在時間域聚焦多光子顯微鏡（temporal focusing-based multiphoton microscopy）中，雷射脈衝在經過光柵分光後，會在成像面重新聚焦並疊合成最窄的頻寬。然而，雷射的脈衝寬度會因為系統本身的校正誤差，所應用的光學元件等外部因素以及樣品本身的干擾而產生變形，並因此降低螢光的激發效率與影像的品質。為了改善雷射在時間上的失真，本論文利用無波前感測器的適應性光學系統（wavefront sensorless adaptive optics system）去補償雷射脈衝寬度在系統中受到干擾而產生的扭曲。透過不同的電壓驅動可調變聚焦鏡（deformable mirror）去改變雷射波前，並利用截取而得的影像作為回饋信號，藉由攀登演算法（hill climbing algorithm）找出最大的區域影像強度，最後將得到的控制訊號作為可調變聚焦鏡變形量的參考，並縮小雷射脈衝的扭曲程度。加入適應性光學系統後，縱向激發呈更加均勻性的再聚焦，雙光子螢光訊號的縱向解析度易保持既有的水準。藉由R6G-doped PMMA 螢光薄膜，雙光子影像的對比變佳，影像強度也提升3.7倍。
本論文亦利用數位微型反射鏡元件（digital micromirror device，DMD）作為繞射元件，並成功運用於時間愈聚焦多光子顯微鏡。實驗結果顯示，以數位微型反射鏡元件為主的系統量測螢光薄膜，其縱向解析度可達4.0 μm，此結果相當於以600條/mm為主的時間域聚焦多光子顯微系統。除了具有光切面能力以外，數位微型反射鏡元件用在系統中可同時產生任意圖形於物鏡聚焦面，達到光罩的功能。

Temporal profile distortions reduce the excitation efficiency and image quality in temporal focusing-based multiphoton excitation (MPE) microscopy. To compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. 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 is the axial excitation symmetrically is refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity.
This thesis also presents an enhanced temporal focusing-based MPE microscope in which the conventional diffraction grating is replaced by a digital micromirror device (DMD). Experimental results from imaging a thin fluorescence film show that the 4.0 μm axial resolution of the microscope is comparable to that of a setup incorporating a 600 lines/mm grating; hence, the optical sectioning ability of the proposed setup is demonstrated. Similar to a grating, the DMD diffracts illuminating light frequencies for temporal focusing, but additionally, it also exactly generates arbitrary patterns on the objective lens’ focal plane.

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