多光子激發螢光顯微術(multiphoton excited fluorescence microscopy，MPEFM)以超快雷射(ultrafast laser)激發非線性光學現象，其超快雷射的波長以近紅外光波段(near-infrared，NIR)作為光源於生物組織具低吸收率的特性，故擁有低光漂白(photobleaching)以及較深的生物穿透深度(penetration depth)等優點，並根據激發光子數目重建樣品之螢光強度影像；另外藉由時間相關單光子計數技術(time-correlated single photon counting，TCSPC)，其根據接收的光子訊號脈衝以及激發雷射脈衝相差的延遲時間紀錄並加以累積光子數目與時間差的直方圖，可直接顯示螢光生命週期(fluorescence lifetime)資訊，而螢光生命週期顯微成像技術(fluorescence lifetime imaging microscopy，FLIM)結合相量分析(phasor analysis)可提供螢光強度影像外的輔助資訊，因此於生物影像、病理研究與疾病檢測都能有廣泛的應用與市場價值。
本論文藉由FLIM技術應用於肺動脈高壓(pulmonary arterial hypertension，PAH)，主要目的為不經過特殊染色直接辨別肺動脈周圍的膠原纖維(collagen)以及平滑肌(smooth muscle)，透過小鼠尾巴肌腱樣品之FLIM分析，有助於得知膠原纖維於不同激發雷射波長與螢光濾片組合之螢光生命週期數值區間，而肺高壓模型以雷射激發波長800 nm和螢光濾片405/150作為實驗最佳組合進行FLIM分析，其中膠原纖維之螢光生命週期數值約為0.6 ns至1.2 ns，而平滑肌之螢光生命週期數值約為1.6 ns至2 ns，最後分別計算控制組、MCT組以及MCT+MAG組的肺動脈厚度百分比，並以Mann─Whitney U檢定來比較兩個獨立組別，其結果表明MCT誘導的PAH動物模型中，MCT+MAG組的肺動脈厚度百分比與MCT組相較之下有顯著降低。

Multiphoton excited fluorescence microscopy (MPEFM) uses an ultrafast laser to induce nonlinear optical phenomena. This laser operates in the near-infrared (NIR) range, which has low absorption in biological tissues, resulting in benefits such as low photobleaching and deeper tissue penetration. MPEFM reconstructs fluorescence intensity images based on the number of signal photons. Time-correlated single photon counting (TCSPC) records the time delay between received photon signals and laser pulses, providing fluorescence lifetime information. Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis offers additional insights beyond fluorescence intensity, making it valuable for biological imaging, pathology, and disease detection. This thesis applies FLIM to pulmonary arterial hypertension (PAH) to identify collagen fibers and smooth muscle around the pulmonary artery without special staining. FLIM analysis of tendon samples helps determining the fluorescence lifetime range of collagen fibers. In the pulmonary hypertension model, FLIM analysis used an 800 nm laser excitation wavelength and a 405/150 nm fluorescence filter, which were optimal parameters. The fluorescence lifetime of collagen fibers was about 0.6 to 1.2 ns, and that of smooth muscle was about 1.6-2 ns. The pulmonary artery wall thickness percentage was calculated for control, MCT, and MCT+MAG groups. The Mann–Whitney U test showed a significant reduction in pulmonary artery smooth muscle thickness in the MCT+MAG group than in the MCT-induced PAH model.

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