廣域多光子激發螢光顯微術(widefield multiphoton excited fluorescence microscopy)利用同時時間域和空間域聚焦 simultaneously spatial and temporal focusing)的特性提供了廣視野的光學切片(optical sectioning)功能，搭配z軸深度的掃描即可得到樣品的三維影像。實驗室目前已利用超快雷射放大器、正立式顯微鏡以及快速的高敏感度EMCCD造相機為主要架構，建立一套廣域多光子激發螢光顯微鏡，目前系統的取像幀率可高達每秒20幀影像以上，縱向解析度約為2.5 μm，可應用於活體生物樣品的即時觀測。
任何光學系統的校正誤差、環境干擾以及生物樣品本身的不均勻度都會造成像差(aberration)進而影響整體成像品質。適應性光學系統(adaptive optics system，AOS)可藉由波前感測器回饋(feedback)訊號，利用波前致動器來動態補償受干擾的波前，使三維顯微系統達到較高的空間解析度並得到較深z 軸的影像。此研究論文主要是應用AOS 於廣視野多光子激發螢光顯微術，利用EMCCD 擷取影像之銳利度(sharpness)來當作回饋訊號，藉由訊號擷取(data acquisition，DAQ)卡之FPGA 模組來動態控制37像素之可調變聚焦鏡(deformable mirror)作及時之波前修正。除了補償系統本身校正誤差導致的影像的不均勻性之外，也修正樣品的干擾像差使時間域聚焦效率得到有效的提升，使影像整體的品質達到最佳化。
實驗上採用攀登演算法(hill climbing algorithm)搭配不同的銳利度參數回饋影像光強及高頻資訊，能使螢光球影像光強提升1.3倍，而在神經細胞影像上也能得到初步的提升。

A widefield multiphoton excited fluorescence microscopy based on simultaneously spatial and temporal focusing technique can provide sequential two-dimensional optical sectioning images via z-axis scanning, and then convert them to construct a three-dimensional (3D) image. Utilizing the strong instantaneous intensity of an ultrafast laser amplifier, the imaging of an upright microscope, and the ultrasensitive detection of a high-speed EMCCD camera as the framework, the widefield multiphoton excited fluorescence microscope has been developed and achieves an axial resolution of 2.5 μm with a frame rate of 20 Hz. Hence, it is useful for in vivo imaging in small animals’ functional activities such as neuron signal transmission and liver metabolism.
The calibration error of an optical system, the disturbances from surrounding environment, and the wavefront distortion from turbid biospecimen will induce optical aberrations to degrade image quality. Therefore, an adaptive optics system (AOS) can be adopted for the wavefront compensation in order to provide high spatial resolution and deep penetration depth 3D images. This thesis is attempted to integrate the AOS into the developed widefield multiphoton excited fluorescence microscope. According to the distorted wavefront information via an EMCCD camera with image sharpness algorithms, a 37-element deformable mirror controlled by the FPGA module in a data acquisition (DAQ) board is used to correct the wavefront distortion in real time. The AOS compensated the optical system misalignment to improve the uniformity of the images. Besides, the distortion from biospecimen is reduced to ensure optimal temporal focusing.
A hill climbing algorithm with different sharpness parameters according to the high spatial frequency of the image is utilized as the feedback control information. Currently, the AOS has successfully enhanced the fluorescence intensity of fluorescence micro-beads with 1.3 folds and also improved the quality of neuron images.

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