of METAVIR and Ishak tests.

Large-scale and cost-effective manufacturing of ceramic micro devices based on tape stacking requires the development of inspection systems to perform high-resolution in-process quality control of embedded manufactured cavities, metal structures and defects. In this work, alumina ceramic samples are evaluated by optical coherence tomography (OCT) operating at 1.3μm wavelength and some dimensional data are obtained by dedicated image processing and segmentation. Layer thicknesses can be measured and laser-machined channels can be verified embedded at around 100μm depth. Moreover, detection of internal defects is enabled. Monte Carlo ray tracing simulations are employed to analyze the abilities of OCT in imaging of the embedded channels. The light scattering mechanism is studied for the alumina ceramics, and different scattering origins and models are discussed. The scattering parameters required as input data for simulations are evaluated from the integrating sphere measurements of collimated and diffuse transmittance spectra using a reconstruction algorithm based on refined diffusion approximation approach. / <p>QC 20120628</p>

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ransmitted-light coherence-controlled holographic microscope (CCHM) based on an off-axis achromatic and space-invariant interferometer with a diffractive beamsplitter has been designed, constructed and tested. It is capable to image objects illuminated by light sources of arbitrary degree of temporal and spatial coherence. Off-axis image-plane hologram is recorded and the image complex amplitude (intensity and phase) is reconstructed numerically using fast Fourier transform algorithms. Phase image represents the optical path difference between the object and the reference arms caused by presence of an object. Therefore, it is a quantitative phase contrast image. Intensity image is confocal-like. Optical sectioning effect induced by an extended, spatial incoherent light source is equivalent to a conventional confocal image. CCHM is therefore capable to image objects under a diffusive layer or immersed in a turbid media. Spatial and temporal incoherence of illumination makes the optical sectioning effect stronger compared to a confocal imaging process. Object wave reconstruction from the only one recorded interference pattern ensures high resistance to vibrations and medium or ambience fluctuations. The frame rate is not limited by any component of the optical setup. Only the detector and computer speeds limit the frame rate. CCHM therefore allows observation of rapidly varying phenomena. CCHM makes the ex-post numerical refocusing possible within the coherence volume. Coherence degree of the light source in CCHM can be adapted to the object and to the required image properties. More coherent illumination provides wider range of numerical refocusing. On the other hand, a lower degree of coherence makes the optical sectioning stronger, i.e. the optical sections are thiner, it reduces coherence-noise and it makes it possible to separate the ballistic light. In addition to the ballistic light separation, CCHM enables us to separate the diffused light. Multi-colour-light

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Optics are a powerful probe of chemical structure that can often be linked to theoretical predictions, providing robustness as a measurement tool. Not only do optical interactions like second harmonic generation (SHG), single and two-photon excited fluorescence (TPEF), and infrared absorption provide chemical specificity at the molecular and macromolecular scale, but the ability to image enables mapping heterogeneous behavior across complex systems such as biological tissue. This thesis will discuss nonlinear and linear optics, leveraging theoretical predictions to provide frameworks for interpreting analytical measurement. In turn, the causal mechanistic understanding provided by these frameworks will enable structurally specific quantitative tools with a special emphasis on application in biological imaging. The thesis will begin with an introduction to 2nd order nonlinear optics and the polarization analysis thereof, covering both the Jones framework for polarization analysis and the design of experiment. Novel experimental architectures aimed at reducing 1/f noise in polarization analysis will be discussed, leveraging both rapid modulation in time through electro-optic modulators (Chapter 2), as well as fixed-optic spatial modulation approaches (Chapter 3). In addition, challenges in polarization-dependent imaging within turbid systems will be addressed with the discussion of a theoretical framework to model SHG occurring from unpolarized light (Chapter 4). The application of this framework to thick tissue imaging for analysis of collagen local structure can provide a method for characterizing changes in tissue morphology associated with some common cancers (Chapter 5). In addition to discussion of nonlinear optical phenomena, a novel mechanism for electric dipole allowed fluorescence-detected circular dichroism will be introduced (Chapter 6). Tackling challenges associated with label-free chemically specific imaging, the construction of a novel infrared hyperspectral microscope for chemical classification in complex mixtures will be presented (Chapter 7). The thesis will conclude with a discussion of the inherent disadvantages in taking the traditional paradigm of modeling and measuring chemistry separately and provide the multi-agent consensus equilibrium (MACE) framework as an alternative to the classic meet-in-the-middle approach (Chapter 8). Spanning topics from pure theoretical descriptions of light-matter interaction to full experimental work, this thesis aims to unify these two fronts. <br>