分子影像在生醫工程中是一項重要的技術。其方法可以分類為兩種：一種為以非光學為基礎的技術，另外一種則是以光學為基礎的技術，然而，後者可以擁有低侵入性和高影像解析度。 在生物體中，分子的螢光放射光譜扮演著重要的角色，可以提供有用的生物資訊。然而，由於螢光放射光譜較寬，不同分子的光譜容易重疊而造成嚴重的光譜交錯現象，導致在分子影像上產生誤判。為了解決此問題，原本應用於太遙的超光譜技術被應用在生醫影像上，此技術同時記錄了分子的空間與光譜資訊，配合光譜的分析將能解決光譜交錯的問題而提昇分子影像的準確性。 我們研發了一套雷射單點掃描雙光子螢光超光譜顯微術，結合雙光子顯微術的良好光學切片能力，和較深的穿透深度，此系統架構適合用在厚樣本上以及活體內的研究。本系統採用了非反掃描以及平行接收的架構，非反掃描架構可以增加訊號的收集效率，而利用二維CCD進行空間及光譜訊號的平行接收，可以提升取樣速度以及光譜解析度。在本論文中將會詳細描述超光譜顯微術系統的構造，同時，利用混合式螢光微粒球與黃金葛葉子，來驗證此系統的取像速度及解析能力。 Molecular imaging is a popular technique in biomedical engineering. It can be classified into two methods, one is non-optics–based and the other one is optics-based. However, the latter can provide non-invasive investigation and better spatial resolution. Fluorescence emission spectrum provides valuable information of molecules and plays an important role in molecular imaging. However, the emission spectrum overlap of different molecules usually causes serious crosstalk which would lead to errors in molecular imaging. To solve this problem, hyperspectral imaging techniques are developed and used to record both the spatial and spectral information of the molecules simultaneously. We developed a two-photon hyperspectral microscopy (TPHM) based on the laser-scanning point excitation, non-de-scanned, and parallel recording geometry. Integrated with the optical sectioning power and higher penetration depth of the two-photon microscopy, this system is suitable for thick tissue or in vivo imaging. The non-de-scanned geometry helps to increase the collection efficiency, while the parallel recording of the spatial-spectralinformation with a 2D CCD can improve the frame rate and spectral resolution. In this thesis, the architecture and the experimental results of this hyperspectral microscopic system will be described in details. The characteristics of this system was demonstrated by using mixed fluorescence microspheres and fresh Epipremnum aureum leaves as samples.
