非線性多光子激發螢光顯微術，在搭配高數值孔徑(numerical aperture，NA)與超快雷射能夠讓脈衝能量集中於聚焦空間上，形成高能量密度的電場，但由於雷射脈衝在經過光學元件如透鏡、菱鏡等會產生色散的現象，導致脈衝的寬度變寬，使瞬時能量分散而降低激發樣品的效率，其中光學色散的二階項(group delay dispersion，GDD)對色散影響是最為明顯，本文實驗的系統中，著重於脈衝測量與脈衝壓縮系統的設計，與基於先前實驗室建立的多光子激發螢光顯微術(multiphoton excited fluorescence microscopy，MPEFM)中應用，實現掃描樣品同時補償脈衝的相位變化使激發效率有所提升，基於先前系統技術下，開發結合飛秒雷射發展具有廣視域激發能力的時域聚焦多光子顯微術(temporal focusing-based multiphoton microscopy，TFMPM)。
本文主要論述關於脈衝量測系統如干涉式自相關儀(interferometric autocorrelation，FRAC)、頻域光學解析開關(frequency resolved optical gating，FROG)等測量雷射相位或脈衝資訊等，再經實驗室演算法如DOES(direct optical-dispersion estimation by spectrogram)後計算脈衝之GDD變化，搭配PI控制器與線性可調變式聚焦鏡(linear deformable mirror，LDM)後進行脈衝補償。並在搭配飛秒級雷射下至TFMPM系統的應用，及能夠透過液態透鏡調控軸向掃描和基於壓電片實作脈衝壓縮測試，或是使用空間光調變器(spatial light modulator，SLM)可呈現光束整形之效果，與系統整合後可實現樣品之均勻度以及多種功能性整合可能。

Ultrafast laser pulses can generate instantaneous energy in optical applications, nonlinear multiphoton excitation fluorescence microscopy to excite fluorescence without damaging the sample. However, when laser pulses pass through optical elements, they experience broadening of pulse width, leading to energy dispersion and reduced efficiency. Among the various types of dispersion, the second-order term known as group delay dispersion (GDD) has the most significant impact. In this study, the focus is on the design of the pulse measurement system and compression system, such as interferometric autocorrelation (FRAC) and frequency-resolved optical gating (FROG), combined with the previously established multiphoton excited fluorescence microscopy (MPEFM). After the technique of direct optical dispersion estimation by spectrogram (DOES), the changes in GDD of the pulse are calculated, and pulse compensation is performed using a proportional-integral (PI) controller and linear deformable mirror (LDM). By compensating for the spectral phase variations of the pulse while scanning the sample, the excitation efficiency is improved. Additionally, we have developed temporal focusing-based multiphoton microscopy (TFMPM) to achieve wide-field excitation. Meanwhile, we have developed the use of a spatial light modulator (SLM) to achieve beam shaping. The SLM allows for the manipulation and control of the spatial profile of the laser beam. And we have also tested the pulse compression performance using a piezoelectric bender. it becomes possible to improve sample uniformity and facilitate the integration of multiple functionalities.

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