開發「多模式多光子顯微影像技術」於生物組織成像之應用

Abstract

開發具有化學專一性且非破壞性的三維影像技術以及其在基礎生物研究與醫學上的應用一直是科學家所感興趣的。由於非線性光學影像技術具有高靈敏度、高解析度、低破壞性、能夠三維成像、與更深成像深度等優點，因此多光子影像技術已被廣泛應用於各種生物醫學相關議題。其中，雙光子激發螢光 (Two-photon excited fluorescence, TPEF)成像技術常用來觀測活細胞內各種胞器或蛋白質，而二倍頻 (Second-harmonic generation, SHG)影像方法則經常用於研究具有非中心對稱之結構蛋白(例如膠原蛋白)。同調反史托克拉曼散射 (Coherent anti-Stokes Raman scattering, CARS) 顯微技術為最新崛起的非線性光學影像技術，此技術除了具備其他非線性光學影像方法之優點外，無須染色便具有化學鑑別力，使其在生醫研究上具有更好的發展潛力。 由於各種非線性光學訊號來自不同的產生機制及不同成分，結合多種模式的光學影像技術可在單一平台上提供更多資訊，並具有專一性地觀測各物種在空間上結構與形貌的分布與改變，幫助我們了解生物組織的病理狀態。而目前結合三種非線性光學技術，且完全無須染色之多模式多光子顯微影像方法在文獻上並不常見。針對此目標，我們開發一套可在同一樣品平台上同時收取生物組織內不同成分產生之雙光子激發螢光、二倍頻與同調反史托克拉曼散射三種非線性光學訊號的多模式光學影像系統，並將拉曼顯微光譜技術整合於同ㄧ平台。在此研究中除了將介紹實驗之原理之外，並將發表應用此技術於皮膚組織及動脈血管壁成像的結果，我們成功地觀測到不同皮膚層之細微結構也觀測到動脈血管壁組織在動脈粥狀硬化 (Atherosclerosis) 不同病理階段時結構與成分的改變，此結果可幫助解釋其他生醫影像技術獲得之結果。最後，我也將討論多模式多光子影像技術在其他生醫應用之潛力。

Multiphoton microscopy has found wide-spreading biomedical applications due to its capability in producing three dimensional images of optically thick specimens with great contrast and high spatial resolution. Among various kinds of multiphoton microscopy, two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) are the two most extensively employed modalities in imaging biomedical samples. TPEF has been applied in live cell imaging through the excitation of intracellular auto-fluorescent components whereas SHG, arising only from media lacking a center of symmetry, has been extensively employed to visualize structural proteins such as collagen. Very recently, another powerful nonlinear microscopy technique utilizing coherent anti-Stokes Raman scattering (CARS) has been extensively exploited in biomedical research because of its chemical specificity based on signals inherent and native to particular molecular vibrations. Although composite imaging modalities by the combination either of SHG and TPEF or of CARS and TEPF have been successfully demonstrated to provide structural and functional information of cells or tissues, a multi-modality imaging system based on three nonlinear optical processes has never been realized in an entirely label-free manner. Because these nonlinear optical signals are produced from distinct mechanisms and/or derived from different molecular species, an integrated multi-nonlinear microscope will not only provide complementary information but also allow better differentiation and co-localization among different tissue constituents – an unique capability which is difficult to achieve with single imaging modality alone. In this thesis, I will detail the instrumentation design and the operation principle of the newly-developed integrated multi-modality, multiphoton microscopy system. I will then illustrate the capability of the system by imaging a model skin (porcine skin) and human aorta with atherosclerotic lesions. My results suggest that the new imaging system possesses a great potential for the characterization of the physiological and pathological statuses of intact and unstained tissues.