多光子顯微術在生物與醫學領域有多樣性的應用，在生醫領域中，有需多研究欲觀察活體生物細胞的動態變化，為此，需要更快速的多光子顯微術。基於飛秒雷射的發明，利用其極高瞬間功率，可在聚焦點上激發光學非線性效應，並搭配光偵測器、共振震鏡掃描器(resonance galvanometer scanner，RGS)、震鏡掃描器及電動可變焦透鏡，建構一套掃描式快速三維(three-dimensional，3D)多光子激發顯微鏡系統。
　　本論文用Cambridge Technology公司出產的震鏡掃描器及RGS做二維平面掃描，共振震鏡以8 kHz的頻率做弦波的震盪，為了補償其非線性的角位移，使用同公司出產之pixel clock電路板，提供修正後的像素點擷取觸發信號，藉此得到無失真的30 Hz幀速率的掃描影像，最後再加入電動可變焦透鏡做縱軸的掃描，完成3D掃描。在光源部分，使用KMLabs公司出產的鈦藍寶石飛秒高快雷射作為激發光源，利用其95 MHz的高重複率，在每個像素點做多次的激發，並利用高速高倍率的跨阻放大電路將電流轉換成電壓，藉此以較低的雷射功率取得高訊噪比的影像，減少雷射對生物細胞的傷害。在這個系統中，像素點取樣速率高達15 MHz，為應付如此快速的取樣速度，我們使用NI公司出產之可程式化閘陣列(field-programmable gate array，FPGA)搭配高速資料擷取模組，其取樣速率最高可達250 MHz，並利用LabVIEW軟體設計整體系統程式介面，處理所有類比數位訊號，完成整個系統的信號擷取和硬體控制。

Multiphoton excitation microscopy has been applied to many researches, including the field of biology and medicine. In biomedical science, there are lots of researches related to the variation of living cells. Therefore, a multiphoton excitation microscope with a high frame rate is needed to be built. By using a femtosecond laser which provide a high peak power within an ultrashort time interval, the nonlinear optical effect can be excited at the focal point. Moreover, with photon detectors, a galvanometer scanners, a resonance galvanometer scanner (RGS), and an electrically tunable lens, a three-dimensional (3D) rapid scanning multiphoton excitation microscope system can be realized.
In this thesis, we use a galvanometer scanner and a resonance galvanometer scanner (Cambridge Technology Inc.) to achieve two-dimensional scan. The motion of resonance galvanometer scanner generates sinusoidal waves with 8 kHz resonant frequency. To compensate the nonlinear displacement causes the distortion of image, we adopt a pixel clock board (Cambridge Technology Inc.) to produce a compensated pixel trigger so that a 30 Hz frame rate imaging system can be achieved. After that, an electrically tunable lens was inserted to perform z-axis scanning to obtain a 3D image. In the light source part, a Ti:sapphire femtosecond laser manufactured by KMLabs was used. Having 95 MHz repetition rate can provide multiple excitation pluses within each pixel. A high speed and high gain transimpedance circuit is used as a current-to-voltage converter. Therefore, lower power can be utilized to get high signal-to-noise ratio (SNR) images so that the damage to biosamples can be reduced. Since the highest pixel rate is up to 15 MHz, we adapt a field-programmable gate array (FPGA) card with a high speed A/D module, which features 250 MS/s sampling rate as the resolution. Finally, the control program in LabVIEW is developed to handle the user interface and all the signals in the system.

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