光譜解析單像素重建顯微術於雙光子激發螢光與拉曼造影之研究

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姓名	羅慶隆(Ching-Lung Luo) 查詢紙本館藏	畢業系所	光電科學與工程學系
論文名稱	光譜解析單像素重建顯微術於雙光子激發螢光與拉曼造影之研究 (Spectrally-resolved single-pixel reconstructed microscopy for two photon excitation fluorescence and Raman imaging)
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摘要(中)	本論文中設計一款激發波長可調式的時域聚焦雙光子激發顯微鏡，利用掃描鏡、透鏡與光柵的搭配而實現激發波長可調的範圍為730至1000 nm，並使用數位微鏡裝置 (Digital Micromirror Device, DMD) 取代光柵以將單像素成像 (Single-Pixel Imaging, SPI) 引入系統，在實驗中透過模擬優化激發光源入射DMD的角度，使得全激發波長可以依照相同激發光路行進並達到最佳繞射效率。藉由此系統與光譜解析系統的結合，螢光染料分子都可使用其最佳激發波長進行激發，使得有最大雙光子激發效率，並從而獲取螢光光譜與光譜解析影像。在多光子激發螢光顯微術系統中，可以從多色螢光細胞中解析光譜影像，其中系統的光譜解析度可達到0.45 nm，且也利用螢光染料在不同環境中最大放射波長擁有偏移的特性，來量測螢光波長偏移量與溶液環境的關係。另也建構單像素成像之拉曼光譜系統結合，來量測循環腫瘤DNA (circulating tumor DNA, ctDNA) 之表面增強拉曼散射 (surface-enhanced Raman scattering, SERS) 影像，藉由此系統可以取得拉曼光譜並且解析各拉曼峰的光譜影像，其中系統的光譜解析度可達到1.41 cm-1，並藉由ctDNA於SERS基板上的拉曼訊號來分析SERS效應之空間分佈情形，以證實SERS基板的均勻增益特性。
摘要(英)	In this thesis, we designed a temporal focusing two-photon excitation microscope with tunable excitation wavelength. The combination of scanning mirror, lenses, and grating realized the tunable excitation wavelength in the range of 730 to 1000 nm. A digital micro-mirror device (DMD) was adopted to replace the diffraction grating in the conventional temporal focusing two-photon excitation microscope for implementing single-pixel imaging (SPI) approach in the presented system. The angle of excitation light at the DMD was optimized according to the simulation result, so that the excitation light for the wavelength from 730 to 1000 nm can have the same excitation light path and achieve the optimal diffraction efficiency. All fluorescent molecules can be excited by their optimal excitation wavelength to have the maximum two-photon excitation efficiency. By combined with the spectrally-resolved detection system, fluorescence spectra and spectrally-resolved images of the specimens can be obtained. Spectrally-resolved images of multifluorophore-labeled cells through the multiphoton excitation fluorescence microscopy system demonstrated that the spectral resolution of the system could reach 0.45 nm. Moreover, the variation in the emission spectrum of fluorescent molecules in different environments was used to determine the relationship between the maximum emission wavelength and solvent environment. In addition, a single-pixel imaging Raman spectroscopy system was also constructed to measure the SERS images of circulating tumor DNA (ctDNA), through which the Raman spectrum and spectrally-resolved images of each Raman peak could be obtained with a spectral resolution of 1.41 cm-1. Finally, the spatial distribution of SERS effect was analyzed by the Raman signals of ctDNA on the SERS substrate to verify the uniform gain characteristics of SERS substrate.
關鍵字(中)	★ 單像素成像 ★ 時域聚焦雙光子激發顯微鏡 ★ 光譜解析 ★ 拉曼光譜 ★ 表面增強拉曼散射 ★ 循環腫瘤DNA	關鍵字(英)	★ Single-pixel Imaging ★ Temporal Focusing Two-photon Excitation Microscope ★ Spectrally-resolved ★ Raman Spectrum ★ Surface-enhanced Raman Scattering ★ Circulating Tumor DNA
論文目次	摘要 I Abstract II 致謝 IV 目錄 V 圖目錄 VIII 第一章緒論 1 1-1前言 1 1-2文獻回顧 3 1-2-1 多光子激發螢光顯微術 3 1-2-2 拉曼掃描光譜 4 1-2-3 單像素成像技術之應用 5 1-3 研究動機 6 1-4 論文架構說明 7 第二章基礎原理 8 2-1 多光子激發螢光光譜 8 2-2 時域聚焦多光子激發螢光影像 9 2-3 拉曼光譜學 10 2-4 單像素成像 10 2-5 軸向激發限制 11 2-6 影像相關係數 13 第三章實驗方法與系統架構 16 3-1 單像素時域聚焦多光子激發螢光顯微術 16 3-2 單像素拉曼系統 22 3-3單像素重建影像之投影圖形 23 3-4深度學習架構 25 3-5樣品製備 27 第四章實驗結果與分析 30 4-1 光譜解析單像素重建顯微術之解析度分析 30 4-1-1 空間解析度 30 4-1-2 光譜解析度 31 4-1-3 軸向激發限制 33 4-2 影像重建與光譜分析 36 4-2-1 細胞螢光光譜與影像 36 4-2-2 鹼基胸腺嘧啶(Thymine)拉曼量測光譜與影像 38 4-2-3 表面增強拉曼量測光譜與影像 39 4-3螢光變化之光譜解析 41 4-3-1螢光染料於不同環境下之分析 41 4-3-2活體細胞螢光變化之分析 43 4-4探討深度學習對影像重建之影響 45 4-5影像結構相似性分析 46 第五章結論 47 參考文獻 49
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指導教授	簡汎清賴昆佑	審核日期	2022-9-29
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