Digital in-line holography (DIH) has been applied extensively to characterizations of a variety of particles, including seeding particles in flow measurements, droplets or bubbles in multiphase flows, microorganisms in the ocean, etc. Unlike point-wise and two-dimensional (2D) diagnostic techniques, in one realization DIH provides three-dimensional (3D) measurements of particles located in a detection volume. The particle size, shape and position information is encoded in the interference patterns recorded in a hologram, which must be processed to extract desired particle characteristics. In this study, the HYBRID method is developed as a hologram processing method. Compared with various existing methods, it features automatic selection of thresholds for image segmentation, applicability to arbitrary-shaped particles and validated measurement uncertainties. A refinement procedure, for use in conjunction with the HYBRID method, is further developed to identify and correct erroneously detected particles due to particle overlapping in the in-plane (x-y) directions. The performance and significance of the refinement are demonstrated by applications to calibration holograms of solid particles and experimental holograms of liquid breakup. Finally, DIH with the HYBRID method, as a 3D diagnostic tool, is applied to characterize multiphase drop fragmentation. In particular, the bag breakup of Newtonian and non-Newtonian drops is investigated. A double-pulsed, double-exposure DIH system is configured to record sequential holograms which enable velocity measurements. Breakup morphologies are visualized by 2D images reconstructed from holograms recorded of various breakup phases, and significant difference is found between breakups of Newtonian and non-Newtonian drops. Comparison with phase Doppler anemometry (PDA) measurements confirms the accuracy of size measurement by DIH. It is shown that droplets due to breakup of the bag and those due to breakup of the rim have distinctly different size distributions. The 3D morphology of the intermediate rim is unprecedentedly determined using DIH, from which the volume ratio of the rim is found to be approximately 90%. The axis-symmetry of the bag breakup process is utilized to examine the accuracy of velocity measurements. Consistent with uncertainty quantification results, the out-of-plane velocity has higher uncertainty than in-plane velocities. To improve the out-of-plane accuracy, a cross-beam two-view DIH system is configured to provide high-accuracy 3D velocity measurement. The achieved high-fidelity z-displacements in turn serve as benchmarks for uncertainty quantification of the HYBRID method in the single-beam configuration.;Digital holographic microscopy (DHM), as a method to realize quantitative phase contrast imaging, is mostly applied to in vitro studies of various living cells, whose topological features are derived from the phase of the reconstructed complex amplitude. However, few have reported in vivo applications of DHM to a vertebrate model organism. In this study, DHM is applied to in vivo developmental imaging and quantitative analysis of the zebrafish embryo, which is a popular vertebrate model organism. An off-axis DHM system is configured and calibrated to quantify dimensions of observed structures and cells. The reconstructed amplitude images reveal morphological structures and different cell types inside developing zebrafish embryos at various developmental stages. Using the developed DHM system, the blood flow rate and heart beat rate are quantified to study the effects of elevated D-glucose (abnormal condition) on circulatory and cardiovascular systems of zebrafish embryos. To demonstrate the potential of DHM as a quantitative tool for high throughput screening applications, the post-processing algorithms are implemented in an automated manner. It is shown that DHM is an excellent tool for visualizing cellular dynamics of organogenesis of zebrafish embryos in vivo..
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