折射式與繞射式紙片光螢光顯微鏡之設計與性能分析

Abstract

螢光顯微鏡於生醫領域已廣泛被用於觀察生物樣本，然而傳統螢光顯微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。為了實現對活體生物樣本的卓越三維成像，需要精細的光學切片、高速圖像採集和低光損傷。近年來各種光學切片技術相繼被提出，共焦顯微術為光學切片能力的黃金標準，利用針孔阻絕失焦平面的資訊，達到優異光學切片能力，然而點掃描的機制限制其成像速率，且具有高光損傷的缺點；結構光照明技術不須橫向掃描，但存在結構光相移誤差的問題；紙片光螢光顯微鏡 (LSFM) 既不需橫向掃描，光損傷也降到最低，因此成為觀測生物發育過程的主要技術。本論文架設兩種基於折射式光學元件的紙片光螢光顯微鏡 (RLSFM)，分別為高斯紙片光螢光顯微鏡 (G-LSFM) 與貝索紙片光螢光顯微鏡 (B-LSFM)，並針對其性能進行量測與展示。跳脫傳統折射式光學元件，本論文另外使用兩種繞射光學技術開發繞射式紙片光螢光顯微鏡 (DLSFM)。使用體積全相片 (Volume hologram) 取代照明端的多個光學元件，如圓柱透鏡、物鏡、空間光調製器等，體積全相術具有單一繞射、緊湊的尺寸和設計靈活等特性，是商業發展上的趨勢；第二種繞射光學元件為超穎透鏡 (Metalens)，使用超穎透鏡作為偵測端的接收物鏡，其奈米等級的尺寸有助於大幅降低偵測端的工作長度。經過實驗證明本論文所架設之RLSFM中，橫向解析度為0.27 μm，而G-LSFM系統與B-LSFM系統分別具有3 μm及5.08 μm的光學切片能力；在設計開發的DLSFM部份，具有3.92 μm光學切片能力及2.46 μm的橫向解析度，能拍出高對比度的影像。為了全面性驗證系統之效能，本論文對秀麗隱桿線蟲進行活體成像，拍攝其卵母細胞與胚胎細胞的生長關係，以及性腺的發育情形。

Fluorescence microscopy is widely utilized in the field of biomedicine to observe biological sample. However, traditional fluorescence microscopy lacks the ability of depth resolution, and it is difficult to obtain high-resolution three-dimensional images from thick biological samples. To achieve superior three-dimensional (3D) imaging of living biological samples, the main requirements are fine optical sectioning, high-speed image acquisition, and low photo-damage. In recent years, various optical sectioning techniques have been proposed, among them Confocal microscopy is considered the gold standard. It uses a pinhole to remove out-of-focus information and achieves excellent optical sectioning capability. However, due to the point-by-point scanning mechanism, it sacrifices the acquisition rate and leads to high photo-bleaching. Structured light illumination doesn't require lateral scanning, but it faces problems of phase difference error between structured illumination images. Light sheet fluorescence microscopy (LSFM) doesn’t require lateral scanning. In addition, it produces minimal photo-damage, which makes it suitable for observing biological development processes. In the theses, we present two light sheet fluorescence microscopy systems and characterize their imaging performance. Based on refractive optical elements (RLSFM), a Gaussian light sheet fluorescence microscopy (G-LSFM) and a Bessel light sheet fluorescence microscopy (B-LSFM) have been developed and are utilized for biological imaging. Without the traditional refractive optical elements, the thesis presents two diffractive optical techniques to develop a diffractive light sheet fluorescence microscopy (DLSFM). In the illumination part, a volume hologram has been used to replace optical elements such as a cylindrical lens, an objective lens, and a special light modulator to make a compact illumination geometry. Volume hologram has the characteristics of single diffraction order, compact size, and design flexibility, which can help in the commercial development of the microscope. The second type of diffractive optical elements is metalens, which is utilized as the receiving objective lens of the detection part, whose nanoscale dimensions drastically reduces the working distance of the detection part. Experiments show that in the RLSFM set up, the lateral resolution of 0.27 μm can be obtained, while the optical sectioning capabilities of the G-LSFM system and the B-LSFM system are around 3 μm and 5.08 μm, respectively. With our DLSFM system, the optical sectioning capability is 3.92 μm and the lateral resolution of 2.46 μm can be achieved, which can help in acquiring high-contrast images. In order to comprehensively verify the performance of the systems, the thesis uses Caenorhabditis elegans as live biological samples to observe oocytes, embryos, and the development of gonads.