| Summary: | 博士 === 國立成功大學 === 工程科學系 === 106 === In this dissertation, the multiphoton excitation (MPE) system, high-speed three-dimensional (3D) microscopy system have been investigated and developed, and applied on the biomedical research. The research topics of this dissertation can be divided into two major issues. First of all, the lab-made laser processing direct writing technique and multiphoton protein crosslinking mechanism are used to fabricate various extracellular matrixes (ECM) micro-scaffolds. The 3D micro-structure provides a 3D growth and migration of cells in the niche microenvironments. From the experimental results, it is verified that the 3D ECM microstructure can be constructed through this system to guide cell growth, and control the growth region of cell in a specific 3D spatial distribution.
In the second part, the main purpose is focused on constructing the high-speed 3D microscopic multiphoton imaging systems. The research and development stage can be broadly divided into three parts: 1) Improvement of the conventional wide-field temporal focusing (TF) technology. A digital micromirror device (DMD) is used to replace the conventional blazed grating, and constructs a TF system for the wide-field image excitation. This new system is the DMD-based TF system, and at the same time, it also act as a digital light processor (DLP) with structured illumination ability; 2) In the image receiving terminal, the light field (LF) technology, that can capture dynamic images at high speed. Through a single microlens array (MLA), the direction and position of incident light can be separated and recorded on the image sensor. The four-dimensional matrix can be used to describe the state of travelling light. Fourier slice theorem is used to reconstruct the images at each focal plane. With the integration of computer vision, each focal plane image can be digitally-refocused. The raw data can be used to calculate and reconstruct the 3D surface morphology; 3) Integrate the MPE technology and LF system of the multi-photon DMD wide-field TF technology as a high-speed image receiving microscopy system. Furthermore, the 3D volume excitation can be controlled by adjusting the numerical aperture in the Fourier conjugate focal plane, and reduce the noise of the background at the same time. The LF can record the entire 3D information in a single frame, and then digitally reconstruct the three-dimensional fluorescence image via 3D deconvolution algorithm. The system is examined and verified by the high-speed Brownian motion experiment.
Finally, through the development of the above two major topic, that can be applied to explore the interaction of the biomedical microenvironments at the cell or tissue level, and provide a high-throughput detection platform to observe the biological behavior via the high-speed volumetric imaging. Moreover, the advantages and the potential of these techniques can be applied to the study of neuronal signal imaging.
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