Near Infrared (NIR) and Photoacoustic (PA) Imaging are promising imaging modalities that provides functional information of the soft biological tissues in-vivo, with applica-tions in breast and brain tissue imaging. These techniques use near infrared light in the wavelength range of (600 nm - 900 nm), giving an advantage of being non-ionizing imaging modality. This makes the prolong bed-side monitoring of tissue feasible, making them highly desirable medical imaging modalities in the clinic. The computation models that are deployed in these imaging scenarios are computationally demanding and often require a high performance computing systems to deploy them in real-time. This the-sis presents three computationally e cient models for near-infrared and photoacoustic imaging, without compromising the quality of measured functional properties, to make them more appealing in clinical scenarios. The attenuation of near-infrared (NIR) light intensity as it propagates in a turbid medium like biological tissue is described by modi ed the BeerLambert law (MBLL). The MBLL is generally used to quantify the changes in tissue chromophore concen-trations for NIR spectroscopic data analysis. Even though MBLL is e ective in terms of providing qualitative comparison, it su ers from its applicability across tissue types and tissue dimensions. A Lambert-W function-based modeling for light propagation in biological tissues is proposed and introduced, which is a generalized version of the Beer-Lambert model. The proposed modeling provides parametrization of tissue properties, which includes two attenuation coe cients o and . The model is validated against the Monte Carlo simulation, which is the gold standard for modeling NIR light propagation in biological tissue. Numerous human and animal tissues are included to validate the proposed empirical model, including an inhomogeneous adult human head model. The proposed model, which has a closed form (analytical), is rst of its kind in providing accurate modeling of NIR light propagation in biological tissues. Model based image reconstruction techniques yield better quantitative accuracy in photoacoustic (PA) image reconstruction, especially in limited data cases. An exponen-tial ltering of singular values is proposed for carrying out the image reconstruction in photoacoustic tomography. The results were compared with widely popular Tikhonov regularization, time reversal, and the state of the art least-squares QR based reconstruc-tion algorithms for three digital phantom cases with varying signal-to-noise ratios of data. The exponential ltering provided superior photoacoustic images of better quanti-tative accuracy. Moreover, the proposed ltering approach was observed to be less biased towards regularization parameter and did not come with any additional computational burden as it was implemented within the Tikhonov ltering framework. It was also shown that the standard Tikhonov ltering becomes an approximation to the proposed exponential ltering. The model based image reconstruction techniques for photoacoustic tomography re-quire an explicit regularization. An error estimate minimization based approach was proposed and developed for the determination of regularization parameter for PA imag-ing. The regularization was used within Lanczos bidiagonalization framework, which provides the advantage of dimensionality reduction for a large system of equations. The proposed method was computationally faster than the state of the art techniques and provided similar performance in terms of quantitative accuracy in reconstructed im-ages.The estimate can also be utilized in determining suitable regularization parameter for other popular techniques such as Tikhonov,exponential ltering and `1 norm based regularization methods.

No presente trabalho, realizado com hemoproteínas na forma de pó, o efeito da hidratação foi observado através de espectroscopia fotoacústica. Amostras de carboxi-hemoglobina e carboxi-mioglobina mantidas em diferentes ambientes de umidade relativa (UR), mostraram variações em seus espectros na região da banda de Soret. Para amostras mantidas em baixa hidratação característico do derivado carboxi, em alta hidratação (acima de aproximadamente 90% UR) o espectro era característico do derivado carboxi e na região intermediária o espectro era de uma mistura dos dois derivados. Essa mudança de ligante observada em alta hidratação pode ser explicada supondo que a proteína tem flexibilidade e atinge um estado conformacional que possibilita a entrada e saída do ligante. Em baixa hidratação a estrutura da proteína é rígida e tal que o acesso ao grupo heme está fechado, impossibilitando a troca do ligante. Essa explicação é coerente com vários resultados experimentais que indicam a existência de duas estruturas para essas hemoproteínas em solução / At the present work accomplished with powder of hemoproteins, the hydration effect was observed through photoacoustic spectroscopy. Samples of caroxyhemoglobin and carboxymyglobin kept at different relative humidity (RH) environments, showed variations in their spectra in the Soret bad region. For the samples which were kept at low hydration (Bellow about 33% RH) the spectrum was characteristic of carboxy derivative, whereas at high hydration (above about 93% RH) the spectrum was characteristic of oxy derivative, and in the the intermediate region the spectrum was a mixture of both derivatives. This ligand change observed at high hydration, may be explained assuming that the protein has flexibility, and reach a conformational state which enables the ligand to GO in and out. At low hydrations the protein structure is rigid and such that the access to the heme group is closed becoming impossible the ligand change. This explanation agrees with several experimental results that point to the existence of two structures to these hemoproteins in solution

Orientador: Gerson Antonio Santarine / Banca: Roberto Naves Domingos / Banca: Osvaldo Missiato / Resumo: A espectrometria fotoacústica sofreu um grande avanço nos últimos anos, como técnica de análise de materiais e sistemas. Os calorímetros que utilizam o fenômeno fotoacústico se desenvolveram para análises que não podem ser feitas por outros procedimentos, como, a reflectância difusa, reflectância total atenuada e espalhamento Raman. O espectrômetro elaborado nesse trabalho pode ser utilizado para a determinação de energia de laser e calores específicos de materiais biológicos sólidos. Foi confeccionado com materiais de baixo custo e existentes no mercado nacional. Foi testado com sucesso para medidas de energia de laser. / Abstract: Photoacoustic spectrometry has undergone a significant development in the last years as a technic of material and system analyses. Calorimeters using photoacoustic phenomenon were developed to provide analyses that cannot be done by other procedures such as diffuse reflectance, total atenuated reflectance and Raman's scattering about. The spectometer constructed in this work can be used to determine laser energy and specific heat of biological solid materials. It was constructed with cheap material easily found in national market. The photoacoustic spectometer was sucessfully tested in measuring laser energy. / Mestre

Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes. Measuring the functional status of the tumour vasculature, including blood flow fluctuations and changes in oxygenation is important in cancer staging and therapy monitoring. A robust method is needed for clinical non-invasive measurement of the oxygen supply and demand in tumours. Current clinically approved imaging modalities suffer high cost, long procedure times and limited spatio-temporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that can provide static images of endogenous haemoglobin concentration and oxygenation. In this work, an integrated framework for quantitative analysis of functional imaging using OT is developed and applied in vivo with preclinical cancer models. Oxygen Enhanced (OE)-OT is established here to provide insight into tumour vascular function and oxygen availability in the tissue. Tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vascular maturity, hypoxia and necrosis, assessed ex vivo. Dynamic Contrast Enhanced (DCE) OT demonstrates the relationship between OE-OT response and tumour perfusion in vivo. Finally, the possibility of using OT data acquired at longer wavelengths to report on tumour water and lipid content is investigated, with a view to future providing intrinsically co-registered imaging of tumour oxygenation and cellular necrosis. These findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease, while combined with DCE-OT, it may offer significant advantage for localised imaging of tumour response to vascular targeted therapies. Further work is needed to establish whether OT can provide a new method to detect tumour necrosis in vivo.

This research is primarily focused on laser-based non-invasive photoacoustic tomography of small animal brains. Photoacoustic tomography, a novel imaging modality, was applied to visualize the distribution of optical absorptions in small-animal brains through the skin and skull. This technique combines the high-contrast advantage of optical imaging with the high-resolution advantage of ultrasonic imaging. Based on the intrinsic optical contrast, this imaging system successfully visualized three-dimensional tissue structures in intact brains, including lesions and tumors in brain cerebral cortex. Physiological changes and functional activities in brains, including cerebral blood volume and blood oxygenation in addition to anatomical information, were also satisfactorily monitored. This technique successfully imaged the dynamic distributions of exogenous contrast agents in small-animal brains. Photoacoustic angiography in small-animal brains yielding high contrast and high spatial resolution was implemented noninvasively using intravenously injected absorbing dyes. In the appendix, the theory of Monte Carlo simulation of polarized light propagation in scattering media was briefly summarized.

This study explored the imaging capability of our frequency-domain photoacoustic (FD-PA) system that utilizes correlation processing alias “photoacoustic radar” and ultrasonic phased array for imaging of soft tissues. The probe imaging capabilities were studied using tissue-mimicking phantoms, tissue samples ex vivo, blood vessels in a human wrist and a rat tumour model in vivo. Our experimental results have shown decent image correlation between our FD-PA and a clinical ultrasound modality. In comparison with ultrasound, we have also demonstrated strong potential of the FD-PA for deep (~15 mm) subsurface imaging with excellent contrast and high signal-to-noise ratio. Additionally, we assessed the potential of silica-coated super paramagnetic iron oxide nanoparticles (SPION) as a novel PA contrast agent. Detection of the nanoparticles up to 24 mm inside an optically tissue-like turbid media and about 5-fold PA signal amplification due to their presence in sheep blood (~1.4 mg/ml) are presented.

This study explored the imaging capability of our frequency-domain photoacoustic (FD-PA) system that utilizes correlation processing alias “photoacoustic radar” and ultrasonic phased array for imaging of soft tissues. The probe imaging capabilities were studied using tissue-mimicking phantoms, tissue samples ex vivo, blood vessels in a human wrist and a rat tumour model in vivo. Our experimental results have shown decent image correlation between our FD-PA and a clinical ultrasound modality. In comparison with ultrasound, we have also demonstrated strong potential of the FD-PA for deep (~15 mm) subsurface imaging with excellent contrast and high signal-to-noise ratio. Additionally, we assessed the potential of silica-coated super paramagnetic iron oxide nanoparticles (SPION) as a novel PA contrast agent. Detection of the nanoparticles up to 24 mm inside an optically tissue-like turbid media and about 5-fold PA signal amplification due to their presence in sheep blood (~1.4 mg/ml) are presented.

Cellular/subcellular imaging of biological tissue is an important tool for understanding disease mechanisms. Many current techniques for subcellular absorption contrast imaging, such as two-photon excited fluorescence (TPEF), require exogenous contrast agents to gain access to many naturally occurring biomolecules. Non-fluorescent biomolecules must have a fluorescent marker (tag) chemically bound in order to be observed by TPEF. Contrast agents and markers, while effective, are not an optimal solution because they can change the local environment in the biological system and require FDA approval for human use. Photoacoustic microscopy (PAM) is an imaging modality with high endogenous absorption contrast and penetration depth due to its ability to detect acoustic waves, which are attenuated much less than light in tissue. However, this technique suffers from poor axial resolution, precluding it from consideration for subcellular imaging. This manuscript describes the author's efforts to improve the axial resolution of traditional PAM by merging it with pump-probe spectroscopy. Pump-probe spectroscopy is a non-linear optical technique that exploits a physical process called transient absorption, providing spatial resolution equivalent to two-photon microscopy and access to molecular-specific traits, such as the ground state recovery time and transient absorption spectrum. These traits provide molecular contrast to the imaging technique, which is highly desirable in a complex, multi-chromophore biological system. In this manuscript, a novel technique called transient absorption ultrasonic microscopy (TAUM) is designed and characterized in detail. A second-generation TAUM system is also described, which improves speed and sensitivity of TAUM by up to 1000-fold. This system is validated by collecting volumes of red blood cells in blood smears and tissue samples. These results constitute the first time single cells have been fully resolved using a photoacoustic microscope. Finally, the TAUM system is modified to measure chromophore ground state recovery times. This technique is validated by measuring the recovery time of Rhodamine 6G, which matches well with published values of the fluorescence lifetime. Recovery times of oxidized and reduced forms of hemoglobin are also measured and shown to statistically differ from one another, suggesting the possibility of subcellular measurements of oxygen saturation in future iterations of TAUM.

This research is primarily focused on laser-based non-invasive photoacoustic tomography of small animal brains. Photoacoustic tomography, a novel imaging modality, was applied to visualize the distribution of optical absorptions in small-animal brains through the skin and skull. This technique combines the high-contrast advantage of optical imaging with the high-resolution advantage of ultrasonic imaging. Based on the intrinsic optical contrast, this imaging system successfully visualized three-dimensional tissue structures in intact brains, including lesions and tumors in brain cerebral cortex. Physiological changes and functional activities in brains, including cerebral blood volume and blood oxygenation in addition to anatomical information, were also satisfactorily monitored. This technique successfully imaged the dynamic distributions of exogenous contrast agents in small-animal brains. Photoacoustic angiography in small-animal brains yielding high contrast and high spatial resolution was implemented noninvasively using intravenously injected absorbing dyes. In the appendix, the theory of Monte Carlo simulation of polarized light propagation in scattering media was briefly summarized.

We investigate the possibility of extracting accurate functional information such as local blood oxygenation level using multi-wavelength photoacoustic measurements. Photoacoustic microscope is utilized to acquire images of microvasculature in smallanimal skin. Owing to endogenous optical contrast, optical spectral information obtained from spectral photoacoustic measurements are successfully inverted to yield oxygenation level in blood. Analysis of error propagation from photoacoustic measurements to inverted quantities showed minimum inversion error in the optical wavelength region of 570-600 nm. To obtain accurate and vessel size independent blood oxygenation measurements, transducers with central frequency of more than 25 MHz are needed for the optical region of 570-600 nm used in this study. The effect of transducer focal position on accuracy of blood oxygenation level quantification was found to be negligible. To obtain accurate measurements in vivo, one needs to compensate for factors such as spectral dependent optical attenuation.