結合光學同調斷層掃描與共焦螢光顯微術之研究

Abstract

光學同調斷層掃描與共焦螢光顯微術為目前兩種最主要的非侵入式光學診斷技術。光學同調斷層掃描，是利用光子在組織內散射特性的變化，提供組織內部之結構性資訊；而共焦螢光顯微術，則藉由量測出之點對點螢光訊號在空間中的分佈情形，達到高解析度功能性造影之目的。由於此兩種技術能提供互補性的影像資訊，並且皆具有微米等級的空間解析度，因此將兩者整合為一具有兩種工作模態之光學影像系統一直是十分熱門且重要的研究主題。 本論文中，我們自製了雙纖衣結構的摻鈰釔鋁石榴石晶體光纖，當此光纖以波長445 nm的藍光雷射二極體作為幫浦光時，可產生中心波長560 nm、頻寬98 nm之放大自發輻射，將其作為光學同調斷層掃描的光源，在空氣中可達到1.52 μm的縱向解析度；同時又以藍光半導體雷射作為共焦螢光顯微術的激發光源，成功建立了一套具有三維空間解析能力之雙模態光學影像系統。我們並利用摻鈰釔鋁石榴石晶體粉末作為樣本，驗證了此系統對樣本同一空間的影像整合與疊合能力，縱向疊合範圍達107 μm。此初期研究成果展現了本系統對於生命科學或是醫學的研究上都極具應用潛力。 我們另外也使用光學同調斷層掃描對培養皿中的肺癌細胞與人體間葉幹細胞進行三維造影。在自製的細胞模型中，我們發現癌細胞與幹細胞在空間中互相接近時，癌細胞有移向幹細胞的現象，顯示出我們所建立的光學同調斷層掃描系統具有檢測非活體細胞模型中之細胞化學趨向性的能力，未來更可結合共焦螢光顯微術對此模型提供更為精確的三維影像與定量分析。

Optical coherence tomography (OCT) and confocal fluorescence miroscopy (CFM) are the two most important noninvasive optical diagnosis techniques nowadays. OCT provides structural information in tissue by detecting variation of scattering property of living sample, where CFM measures the intensity of fluorescence signals point-to-point in three-dimensional (3D) space, and achieves high resolution functional imaging. The two techniques provide not only spatial resolution with micrometer scale but also complementary image information. Therefore, it is an very important research object to integrate the two techniques into a two modal optical imaging system up to now. In this thesis,we use a 445 nm laser diode as excitation light of CFM and pumping source of broadband amplified spontaneous emission (ASE) generated by Ce3+:YAG double-cladding crystal fiber, successfully construct a two modal imaging system with 3D resolving power. By using Ce3+:YAG particle as sample, we verify the ability of two modal image integration, and this preliminary result demotrate the application potential of our system on medical and life science. We also perform 3D imaging on cultured human pulmonary adenocarcinoma cells and mesenchymal stem cells. In a homemade cellular structure, we find that cancer cells have the tendency of approaching stem cells when they get closer. By using fluorescence protein transfected cells as sample, we can make a much more quantitative analysis of this phenomenon with the help of CFM in our system in the future.