Files
Abstract
This dissertation addresses a critical need in intravital microscopy to explore tissue structure and dynamics in the bone. Overcoming the inherent challenges of imaging bone in vivo holds the potential to deepen our comprehension of bone health and unravel the intricate interactions between bone structure and tissues. Our work begins by developing a polarimetric second harmonic generation (pSHG) microscope to determine the dominant orientation of collagen fibers tailored for investigating cranial bone collagen assembly defects observed in a mouse model of hypophosphatasia (HPP). We propose a spatial polarimetric gray-level co-occurrence matrix (spGLCM) method to explore polarization-mediated textural differences in the bone collagen mesh. By comparing machine learning classifiers, we were able to accurately separate unknown images from the two groups with an averaged F1 score of 92.30% by using random forest.
However, multiphoton imaging depth is limited due to loss of signal and degradation of the focus due to tissue distortion. We developed a multi-photon fluorescence microscope with adaptive optics (MPFM-AO) which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensor less approach for correcting low order tissue aberrations. This approach facilitates rapid imaging of subcellular organelles with approximately 400 nanometer resolution, penetrating up to 85 micrometers into highly scattering tissue. We achieved ~1.55x, ~3.58x and ~1.77x intensity increases using AO, and a reduction of the PSF width by ~0.83x, ~0.74x and ~0.9x at the depths of 0, 50 micrometers, and 85 micrometers in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5 micrometers x 67.5 micrometers. Furthermore, we proposed an innovative approach that combines low-order deformable mirror (DM) aberration correction with high-order digital micromirror device (DMD) scattering correction. We demonstrate the synergistic enhancement of imaging performance at depths of 150 micrometers beneath the surface of mouse cranial bone ex vivo. These advancements not only contribute significantly to the evolution of biomedical imaging technologies but also deepen our understanding of tissue microstructures and pathology.
However, multiphoton imaging depth is limited due to loss of signal and degradation of the focus due to tissue distortion. We developed a multi-photon fluorescence microscope with adaptive optics (MPFM-AO) which uses a home-built Shack-Hartmann wavefront sensor (SHWFS) to correct system aberrations and a sensor less approach for correcting low order tissue aberrations. This approach facilitates rapid imaging of subcellular organelles with approximately 400 nanometer resolution, penetrating up to 85 micrometers into highly scattering tissue. We achieved ~1.55x, ~3.58x and ~1.77x intensity increases using AO, and a reduction of the PSF width by ~0.83x, ~0.74x and ~0.9x at the depths of 0, 50 micrometers, and 85 micrometers in living mouse bone marrow respectively, allowing us to characterize mitochondrial health and the survival of functioning cells with a field of view of 67.5 micrometers x 67.5 micrometers. Furthermore, we proposed an innovative approach that combines low-order deformable mirror (DM) aberration correction with high-order digital micromirror device (DMD) scattering correction. We demonstrate the synergistic enhancement of imaging performance at depths of 150 micrometers beneath the surface of mouse cranial bone ex vivo. These advancements not only contribute significantly to the evolution of biomedical imaging technologies but also deepen our understanding of tissue microstructures and pathology.