2016-08-012024-05-16https://scholars.lib.ntu.edu.tw/handle/123456789/669390摘要：本計劃的研究整體目標，在研發設計新型的光電生醫顯微系統:「飛秒激發光學全像顯微術(Femtosecond laser induced optical holographic microscopy, FL-OHM)」。不同於其它傳統的光學生醫顯微系統，此新型光電生醫顯微系統，基於所設計的光學全像濾波器(volume holographic filters)結合非線性飛秒雷射 (femtosecond laser)與一般光學元件，可以提供高速、高解析度與取得深層之光學切片(optical sectioning)生物組織立體(3D)與光譜的資訊。 基於光學全像濾波器獨特之角度高選擇性，與透過波長的高選擇性，並經由 pq-MMA 全像多工技術(multiplexing)整合所有光學全像濾波器，此系統可取提供所觀察物體的三維影像與光譜訊息。再結合近紅外光(near-infrared)之飛秒雷射激發，使此系統更可降低生物訊號之光致漂白(photo-bleaching)、並取得較深層之空間螢光影像。讓此系統得以高速方式，同時提供生物組織的 3D 斷層影像(optical sectioning)及光譜資訊，呈現在數位相機(CCD)上。。 此新型光電生醫影像系統，具備其它現有的系統所沒有的優點包括: 高三維解析度、高運行效率、成本相對低廉的三維顯微系統。除次之外，雖然不在本此計劃的規劃，但若將此系統搭配光學接替系統(relay system)，可衍伸成為新型光學內視鏡。 三年計畫中，分為下列多個目標進行: 目標一: 發展與研製高解析度的光學全像濾波器。此外，將配合此實驗運用光學軟件優化體積全像顯微術的光學效能，並探討更有效率運用體積全像術於三維呈像之結合。測試與量化光學全像濾波系統的性能。此過程的量化參數，包括: 空間解析度、系統操作波長與光譜解析度。也將用已知參數的測試物體來測試系統的性能。 目標二: 建置可取得三維光學斷層的飛秒全像顯微系統 FL-OHM，並適當調整系統的設計，同時優化系統的非線性飛秒照明及三維全像系統的整合。此外，也將用已知參數的三維測試物體，探討螢光(fluorescence)影像，更有效率運用體積全像多工技術。 目標三: 測試與量化 FL-OHM 的性能。此過程的量化參數與目標一相似，包括: 空間解析度、系統操作波長與光譜解析度。更進一步將用老鼠組織(in-vivo or ex-vivo mouse tissue sample)更真實地進行優化與測試系統的性能。<br> Abstract: The overall goal of the research is to develop, fabricate and test a new type of optical microscopic system that requires optical sectioning three-dimensional (3D) images of tissue samples. The proposed system is based on volume holographic filters and nonlinearity of femtosecond laser excitation used in conjunction with conventional optical imaging components to form a femtosecond laser induced optical holographic microscopy (FL-OHM). The high angular selectivity of the volume hologram in the FL-OHM can be used to obtain two-dimensional image information from objects, while the high wavelength selectivity of the hologram in the system provides spectral information of a specific area of the object that is being observed. In addition, the nonlinear excitation using a near-infrared femtosecond pulse laser will have greater penetration depth, and will significantly reduce photo-bleaching effects during imaging fluorescently labeled tissues. Multiple sections of the object are projected using multiplexed holograms in a volume holographic material, and we will use phenanthrenequinone-doped methyl methacrylate (pq-MMA) as the holographic recording materials. The proposed system has many advantages over competing optical imaging techniques for biological tissue samples. These include simultaneous high spatial and spectral image resolution with the same optical system, use of less expensive 3D operation format, and near-infrared pulse excitation for longer penetration depth and less photo bleaching for imaging. The system is capable of near real-time high-speed operation. While beyond the scope of the current proposal, the system can also be combined with a commercially available miniature optical image relay systems for endoscopic operation. The specific aims of this proposal are as follows: Specific aim 1: Develop a design methodology for high spatial and spectral resolution holographic filters. This work will include modeling the performance of volume holograms. Characterize the optimized volume hologram for imaging by quantifying parameters such as spatial resolution, and spectral resolution. We will also optimize polymer materials for volume holography, and investigating excitation wavelength and digital cameras suitable for image multiplexing and transfer. Specific aim 2: Design and fabricate a FL-OHM based on nonlinearity of near-infrared pulse excitation and volume holographic filters for spatial and spectral imaging. This work will include optimizing illumination setup of femtosecond pulse excitation, and integration of 3D holographic imaging. Experiments will be conducted on well-characterized tissue phantoms for fluorescently labeled imaging for evaluation of the FL-OHM. Specific aim 3: Characterize the optimized FL-OHM system by quantifying parameters including x, y, and z resolutions (as described in Specific aim 1). Experiments will be conducted on proof of principle imaging in ex-vivo and in-vivo mouse tissue samples.近紅外光非線性飛秒雷射全像濾波器光學切片老鼠組織螢光影像near-infraredfemtosecond laservolume hologramoptical sectioningex-vivoin-vivomouse tissuefluorescently labeled imaging.