運用超穎透鏡於雙紙片螢光顯微鏡之設計開發

Abstract

螢光顯微鏡廣泛應用於生醫領域，然而傳統螢光顯微鏡缺少深度解析能力，無法從厚生物樣本獲得高解析三維影像，光學切片顯微鏡陸續被開發出來，包括共焦顯微鏡、結構光照明技術與紙片光顯微鏡。共焦顯微鏡有良好的切片能力與橫向解析度，利用針孔阻絕失焦平面資訊，但點掃描的成像機制，導致成像速度有限，且具有高光損傷缺點。結構光照明技術無需點掃描，但存在結構光相移誤差的問題。紙片光顯微鏡成像速度高，且無須影像後處理重建，光損傷也相對低。本論文架設兩種紙片光顯微鏡，分別為近紅外接收與雙邊照明的紙片光顯微鏡，針對其性能進行量測與展示。近紅外波長使用於生物組織樣本上，相較於可見光，能抑制光的吸收與散射，提升光的有效傳播距離與成像解析度，而多向照明對於緻密生物樣本上，改善光線受阻擋產生假影問題，提升照明範圍與成像視野。近紅外紙片光顯微鏡中，於照明波長 491 nm、532 nm 與 880 nm 分別具有 1.7 μm、1.8 μm 及2.9 μm 光學切片能力，近紅外接收成像具有 1.55 μm 的橫向解析度，小鼠肺部成像能於樣本更深處獲得更高對比的影像，提升成像深度。雙紙片光顯微鏡中，使用雙超穎透鏡產生雙邊紙片光照明，超穎透鏡其奈米等級尺寸，有助於提升設備緊緻，設計對位裝置用於架設超穎透鏡於樣本水腔上，量測雙紙片光的平面誤差為 11.5 μm，系統具有 6.8 μm 的光學切片能力，具有 1.38 μm 的橫向解析度，雙邊對比單邊照明獲得均勻光強的螢光球輪廓。

Fluorescence microscopes are widely used in biomedicine. However, traditional fluorescence microscopes lack axial resolution capabilities and cannot obtain high-resolution 3D images from thick biological samples. Optical section microscopes have been developed one after another, including confocal microscopes, structured light illumination technology and light sheet microscope. Confocal microscopes have good section ability and lateral resolution, and use pinholes to block out-of-focus plane information. However, the imaging mechanism of point scanning results in limited imaging speed and has the disadvantage of photo damage. Structured light lighting technology does not require point scanning, but there is a problem of structured light phase shift error. The imaging speed of the light sheet microscope is high, and there is no need for image post-processing and reconstruction, and the photo damage is relatively low. In this thesis, two kinds of light sheet microscopes are set up, namely the near-infrared receiving and double-side illuminating light sheet microscopes, and their performances are measured and demonstrated. Near-infrared wavelengths are used on biological tissue samples. Compared with visible light, it can suppress the absorption and scattering of light, improve the effective propagation and imaging resolution. Multi-directional illumination can improve the blocking of light for dense biological samples, solve the shadow problem, and improve the field of view. In the near-infrared light sheet microscope, the illumination wavelengths of 491 nm, 532 nm and 880 nm have optical sectioning capabilities of 1.7 μm, 1.8 μm and 2.9 μm , and the receiving imaging has a lateral resolution of 1.55 μm. Mouse lung Imaging can obtain higher-contrast images deeper in the sample. In the double-side light sheet microscope, a double metalens is used to generate light sheet illumination. The nano-scale size of the metalens helps to improve the compactness of the equipment. The alignment device is designed to set the metalens on the sample cavity. The position error of the double light sheet measurement is 11.5 μm. The system has an optical sectioning capability of 6.8 μm and a lateral resolution of 1.38 μm . It can obtain a fluorescent ball image with uniform light intensity profile.