| Summary: | 博士 === 國立成功大學 === 光電科學與工程學系 === 103 === Confocal microscopy and multiphoton excitation microscopy have become a very important imaging and analyzing tool for biomedical research. The micron/nanometer-scale spatial resolution and three-dimension imaging ability enable researchers to observe more detail information with multiple combinations of different fluorescent dyes and light excitation mechanism. This thesis first reveals an optical microscope combined with confocal microscopy and multiphoton microscopy. The system is mainly designed for in vivo mouse imaging. The soliton self-frequency shift (SSFS) via a photonic crystal fiber generates longer wavelength soliton. With BiBO crystal frequency doubling technique, it further allows us to flexibly adjust femtosecond pulse wavelength to match the optimal two-photon absorption band of the fluorescent dye in order to provide superior excitation efficiency. By combining the multiphoton excitation mechanism and temporal focusing technique, this thesis also demonstrates a temporal focusing multiphoton microscopy system which has widefield optical sectioning ability and already demonstrates the potential for widely applications. However, temporal profile distortions reduce excitation efficiency and degrade image quality. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only the axial excitation symmetrically is refocused, but the axial resolution with two-photon excited fluorescence (TPEF) intensity is also maintained.
AOS is successfully proven that it can help optical system to get rid of aberrations. However, the integration complexity and control loop speed are serious limitations for practical use. Furthermore, a field programmable gate array (FPGA)-based Shack-Hartmann wavefront sensor (SHWS) is developed for performing 30 Hz real-time wavefront measurement. A lab-made video decoder digitalizes the CCD video signal with an odd field and an even field of one frame, and transmits the data to the FPGA. Only the odd field image is transmitted for reconstructing the wavefront, and then the computations and further customized application are executed in the even field time slot; hence, no additional time delay between two frames is needed. With the FPGA-based SHWS, an easily implementable AOS based on a real-time FPGA platform with state-space multichannel control has been developed, and also integrated into a laser focusing system successfully. The overall system with a 32-channel driving signal for a deformable mirror as input and a Zernike polynomial via the SHWS as output is optimally identified to construct a multichannel state-space model. In real-time operation, the FPGA platform first calculates the Zernike polynomial of the optical wavefront measured from the SHWS as the feedback signal. Then, a state-space multichannel controller designed by the identified model is implemented in the FPGA to drive the DM for phase distortion compensation. The FPGA-based AOS is capable of suppressing low-frequency thermal disturbances.
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