Laser additive remanufacturing technology is an important technology in the field of manufacturing technology innovation and development, which has largely revolutionized the design and manufacturing mode of high-end devices to a large extent, and is a key component of green remanufacturing that can effectively promote the sustainable and sound development of the manufacturing industry. A multiphase model of laser additive remanufacturing with pulsed lasers is developed to analyze the evolution of the molten pool, the trend of the surface tension for different process parameters, and their relation to the molten pool morphology. The results demonstrate that the maximum flow velocity at the surface of the molten pool is inversely proportional to the pulsed laser frequency and to the duty ratio. While the trend of the height of the cladding layer corresponds to the trend of the heat accumulation in the molten pool, the width and penetration depth of the cladding layer are governed by the size of the heat-affected zone. The cladding layer and substrate will have poor metallurgical bonding if the pulsed laser frequency is too high. When the duty ratio is too large, the likelihood of over-melting increases, affecting the substrate properties and increasing the surface roughness, which is detrimental to the surface finish of the cladding layer.

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Fluorescent proteins (FP) have revolutionized our imaging technologies and facilitated visualization of biochemical and physiological processes occurring in complex biological systems, which has opened up a new and unprecedented era in cell biology. Although FPs mainly serve as passive fluorescent labels for reporting gene expression and protein localization, FP-based indicators also serve as indispensable tools for dynamic imaging of cellular signaling including neuronal activities. However, few FP-based indicators provide robust performance for in vivo imaging, and the development of reliable FP-based indicators remains a challenging engineering problem, mainly due to lack of structural information for rational design and effective methodologies of protein engineering. The goal of this thesis work is to tackle the long-standing challenge of engineering FP-based indicators for improved performance. This thesis describes a variety of directed evolution strategies to develop FP-based indicators for neuronal activities. First, I developed a colony-based directed evolution method to screen for improved single FP-based calcium ion (Ca2+) indicators. This novel strategy accelerated the engineering of single FP-based Ca2+ indicator and led to several variants with improved performance and various new colors. This palette of new Ca2+ indicators enables simultaneous monitoring of Ca2+ transients in different cellular compartments or different types of cells, which opens up a new era of colorful Ca2+ imaging. Next, by combining microfluidic technology and colony-based screening, I designed an automated cell sorting approach that enables screening variants of FP-based Ca2+ indicator with throughput up to 300 cells/s. This new approach saved considerable time and effort for evolving a new yellow FP-based Ca2+ indicator, Y-GECO. The end product, Y-GECO1, is a useful tool for Ca2+ imaging in cell cultures and brain slices. Finally, I designed a hierarchical screening method to engineer Archaerhodopsin-based voltage indicators with a focus on improving fluorescent brightness. The latest generation of variants, designated QuasAr1 and QuasAr2, shows superior performance and brightness compared to their predecessors. Together with our collaborators, we demonstrated that QuasAr1 and QuasAr2 enable fully optical electrophysiological interrogation of neuronal circuits in intact brain tissues.