可調高低頻光學切片內視鏡用於三維螢光成像

Abstract

螢光內視鏡能提供高對比生物影像，因此廣泛應用於生醫領域。然而，傳統螢光內視鏡缺少深度解析能力，因此很難從厚生物樣本獲得高解析三維影像。近年來光學切片內視鏡的研究蓬勃發展，各種光學切片法相繼被提出，但還存在待克服的限制。共焦內視鏡有良好的切片能力與橫向解析度，但需要冗長的橫向掃描，成像速度有限；結構光內視鏡無需橫向掃描，但存在結構光相移誤差的問題；紙片光內視鏡成像速度高、無須重建，但有不均勻照明光、空間限制影像最高解析度、複雜系統等缺點。相較於早期開發光學切片系統，高低頻(HiLo)光學切片內視鏡僅需兩幀即可重建出光學切片影像，且系統架構以螢光顯微鏡為基礎，相對簡單。為了產生全空間頻率之光學切片影像並重建生物樣本三維螢光分布，本論文結合微反射鏡裝置產生高低頻光學切片法所需柵狀結構照明，電控可變焦透鏡進行定放大率之軸向掃描，搭配NA= 0.26之自設計內視鏡探頭，達成30 μm之光學切片能力，同時掃描範圍長達810 μm。為了全面性驗證系統之光學切片效能，對標準螢光球、螢光花粉、老鼠心臟肌肉組織、老鼠小腸組織以所開發可調高低頻光學切片內視鏡進行了實際拍攝測試。

Fluorescence endo-microscopy is widely used in biomedical field due to its capability to provide biological images with high contrast. However, conventional fluorescence microscopy is lack of depth resolving power. In recent years, research about optically sectioning endo-microscopy has been flourishing. Several optical sectioning technique has been presented, all of which have their own limitation, though. Confocal endo-microscopy has strong depth discrimination power, but its acquisition time is relatively long due to requirement of lateral scanning. Structured light endo-microscopy, a kind of wide-field optical sectioning microscopy, doesn't require lateral scanning, but it faces problems of phase difference error between structured illumination images. Light sheet endo-microscopy has high acquisition rate but has disadvantages such as nonuniform illumination, resolution limitation due to orthogonal setup, complicated system. Compared to above optically sectioning techniques, HiLo endo-microscopy only requires two frames to reconstruct an optically sectioned image, and its structure is based on fluorescence endo-microscopy, which is relatively simple. To obtain an optical -ly sectioned image with full spatial resolution and reconstruct 3-dimensional (3D) fluorescence distribution of biological samples, the thesis combined digital micro-mirror device to generate grid illumination required by HiLo optical sectioning algorithm, electrically tunable lens to achieve axial scanning with invariant resolution and magnification, and custom-made NA = 0.26 endoscopic probe. The optical sectioning capability is measured to be 30 μm, and axial scanning range is 810 μm. In order to fully demonstrate the optical sectioning performance, standard fluorescent beads, fluorescent pollens, mice heart tissues, mice intestine tissues are imaged using the developed endoscope.