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研究生:林祺登
研究生(外文):Chi-deng Lin
論文名稱:應用線狀結構照明提升雙光子顯微鏡解析度
論文名稱(外文):Resolution Enhancement in Two-photon Microscopy by Applying Structured Line Illumination
指導教授:陳思妤陳思妤引用關係
指導教授(外文):Szu-yu Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:光電科學與工程學系
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:56
中文關鍵詞:雙光子螢光結構照明顯微鏡線狀結構照明
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本研究主要為改良雙光子螢光顯微鏡(Two-Photon Microscopy, TPM),應用線狀光形的干涉條紋當作結構照明(Structured Illumination)以提升解析度。結構照明顯微鏡(Structured Illumination Microscopy)可將頻譜空間拓展至傳統顯微鏡的兩倍,並使解析度提升至傳統顯微鏡的兩倍。而雙光子螢光與激發光強度成平方關係的特點,使雙光子螢光顯微鏡有良好的光學切片能力,可以改善結構照明顯微鏡縱向解析度的不足,且雙光子螢光之結構照明可提升影像的空間頻率為將近原本的三倍。因此可透過此系統架構,不但使解析度提高亦可以擁有光學切片能力。
本研究可分為實驗與模擬兩部分。在模擬方面,依照實驗架構的規格,模擬雙光子螢光顯微鏡以線狀結構照明激發的影像,並將影像經由演算法重建還原,其解析度約提升2.57倍;而在實驗部分,成功架設出應用線狀結構照明之雙光子螢光顯微鏡,以量子點奈米微粒(Quantum Dot Nanosphere)作為空間解析度量測的樣本,並經由演算法將影像重建還原,可得其橫向空間解析度約提升至1.7倍。

The aim of this study is to enhance the spatial resolution in two-photon microscopy (TPM) by applying a line-shaped structured illumination. Since structured illumination microscopy (SIM) expands the effective frequency spectrum space twice the size bigger of a traditional microscopy’s, its spatial resolution is therefore two times better. Combining it with TPM’s optical sectioning ability, which is due to the relation between the emission and excitation intensity, we can increase the vertical spatial resolution, and also raise the image’s spatial resolution up nearly three times better. Therefore, utilizing our system not only improves the lateral resolution, but also provides optical sectioning ability.
This study is separated into simulation and experimental parts. In the simulation part, by using the parameters shown in the spec of the experiment setup, we simulate the whole process of a TPM image being excited by a structured line illumination, and then use the results to reconstruct the image via algorithm, in which, we were able to come up with a improve factor of 2.57. Later in the experimental part, by using quantum dot nanospheres as our samples for lateral resolution measurement purposes, and exciting two-photon fluorescence while applying structured line illumination, we successfully reconstructed the image via algorithm with a lateral resolution improve factor of 1.7. Finally, we compare the experiment results with the simulation, and discuss its advantages.

目錄
摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖索引 vi
第一章 緒論 1
1.1顯微影像簡介 1
1.2超解析顯微鏡 4
1.3結構照明顯微鏡(SIM)發展 6
1.4研究動機與目的 10
1.5論文架構 11
第二章 原理 13
2.1結構照明顯微鏡(SIM)原理 13
2.2雙光子螢光 18
2.3線狀結構照明雙光子顯微鏡(SLITPM)原理 21
第三章 實驗架構建立 28
3.1系統架構 28
3.2實驗架構與結構照明相位之校準 31
3.3實驗樣本備製 34
3.4模擬 36
第四章 實驗結果 44
4.1影像還原流程與實驗結果 44
4.2影像分析與討論 48
第五章 結論 51
第六章 參考資料 53
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