En microscopie, savoir focaliser la lumière à l’échelle micrométrique est déterminant. Dans les tissus biologiques néanmoins, les inhomogénéités du milieu diffusent la lumière, empêchant toute focalisation au-delà d’une profondeur de l’ordre du millimètre. Des techniques de façonnage de front d’onde ont été développées afin de pré-compenser la distorsion du faisceau lumineux induite par la propagation à travers un milieu diffusant. Pour parvenir à focaliser la lumière à l’intérieur même du milieu diffusant, l’enjeu est de mesurer l’intensité lumineuse en profondeur de manière non invasive. Nous proposons d’utiliser l’effet photoacoustique pour sonder cette intensité. Une structure optiquement absorbante éclairée par une impulsion lumineuse émet en effet un signal ultrasonore, dont l’amplitude est proportionnelle à l’intensité lumineuse. Ces ultrasons se propagent de façon quasi-balistique dans les tissus mous et peuvent donc être détectés à l’aide d’un transducteur acoustique externe. Cette mesure permet donc de déterminer l’intensité lumineuse éclairant l’absorbeur. Nous avons montré qu’il était possible d’utiliser l'imagerie photoacoustique pour mesurer la matrice de transmission d’un échantillon diffusant. Cette caractérisation nous permet de focaliser la lumière sur des structures absorbantes et de sonder des propriétés mésoscopiques du milieu diffusant. Nous avons montré que la large bande spectrale des signaux photoacoustiques permet d’améliorer la focalisation. Enfin, nous avons montré que l’utilisation d’une source de lumière cohérente permet de pallier certains artefacts de l’imagerie photoacoustique, ainsi que de franchir la limite de résolution acoustique. / Light focusing is a crucial requirement for high resolution optical imaging. In biological tissue though, refractive index inhomogeneities scatter light, preventing any focusing beyond one millimeter. Wavefront shaping techniques have been recently developed to partially compensate for light scattering after propagation through a scattering medium. These techniques require a measurement of the light intensity at the target point. These techniques hold much promise for performing wavefront correction in order to focus light deep inside scattering media. This would require a non-invasive measure of the light intensity at depth. In this PhD study, we propose to use the photoacoustic effect for such task. An optically absorbing structure under pulsed illumination indeed generates ultrasonic waves, whose amplitude is proportional to the absorbed light intensity. These ultrasounds mostly propagate in a ballistic way, and can therefore be detected with an external transducer. We have shown that photoacoustic imaging could be used to measure the transmission matrix of a scattering sample, enabling to focus light on absorbing structures as well as to retrieve mesoscopic properties of the medium. We have shown that the broadband spectral content of the photoacoustic signals can be harnessed to improve the focusing performances. Finally, we demonstrated that coherent illumination could be used to remove fundamentals artefacts, as well as to break the acoustic resolution limit of conventional deep tissue photoacoustic imaging.

L'imagerie optique des tissus biologiques est un défi du fait de la diffusion de la lumière. Pour sonder les propriétés optiques à quelques cm de profondeur, on peut coupler l'information optique des ultrasons. De cette idée sont nées les imageries acousto-optique et photoacoustique. La première repose sur la modulation de la lumière par des ultrasons balistiques. La seconde se base sur la génération d'ultrasons lors de l'absorption de lumière par un objet. Que ce soit pour l'une ou pour l'autre, l'enregistrement du signal nécessite la mesure de très faibles modulations de phase dans une figure de speckle. L'holographie dynamique est une bonne solution. En effet, les techniques interférométriques sont suffisamment sensibles pour mesurer de telles modulations et l'holographie permet de corriger la nature speckle de la lumière. Dans cette thèse nous démontrons la faisabilité de fabriquer un système d'holographie adaptative basé sur un milieu laser (Nd :YVO4). Un des grands avantages de ce type de milieu est le temps de réponse. On montrera que le rafraîchissement d'un hologramme dans notre cristal peut se faire en moins de 100 ?s, bien inférieur au temps de décorrélation du speckle (? 1ms) qui pourrait grandement perturber les techniques de détection plus lentes lors d'expériences in vivo. Trois montages sont présentés ici, le premier pour la détection acousto-optique par conjugaison de phase, le deuxième pour la détection acousto-optique par adaptation de front d'onde et enfin le troisième pour détection photoacoustique. Dans les trois cas on mesure un temps de réponse entre 15 ?s et 50 ?s, et on utilise le montage imager un échantillon. / Strong scattering properties of biological media make their optical imaging in depth a challenge. A solution to probe the local optical properties is to couple the optical information with ultrasound. Two imaging techniques were born from this idea, acousto-optic imaging and photoacoustic imaging. The first technique is based on the local modulation of light by ballistic ultrasound. The latter relies on the emission of ultrasound following the absorption of light by an object. Whether it is acousto-optic imaging or photoacoustic imaging, the recording of the the signal requires a detection system sensitive to weak phase modulation. In addition, the detection system must be compatible with a speckle pattern. Dynamic holography is a good solution. Indeed, as it is based on interferometry, it is very sensitive to small phase variations and holography can be used to correct the speckle nature of light. In this manuscript, we show the use of an holographic detection system based on a laser medium (Nd:YVO4). One of the main advantage of this type of material is the very fast response time. It will be highlighted that the recording of a hologram inside our crystal can be done in less than 100 μs, much faster than the speckle decorrelation time (≈ 1ms), which is one of the major obstacle towards in vivo imaging. Three optical setups will be presented in this manuscript. The first one is a phase conjugation setup for acousto-optic detection. The second one is a wavefront adaption setup, also for acousto-optic detection. Finally, the third setup is an adaptive vibrometry setup for photoacoustic detection. In each setups the measured response time is between 15 μs and 50 μs.

Billions of people use biomass burning cookstoves in their homes and suffer serious health repercussions. Additionally, global warming is exacerbated by cookstove emissions containing greenhouse gases and particulate matter. Improved cookstoves (ICSs) mitigate the problem, but accurate and affordable emission gas measurements, particularly of Carbon Dioxide (CO2) and Carbon Monoxide (CO), are required in order to confidently declare ICSs cleaner burning than traditional cookstoves. The aim of this research is to assess the suitability of photoacoustic (PA) CO2 detection technology for cookstove emissions monitoring. The designs of several longitudinally resonant, photoacoustic, LED, CO2 sensors of varying levels of functionality are presented. Three aluminum cell designs allowed the detection of a photoacoustic signal: a 4cm long cylinder with a ~1cm diameter (Design 3), a 3.9cm long cylindrical resonator with ~1in diameter and quarter-acoustic-wavelength buffer volumes (Designs 4a,b), and a 3.7cm long cylinder with ~1in diameter (Design 5). All three cell designs operate in the longitudinal resonant mode via the irradiation of gases inside the PA cell with a 4.3um wavelength LED, driven at an on-off frequency in the kHz range by a square wave from an Arduino. A rudimentary lock-in amplifier (LIA) based on the AD630 was considered, but the SR830 LIA was actually used to extract the desired MEMS microphone signal from noise. Designs 3-4b produced PA signals dominated by wall-absorption, but the final design (Design 5) yielded a resonant PA signal proportional to CO2 concentration. It was discovered that photoacoustic gas detection is challenging to design and set up without extensive experience and equipment. Practical lessons learned are shared. Primary limitations with the presented designs are identified as the extremely low power of the 4.3um LEDs, wall absorption due to insufficient collimation of LED radiation, dependence on temperature, and reliance on an expensive, high performance, lock-in amplifier. Further testing and development of designs like Design 5 (short cylinder with large diameter-to-length ratio) is necessary to evaluate their potential for in-field, real-time CO2 concentration measurement. Though LED PA CO2 sensing was demonstrated to be possible, it is concluded that NDIR CO2 sensors are currently better suited for cookstove use. In addition to photoacoustic detection, a method of detecting CO2 concentration by measuring resonant frequency of the gas cell (The Acoustic Method) is presented.

Contamination of peanuts by secondary metabolites of certain fungi, namely aflatoxins present a great health hazard when exposed either at low levels for prolonged times (carcinogenic) or at high levels at once (poisonous). It is important to develop an accurate and rapid measurement technique to trace the aflatoxin and/or source fungi presence in peanuts. Thus, current research focused on development of vibrational spectroscopy based methods for detection and separation of contaminated peanut samples. Aflatoxin incidence, as a chemical contaminant in peanut paste samples, was investigated, in terms of spectral characteristics using FTIR-ATR. The effects of spectral pre-processing steps such as mean-centering, smoothing the 1st derivative and normalizing were studied. Logarithmic method was the best normalization technique describing the exponentially distributed spectral data. Spectral windows giving the best correlation with respect to increasing aflatoxin amount led to selection of fat associated spectral bands. Using the multivariate analysis tools, structural contributions of aflatoxins in peanut matrix were detected. The best region was decided as 3028-2752, 1800-1707, 1584-1424, and 1408-1127 cm-1 giving correlation coefficient for calibration (R2C), root mean square error for calibration (RMSEC) and root mean square error for prediction (RMSEP) of 98.6%, 7.66ppb and 19.5ppb, respectively. Applying the constructed partial least squares model, 95% of the samples were correctly classified while the percentage of false negative and false positive identifications were 16% and 0%, respectively. Aspergillus species of section Flavi and the black fungi, A. niger are the most common colonists of peanuts in nature and the majority of the aflatoxin producing strains are from section Flavi. Seed colonization by selected Aspergillus spp. was investigated by following the chemical alterations as a function of fungal growth by means of spectral readouts. FTIR-ATR was utilized to correlate spectral characteristics to mold density, and to separate Aspergillus at section, species and strain levels, threshold mold density values were established. Even far before the organoleptic quality changes became visually observable (~10,000 mold counts), FTIR distinguished the species of same section. Besides, the analogous secondary metabolites produced increased the similarity within the spectra even their spectral contributions were mostly masked by bulk peanut medium; and led to grouping of species producing the same mycotoxins together. Aflatoxigenic and non-aflatoxigenic strains of A. flavus and A. parasiticus were further studied for measurement capability of FTIR-ATR system in discriminating the toxic streams from just moldy and clean samples. Owing to increased similarity within the collected spectral data due to aflatoxin presence, clean samples (having aflatoxin level lower than 20 ppb, n=44), only moldy samples (having aflatoxin level lower than 300 ppb, n=28) and toxic samples (having aflatoxin level between 300-1200 ppb, n=23) were separated into appropriate classes (with a 100% classification accuracy). Photoacoustic spectroscopy (PAS) is a non-invasive technique and offers many advantages over more traditional ATR system, specifically, for in-field measurements. Even though the sample throughput time is longer compared to ATR measurements, intact seeds can be directly loaded into sample compartment for analysis. Compared to ATR, PAS is more sensitive to high moisture in samples, which in our case was not a problem since peanuts have water content less than 10%. The spectral ranges between: 3600-2750, 1800-1480, 1200-900 cm-1 were assigned as the key bands and full separation between Aspergillus spp. infected and healthy peanuts was obtained. However, PAS was not sensitive as ATR either in species level classification of Aspergillus invasion or toxic-moldy level separation. When run for separation of aflatoxigenic versus non-aflatoxigenic batches of samples, 7 out of 54 contaminated samples were misclassified but all healthy peanuts were correctly identified (15 healthy/ 69 total peanut pods). This study explored the possibility of using vibrational spectroscopy as a tool to understand chemical changes in peanuts and peanut products to Aspergillus invasion or aflatoxin contamination. The overall results of current study proved the potential of FTIR, equipped with either ATR or PAS, in identification, quantification and classification at varying levels of mold density and aflatoxin concentration. These results can be used to develop quality control laboratory methods or in field sorting devices. / Ph. D.

A range of microgels with two different functionalities were synthesized, and their utility in novel bioapplications was examined. Cationic microgels with varying properties were developed by tuning synthesis conditions. Their size and primary amine content was analyzed, and one microgel system was selected as a model construct. Its primary amine groups were conjugated to two dyes with properties favorable for utilization as contrast agents in photoacoustic imaging. The concentration of contrast agent in single particles was determined. The implications of a high local dye concentration in the generation of high intensity photoacoustic signals, are discussed. The second bioapplication involved the targeted delivery of fibrinolytics to fibrin clots, in order to bring about dissolution of abnormal thrombi. For this purpose, core/shell microgels with carboxylic acid groups in their shells were synthesized in three size ranges. Following this, their dimension based differential localization in and around porous fibrin clots was examined. Fibrin-specific peptides were then conjugated onto the shells of these particles and the conjugates were shown to demonstrate strong interactions with the fibrin clots. The microgels conjugated to the peptide with the highest binding affinity to fibrin, were observed to bring about disruption of fibrin clots, merely through interference in the dynamic interactions among clot fibers, due to the equilibrium nature of the fibrin polymer. The implications of these novel results and future studies required to facilitate a better understanding of the phenomena involved, are discussed.

L'imagerie photoacoustique est une modalité d'imagerie fonctionnelle basée sur la génération d'ondes acoustiques par des tissus soumis à une illumination optique (impulsion laser). L'utilisation de différentes longueurs d'ondes optiques permet la discrimination des milieux imagés. Cette modalité est prometteuse pour de nombreuses applications médicales liées, par exemple, à la croissance, au vieillissement et à l'évolution de la vascularisation des tissus. En effet, l'accès à l'oxygénation du sang dans les tissus est rendu possible par l'imagerie photoacoustique. Cela permet, entre autres applications, la discrimination de tumeurs bénignes ou malignes et la datation de la mort tissulaire (nécrose). Ce travail de thèse a pour objectif principal la construction d'une chaîne de traitement des données photoacoustiques multispectrales pour le calcul de l'oxygénation du sang dans les tissus. Les principales étapes sont, d'une part, la discrimination des données (clustering), pour extraire les zones d'intérêt, et d'autre part, la quantification des différents constituants présents dans celles-ci (unmixing). Plusieurs méthodes non supervisées de discrimination et de quantification ont été développées et leurs performances comparées sur des données photoacoustiques multispectrales expérimentales. Celles-ci ont été acquises sur la plateforme photoacoustique du laboratoire, lors de collaborations avec d'autres laboratoires et également sur un système commercial. Pour la validation des méthodes développées, de nombreux fantômes contenant différents absorbeurs optiques ont été conçus. Lors du séjour de cotutelle de thèse en Italie, des modes d'imagerie spécifiques pour l'imagerie photoacoustique 2D et 3D temps-réel ont été développés sur un échographe de recherche. Enfin, des acquisitions in vivo sur modèle animal (souris) au moyen d'un système commercial ont été réalisées pour valider ces développements. / Photoacoustic imaging is a functional technique based on the creation of acoustic waves from tissues excited by an optical source (laser pulses). The illumination of a region of interest, with a range of optical wavelengths, allows the discrimination of the imaged media. This modality is promising for various medical applications in which growth, aging and evolution of tissue vascularization have to be studied. Thereby, photoacoustic imaging provides access to blood oxygenation in biological tissues and also allows the discrimination of benign or malignant tumors and the dating of tissue death (necrosis). The present thesis aims at developing a multispectral photoacoustic image processing chain for the calculation of blood oxygenation in biological tissues. The main steps are, first, the data discrimination (clustering), to extract the regions of interest, and second, the quantification of the different media in these regions (unmixing). Several unsupervised clustering and unmixing methods have been developed and their performance compared on experimental multispectral photoacoustic data. They were acquired on the experimental photoacoustic platform of the laboratory, during collaborations with other laboratories and also on a commercial system. For the validation of the developed methods, many phantoms containing different optical absorbers have been produced. During the co-supervision stay in Italy, specific imaging modes for 2D and 3D real-time photoacoustic imaging were developed on a research scanner. Finally, in vivo acquisitions using a commercial system were conducted on animal model (mouse) to validate these developments.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O objetivo deste trabalho é a síntese e investigação estrutural e óptica de amostras SrGa2O4 dopados com 1% de íons Ni2+. Estas amostras foram sintetizados por reação do estado sólido convencional, utilizando como materiais de partida de alta pureza Ga2O3, SrCO3 e NiO em quantidades estequiométricas. As amostras foram caracterizadas estruturalmente pelo método de difração de raios - X( XRD ) e as medições de difração mostraram que as amostras têm uma única fase monoclínica. Os padrões de XRD também foram refinados pelo método de Rietveld, que permitiu a determinação dos parâmetros de célula unitária. A Caracterização óptica das amostras puras e dopadas SrGa2O4 foram realizadas as medições a partir de fotoluminescência, de excitação e de absorção fotoacústica, à temperatura ambiente. Os espectros de emissão mostraram três bandas de emissão localizadas em 557 nm, 661 nm e 844 nm e foram identificadas essas bandas, respectivamente, com as seguintes transições eletrônicas :1T2 (1D) → 3A2 (3F), 3T1 (3F)→ 3A2 (3F) e 1T2 (1D) → 3T2 (3F). Os espectros de excitação mostraram seis bandas de absorção associadas às transições electrônicas do nível 3A2 (3F) para o 3T1 (3P) , T1 (3P), 1A1 (1G), 1T2 (1D), 3T1 (3F), 1E (1D) e 1T2, 1E (1G). Medidas de absorção fotoacústica também foram realizados com o fim de verificar as transições ópticas observadas nos espectros de excitação e de identificar novas bandas de absorção óptica. Os resultados demonstraram que os íons de Ni2+ ocupam dois locais octaédricos diferentes na amostra SrGa2O4 dopado. A partir das transições ópticas observadas nos espectros de excitação e fotoacústica, determinou-se o parâmetro de cristal de campo, dq, e parâmetros Racah, B e C. A proporção Dq / B ≈ 1.2 para ambos os locais são típicos para Ni2+ íons inseridos em redes de óxido e em coordenação octaédrica. / The aim of this work is the synthesis and structural and optical investigation of SrGa2O4 samples doped with 1% of Ni2+ ions. These samples were synthesized by conventional solid-state reaction using as starting materials high purity SrCO3, Ga2O3 and NiO in stoichiometric quantities. Samples were structurally characterized by X-ray diffraction (XRD) method and the diffraction measurements showed that the samples have a single monoclinic phase. The XRD patterns were also refined by the Rietveld method which allowed the determination of the unit cell parameters. Optical characterization of the pure and doped SrGa2O4 samples were performed from photoluminescence, excitation and photoacoustic absorption measurements at room temperature. Emission spectra showed three emission bands localized at 557 nm, 661 nm and 844 nm and these bands were identified, respectively, with the following electronic transitions: 1T2 (1D) → 3A2 (3F), 3T1 (3F)→ 3A2 (3F) e 1T2 (1D) → 3T1 (3F). Excitation spectra showed six absorption bands associated to the electronic transitions from the level 3A2 (3F) to the 3T1 (3P), 1A1 (1G), 1T2 (1D), 3T1 (3F), 1E (1D) e 1T2, 1E (1G) levels. Photoacoustic absorption measurements were also carried out in order to verify the optical transitions observed in the excitation spectra and to identify new optical absorption bands. The results showed that Ni2+ ions occupy two different octahedral sites in the SrGa2O4 doped sample. From the optical transitions observed in the excitation and photoacoustic spectra, we determined the crystal-field parameter, Dq, and Racah parameters, B and C. The ratio Dq/B ≈ 1.2 for both sites are typical for Ni2+ ions inserted in oxide lattices and in octahedral coordination.

Aerosols contribute the largest uncertainty to estimates of radiative forcing of the Earth’s atmosphere, which are thought to exert a net negative radiative forcing, offsetting a potentially significant but poorly constrained fraction of the positive radiative forcing associated with greenhouse gases. Aerosols perturb the Earth’s radiative balance directly by absorbing and scattering radiation and indirectly by acting as cloud condensation nuclei, altering cloud albedo and potentially cloud lifetime. One of the major factors governing the uncertainty in estimates of aerosol direct radiative forcing is the poorly constrained aerosol single scattering albedo, which is the ratio of the aerosol scattering to extinction. In this thesis, I describe a new instrument for the measurement of aerosol optical properties using photoacoustic and cavity ring-down spectroscopy. Characterisation is performed by assessing the instrument minimum sensitivity and accuracy as well as verifying the accuracy of its calibration procedure. The instrument and calibration accuracies are assessed by comparing modelled to measured optical properties of well-characterised laboratory-generated aerosol. I then examine biases in traditional, filter-based absorption measurements by comparing to photoacoustic spectrometer absorption measurements for a range of aerosol sources at multiple wavelengths. Filter-based measurements consistently overestimate absorption although the bias magnitude is strongly source-dependent. Biases are consistently lowest when an advanced correction scheme is applied, irrespective of wavelength or aerosol source. Lastly, I assess the sensitivity of the direct radiative effect of biomass burning aerosols to aerosol and cloud optical properties over the Southeast Atlantic Ocean using a combination of offline radiative transfer modelling, satellite observations and global climate model simulations. Although the direct radiative effect depends on aerosol and cloud optical properties in a non-linear way, it appears to be only weakly dependent on sub-grid variability.

Photoacoustic tomography (PAT), as well as thermoacoustic tomography (TAT), utilize electromagnetic radiation in its visible, near infrared, microwave, and radiofrequency forms, respectively, to induce acoustic waves in biological tissues for imaging purposes. Combining the advantages of both the high image contrast that results from electromagnetic absorption and the high resolution of ultrasound imaging, these new imaging modalities could be the next successful imaging techniques in biomedical applications. Basic research on PAT and TAT, and the relevant physics, is presented in Chapter I. In Chapter II, we investigate the imaging mechanisms of TAT in terms of signal generation, propagation and detection. We present a theoretical analysis as well as simulations of such imaging characteristics as contrast and resolution, accompanied by experimental results from phantom and tissue samples. In Chapter III, we discuss the further application of TAT to the imaging of biological tissues. The microwave absorption difference in normal and cancerous breast tissues, as well as its influence on thermoacoustic wave generation and the resulting transducer response, is investigated over a wide range of electromagnetic frequencies and depths of tumor locations. In Chapter IV, we describe the mechanism of PAT and the algorithm used for image reconstruction. Because of the broad bandwidth of the laser-induced ultrasonic waves and the limited bandwidth of the single transducer, multiple ultrasonic transducers, each with a different central frequency, are employed for simultaneous detection. Chapter V further demonstrates PAT’s ability to image vascular structures in biological tissue based on blood’s strong light absorption capability. The photoacoustic images of rat brain tumors in this study clearly reveal the angiogenesis that is associated with tumors. In Chapter VI, we report on further developing PAT to image deeply embedded optical heterogeneity in biological tissues. The improved imaging ability is attributed to better penetration by NIR light, the use of the optical contrast agent ICG (indocyanine green) and a new detection scheme of a circular scanning configuration. Deep penetrating PAT, which is based on a tissue’s intrinsic contrast using laser light of 532 nm green light and 1.06 µm near infrared light, is also presented.

Electron transfer, ET, and proton-coupled electron transfer, PCET, reactions are central to biological reactions involving catalysis, energy conversion and energy storage. The movement of electrons and protons in either a sequential or concerted manner are coupled in a series of elementary reaction steps in respiration and photosynthesis to harvest and convert energy consumed in foodstuffs or by absorption of light into high energy chemi-cal bonds in the form of ATP. These electron transfer processes may be modulated by conformational dynamics within the protein matrix or at the protein-protein interface, the energetics of which are still not well understood. Photoacoustic calorimetry is an estab-lished method of obtaining time-resolved reaction enthalpy and volume changes on the nanosecond to microsecond timescale. Photoacoustic calorimetry is used here to probe 1) the energetics and volume changes for ET between the self-assembled anionic uroporphy-rin:cytochrome c complex and the role of the observed volume changes in modulating ET within the complex, 2) the enthalpy and volume change for the excited state PCET reac-tion of a tyramine functionalized ruthenium(II) bis-(2,2'-bipyridine)(4-carboxy-4'-methyl-2,2'-bipyrine) meant to be a model for the tyrosine PCET chemistry carried out by cyto-chrome c oxidase and photosystem II, 3) the enthalpy and volume changes related to car-bon monoxide and tryptophan migration in heme tryptophan catabolic enzyme indoleam-ine 2,3-dioxygenase.