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This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

Photoacoustic computed tomography (PAT) has great potential for application in the biomedical field. It best combines the high contrast of electromagnetic absorption and the high resolution of ultrasonic waves in biological tissues. In Chapter II, we present time-domain reconstruction algorithms for PAT. First, a formal reconstruction formula for arbitrary measurement geometry is presented. Then, we derive a universal and exact back-projection formula for three commonly used measurement geometries, including spherical, planar and cylindrical surfaces. We also find this back-projection formula can be extended to arbitrary measurement surfaces under certain conditions. A method to implement the back-projection algorithm is also given. Finally, numerical simulations are performed to demonstrate the performance of the back-projection formula. In Chapter III, we present a theoretical analysis of the spatial resolution of PAT for the first time. The three common geometries as well as other general cases are investigated. The point-spread functions (PSF's) related to the bandwidth and the sensing aperture of the detector are derived. Both the full-width-at-half-maximum of the PSF and the Rayleigh criterion are used to define the spatial resolution. In Chapter IV, we first present a theoretical analysis of spatial sampling in the PA measurement for three common geometries. Then, based on the sampling theorem, we propose an optimal sampling strategy for the PA measurement. Optimal spatial sampling periods for different geometries are derived. The aliasing effects on the PAT images are also discussed. Finally, we conduct numerical simulations to test the proposed optimal sampling strategy and also to demonstrate how the aliasing related to spatially discrete sampling affects the PAT image. In Chapter V, we first describe a prototype of the RF-induced PAT imaging system that we have built. Then, we present experiments of phantom samples as well as a preliminary study of breast imaging for cancer detection.

Pancreatic cancer remains a major unsolved health problem, with conventional cancer treatments having little impact on disease course. The objective of this thesis is to create innovative tools to better understand and improve chemotherapeutic treatment of pancreatic cancer. Towards this end, nanosystems were designed with a dual purpose: to carry chemotherapeutic drugs and act as photoacoustic imaging contrast agents. The overarching hypothesis is that these nanosystems can provide enhanced therapy for pancreatic cancer and enable visualization of drug delivery. Demonstrated in this dissertation is the design, synthesis, and characterization of two such nanosystems built to carry the chemotherapeutic agent gemcitabine while acting as a photoacoustic imaging contrast agent. The nanosystems were also shown to be multifunctional with possible application as photothermal therapy agents and cellular functional sensors. Although future research is required to fully investigate the clinical potential of these systems for pancreatic cancer, the work presented in this dissertation is a step towards creation of multifunctional nanosystems that will enable non-invasive, in vivo photoacoustic imaging of drug delivery. / text

Um mimetizador de tecido, mais conhecido como phantom, é um objeto que mimetiza tecidos biológicos e são importantes para caracterização e calibração de equipamentos de imagens médicas como ultrassonografia, e no desenvolvimento de novas modalidades de imagens como a fotoacústica. Este trabalho aborda o desenvolvimento de um gel à base de óleo mineral e polímeros para phantom para aplicações em técnicas acústicas e ópticas. Os polímeros utilizados foram o elastômero tribloco tipo estireno-etileno/butileno-estireno (SEBS) e o polietileno de baixa densidade (PEBD). Foram confeccionados três grupos géis poliméricos com porcentagem de SEBS entre 5%-15% de SEBS, 0%-9% de PEBD. Os géis foram caracterizados acusticamente pela velocidade do som e coeficiente de atenuação através de transdutores de imersão com frequências entre 2,25 MHz-10 MHz, e opticamente entre 400-1200 nm pelos coeficientes de absorção, espalhamento e Albedo. Foi observada velocidade do som entre 1458,6 ± 3,1 m/s e 1480,7 ± 1,9 m/s, sendo compatíveis com valores para gordura; coeficiente de atenuação entre 0,6 ± 0,1 dB/cm a 2,25 MHz e 11,3 ± 0,1 dB/cm a 10 MHz, compatíveis para tecidos moles; coeficiente de absorção em 532 nm entre 0,11-2,62 cm-1 e em 1064 nm entre 0,09-1,70 cm-1, e uma banda de absorção em torno de 930 nm com gordura; o coeficiente de espalhamento em 532 nm entre 0,15 -3,96 cm-1 e em 1064 nm entre 0,17- 3,20 cm-1, valores inferiores para tecidos moles. O coeficiente Albedo mostrou que os géis apresentam caráter absorvedor entre 400-1200 nm. Foi desenvolvido um phantom para imagem por fotoacústica com um dos géis estudados (7%SEBS/5%PEBD) e com uma inclusão com pigmento de urucum e foram feitas imagens fotoacústicas em 532 nm e 1024 nm. Foi observado o sinal fotoacústico mais intenso para a imagem em 532 nm. Com este trabalho pode-se obter uma boa caracterização acústica e óptica de géis formados a partir de polímeros do tipo SEBS em conjunto com o PEBD ainda não descritos na literatura. Os materiais desenvolvidos se mostraram bons mimetizadores para tecidos compostos de gordura e com potencial para aplicações em fotoacústica. / Phantoms are structures composed by materials that mimic specific properties of biological tissues and they are commonly used to calibrate and characterize current medical imaging techniques such as ultrasound and optical imaging, and new imaging modalities such as photoacoustics. In this dissertation we developed an oil-based tissue mimicking gel material with mineral oil, triblock copolymer styrene-ethylene/butylene styrene (SEBS) and low-density polyethylene (LDPE). The gel phantoms were prepared mixing SEBS and LDPE in mineral oil at room temperature, varying the SEBS concentration between 5%15%, and low-density polyethylene (LDPE) between 0%-9% and glass microspheres. Acoustic properties such speed of sound and attenuation coefficient were measured using five unfocused ultrasound transducers with frequencies ranging between 2.2510 MHz. Optical properties such albedo, scattering and absorption coefficients ranging from 400-1200 nm were measured. Speed of sound from 1458.6 ± 3.1m/s and 1480.7 ± 1.9 m/s, and attenuation from 0.6 ± 0.1 dB/cm at 2.25 MHz and 11.3 ± 0.1 dB/cm at 10 MHz were observed. Absorption coefficient at 532 nm between 0.11-2.62 cm-1; at 1064 nm between 0.09-1.70 cm-1 were observed. Peak absorption around 930 nm was observed for all gels. Scattering coefficient at 532 nm between 0.15 -3.96 cm-1 and at 1064 nm between 0.17-3.20 cm-1 were found. Albedo coefficient showed that gels are absorptive characteristic for the selected range of wavelength. A phantom made with a 7% SEBS/5% LDPE gel containing an optical-absorber spherical inclusion made with the same material and annatto were developed. Photoacoustic spectroscopic images of the phantom were acquired using a laser operating at 532 nm and 1064 nm. The photoacoustic signal from the inclusion showed the highest intensities at 532 nm with as expected according to the measured absorption spectrum of annatto. With this dissertation we obtained a suitable acoustic and optical characterization of the SEBS/LDPE gels that was not described in the literature. The materials developed seem suitable to mimic fat tissue and have potential for applications in photoacoustics.

- An extensive summary in French is available in Appendix E on page 189 -

The application of photoacoustic (PA) phenomena to medical imaging has been investigated for more than a decade. To implement this modality, one may choose between two types of laser sources, pulsed or continuous wave (CW). This selection affects all features of the imaging technique. Nowadays pulsed lasers are the state-of-the-art technique in the PA research. In this work, various features of the alternative frequency-domain (FD) PA were investigated. An axially symmetric transfer function model of PA wave generation and a Krimholtz-Leedom-Matthaei (KLM) transducer model were developed and used to analyze the experimental results. The controllable parameters of the FD-PA were optimized to improve the signal-to-noise ratio (SNR), contrast, axial resolution and depth detectivity. For example, it was shown that employing the optimal chirp bandwidth can enhance the SNR by more than 10 dB. In addition to the image produced by the cross-correlation amplitude, the phase of the correlation signal was used as a separate channel. A statistical method was introduced to generate an image from this phase channel, and also to filter the PA amplitude channel. A study was also performed to compare FD PA and the prevalent pulsed method. Various features of both methods were examined experimentally using a dual-mode PA system and under the condition of maximum permissible exposure (MPE). The SNRs of both methods were evaluated theoretically and experimentally. It was shown that at low frequencies, both modalities generate comparable SNRs, and at high frequencies pulsed PA produces superior SNRs and depth detetivity. However, by increasing the laser power and decreasing the chirp duration within the safety limits, the SNR and depth detectivity of the FD-PA method are enhanced considerably. The main cause to achieve lower experimental SNRs than theoretical predictions for pulsed PA response was shown to be the oscillating baseline, which can be partially eliminated by filtering.

The application of photoacoustic (PA) phenomena to medical imaging has been investigated for more than a decade. To implement this modality, one may choose between two types of laser sources, pulsed or continuous wave (CW). This selection affects all features of the imaging technique. Nowadays pulsed lasers are the state-of-the-art technique in the PA research. In this work, various features of the alternative frequency-domain (FD) PA were investigated. An axially symmetric transfer function model of PA wave generation and a Krimholtz-Leedom-Matthaei (KLM) transducer model were developed and used to analyze the experimental results. The controllable parameters of the FD-PA were optimized to improve the signal-to-noise ratio (SNR), contrast, axial resolution and depth detectivity. For example, it was shown that employing the optimal chirp bandwidth can enhance the SNR by more than 10 dB. In addition to the image produced by the cross-correlation amplitude, the phase of the correlation signal was used as a separate channel. A statistical method was introduced to generate an image from this phase channel, and also to filter the PA amplitude channel. A study was also performed to compare FD PA and the prevalent pulsed method. Various features of both methods were examined experimentally using a dual-mode PA system and under the condition of maximum permissible exposure (MPE). The SNRs of both methods were evaluated theoretically and experimentally. It was shown that at low frequencies, both modalities generate comparable SNRs, and at high frequencies pulsed PA produces superior SNRs and depth detetivity. However, by increasing the laser power and decreasing the chirp duration within the safety limits, the SNR and depth detectivity of the FD-PA method are enhanced considerably. The main cause to achieve lower experimental SNRs than theoretical predictions for pulsed PA response was shown to be the oscillating baseline, which can be partially eliminated by filtering.

We demonstrate intracellular delivery of various molecules by inducing controlled and reversible cell damage through pulsed laser irradiation of carbon black (CB) nanoparticles. We then characterized and optimized the system for maximal uptake and minimal loss of viability. At our optimal condition 88% of cells exhibited uptake with almost no loss of viability. In other more intense cases it was shown that cell death could be prevented through addition of poloxamer. The underlying mechanism of action is also studied and our hypothesis is that the laser heats the CB leading to thermal expansion, vapor formation and/or chemical reaction leading to generation of acoustic waves and then there is energy transduction to the cell causing poration of the cell membrane. We also delivered anti-EGFR siRNA to ovarian cancer cells. Cells exposed to a laser at 18.75 mJ/cm2 for 7 minutes resulted in a 49% knockdown of EGFR compared to negative control. We established an alternative way to deliver siRNA to knockdown proteins, for the first time using laser CB interaction.