整合自動調焦與過飽和螢光深度切片功能之高解析顯微鏡設計開發

Development and design of combining tunable focusing and saturated excitation high resolution microscope

羅啟豪(Chi-Hao Luo)

指導教授 ： 黃光裕

共同指導教授 ： 駱遠(Yuan Luo)

國立臺灣大學/工學院/機械工程學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103501

摘要

螢光顯微鏡於生醫領域以廣泛被用於觀察生物樣本，而傳統螢光顯微鏡受缺少深度解析能力且受限於光學繞射極限，因此無法觀察度大於10~30 μm之厚生物樣本以及樣本細微資訊。近年於具光切片能力及超解析顯微術研究蓬勃發展，其中共軛焦顯微術為具光切片能力顯微術中的黃金標準；各種超解析方法亦被相繼提出。激放射耗乏顯微術於影像解析度有卓越表現且亦有光學切片能力，能應用於厚生物樣本中，但光學架構中須使用兩種不同波長之雷射光源，成本高且結構複雜。隨機光學重建顯微術能有效定位目標螢光位置協助研究人員判斷，但由於使用的照明方式為寬場照明，應用於厚生物樣本時會導致影像模糊無法判斷。而過飽和及差分過飽和螢光顯微術相較於上述兩種方式有著較簡易的光學系統架設，且能應用於厚生物樣本上。為了能夠以更加穩定獲取生物樣本三維資訊，本論文結合過飽和螢光顯微術與電控變焦透鏡進行高解析等放大率軸向掃描，搭配振鏡以及數值孔徑為0.8 之物鏡，拍攝螢光球做實際測試，並比較兩種過飽和螢光顯微術搭配使用所帶來的差異。結果於共軛焦顯微術達成3.14 μm之軸向解析度，拍攝解析度試片以及200 nm螢光球分別獲得244 nm以及413 nm之橫向解析度。然而差分過飽和螢光顯微術將橫向解析度更進一步提升至366 nm，軸向掃描範圍則可達35 μm。

關鍵字

共軛焦顯微術 ； 過飽和螢光顯微術 ； 激發光調製 ； 電控變焦透鏡 ； 軸向掃描顯微鏡 ； 高解析螢光影像

並列摘要

Fluorescence microscopes are widely used to observe biological samples in the field of biomedicine. However, traditional fluorescent microscopes lack deep resolution capabilities and are limited by the optical diffraction limit, so they cannot observe thick biological samples and samples with a degree greater than 10~30 μm and detailed information about the samples. In recent years, researches on optical sectioning capabilities and super-resolution microscopy have developed vigorously. Among them, confocal microscopy is the gold standard in optical sectioning capabilities; various super-resolution methods have also been proposed. Stimulated emission depletion microscopy (STED) has excellent performance in image resolution and optical slicing capabilities. It can be used in biological samples. However, two laser light sources of different wavelengths must be used in the optical architecture, which is costly and complex. Stochastic Optical Reconstruction Microscopy (STORM) can effectively locate the target fluorescent position to help researchers judge, but because the illumination method used is wide-field illumination, when applied to thick biological samples, the image will be blurred and cannot be judged. Compared with the above two methods, Saturated Excitation microscopy (SAX) and differential Saturated Excitation microscopy (dSAX) has a simpler optical system setup, and can be applied to thick biological samples. In order to obtain the three-dimensional information of biological samples more stably, we combines SAX microscopy and electrical tunable lens to perform high-resolution axial scanning with constant magnification. With galvanometers and an objective lens with a numerical aperture of 0.8, the fluorescent beads is taken for practical testing. And compare the differences between SAX and dSAX microscopy. As a result, an axial resolution of 3.14 μm was achieved by confocal microscopy, and the lateral resolution of 244 nm and 413 nm were obtained by taking resolution target image and the 200 nm fluorescent beads, respectively. And in differential supersaturation fluorescence microscopy, the lateral resolution is further improved to 366 nm, and the axial scanning range reaches 35 μm.

並列關鍵字

Confocal ； Saturated excitation microscopy ； Light Modulation ； Electrically tunable Lens ； Axial Scanning Microscope ； High Resolution Fluorescence Image

參考文獻

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