Ultrasound & Photoacoustic Microscopy for Biomedical Applications

Abstract

<p>Ultrasound and photoacoustic microscopy hold great promise for deep tissue imaging with high resolution, offering different applications in biological research. The primary aim of this thesis is to perform correlative photoacoustic and ultrasound microscopy experiments on various biological samples. <p>This thesis discusses multiple imaging modes, including A-scan, B-scan, and C-scan, each offering an unique perspectives on the sample data visualization. This includes the development of a novel approach to combine and visualize SAM (Scanning Acoustic Microscopy) and PAM (Photoacoustic Microscopy) data simultaneously, using tailored color and transparency mappings. Advanced visualization tools like MATLAB and 3D Slicer were also employed for biomedical image processing, enabling the integration of multiple datasets for comprehensive 3-D rendering. This approach reveals the concentration and distribution of pigment map has been revealed as 3-D PAM image. The integration of SAM and PAM images on top of each other gives a comprehensive visualization method and it can be explored for further studies such as effects of seasons, effect of any external stimuli on the pigments or studying several diseases, which affects the pigments. The pigment can also be quantified using photoacoustic microscopy and this can provide insights about stress levels and overall health of marine species. <p>Different applications in plant biology harnessing the endogenous pigment anthocyanin have been explored such as blueberry, rhubarb, <i>Drosera capensis</i> using PAM and SAM. We experimented with multilayered agarose embedding for microparticles, which aims to create a boundaryless medium and reduce signal artifacts. For samples like salmon skin and shrimp shells, a 'lifted samples' technique using polyamide thin film was employed. We also present a multi-layered method of sample preparation where the majority of microparticles are lifted, floating, and almost lying in the same plane. Overall, the emphasis is on consistency in preparation methods to ensure reproducibility across experiments. Although, this imaging modalities has been applied for applications related to human beings for example blood oxygenation but has not been applied to marine biology and botanical applications. Different pigments such as anthocyanin and astaxanthin have not been explored using this microscopy technique. <p>The research results are presented in four original scientific papers. Paper I focuses on the acoustic imaging where coded waveforms such as Ricker and chirp waves are used, and a performance comparison is made on different parameters for a commercial polyvinylidene fluoride (PVDF) and in-house fabricated poly (vinylidene fluoride-trifluoro ethylene) also known as P(VDF-TrFe) transducer. B- and C-scan images were obtained from the recorded time series and several parameters such as resolution, signal-to-noise-ratio, point spread function and depth imaging capabilities of both the transducer were compared. In Paper II, laser generated ultrasound for polystyrene microspheres of different sizes was performed in our lab. The COMSOL simulation for the experimental setup where all the components involved in light delivery to acoustic detection was simulated. The experimental and simulation results were in close agreement with each other. The presence of Scholte wave was detected both experimentally and in simulation. The Eigenvalues in the frequency spectrum aligned in a close agreement in both the frequency spectrums. In Paper III, the estimation of hardness using thin films calibration where a reflection coefficient is found out and utilized it for finding the hardness of the shrimp shells is shown. as to the best of our knowledge, we have used ultrasound to estimate the acoustic impedance of shrimp’s shell for the first time. Astaxanthin pigment has been used for an endogenous contrast agent in shells and antennae in PAM image generation. This work is, to our understanding, the first experimental application of PAM for shrimps. This thesis is a step forward in PAM’s potential in imaging of marine organisms. Paper IV discusses the application of SAM and PAM for salmon skin. Ultrasound and photoacoustic images are overlayed on top of each other with a tailored colormap and alpha map in MATLAB. The 3-D mapping reveals the concentration and density of the pigments from three different datasets. A comparison of PAM and optical image is shown showing high resolution imaging with deep penetration depth. A histogram for calculation of voxels contributing to the PAM and SAM signal is shown. <p>The unique ability of PAM for providing morphological and functional information paves the way for understanding the complex marine organism interactions. This thesis studies different approaches of sample preparation, building an experimental setup, simulating the experiments, analyzing the data and visualization for different SAM and PAM applications.