多光子激發螢光顯微術(multiphoton excited fluorescence microscopy，MPEFM)能夠提供良好的空間解析之激發能力，並可重建三維螢光影像，並且透過雷射脈衝時域能量集中的特性，可透過低平均功率之高瞬時能量，以不傷害樣品的情況激發其螢光訊號，能夠有效降低樣品損壞的風險。然而當雷射脈衝經過光學元件以及樣品時，會因雷射脈衝不同波長的相位變化產生二階群色散(group delay dispersion，GDD)而降低雷射脈衝之時域能量集中效率，使其無法達到理想的多光子激發螢光效率，因此需發展一套能夠同時量測雷射脈衝的相位變化，並補償其相位變化以最佳化其時域能量集中之特性。
本論文著重於發展雷射脈衝量測系統，包含干涉式自相關量測(interferometric autocorrelation，FRAC)以及頻率解析光學開關(frequency resolved optical gating，FROG)等，FRAC主要為透過量測雷射分光後在不同光程差疊合激發的二倍頻訊號之強度曲線變化，並推得其脈衝寬度；而FROG主要為透過量測雷射在不同光程差時的自相關頻譜，搭配其相位回推演算法可收斂出雷射脈衝的電場資訊，其中我們透過修改FROG之相位回推演算法可提升約1.2倍的FROG分析速度與約1.7倍的收斂穩定性，並且在實際應用於多光子激發螢光顯微系統時將FROG量測系統結合菱鏡壓縮系統(prism-based compressor)以及PI控制理論，可實現自動量測雷射脈衝並補償其光學時域色散的整合系統，搭配PI控制器可實現2～3步收斂的快速補償效果，最後透過多光子激發螢光影像觀察其影像強度的變化，能夠提升約1.6倍的螢光激發效率。

In this thesis, we have introduced a self-built multiphoton excitation fluorescence microscopy (MPEFM), which can be used to measure and reconstruct the 3-dimensional resolved image to biotissue in vivo. A fast and simple dispersion scanner GRENOUILLE and dispersion estimation method with fast and highly stable computation timing are proposed. The optical dispersion information is directly estimated based on the frequency-resolved optical gating (FROG) trace without the requirement of the pulse-retrieval iterative algorithm. Instead of using the iterative reconstruction algorithm which relies on the initial electrical filed assumption, the group delay dispersion (GDD) can be directly determined based on the marginals of frequency and delay which are related to the laser spectrum and intensity autocorrelation. The estimated GDD is confirmed with the spectral phase distribution of the reconstructed electrical field based on the principal component generalized projections algorithm (PCGPA) for FROG. The fast and direct estimation method can improve the computation time over 13 times faster than traditional iterative algorithm. The fast and direct estimation with stable computation time is suitable for providing the dispersion as feedback information in a control closed-loop for ultrafast pulse compression. The fast estimated GDD value is sent to the linear control system consists of the prism-based compressor and PI (proportional-integral) controller. The dispersion is shown to be compensated within three control loops. Finally, we apply the compensated pulse into MPEFM to show the fluorescence images quality improvement of both the intensity and contrast after the dispersion is compensated.

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