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研究生:鄒政浩
研究生(外文):Cheng-Hao Tsou
論文名稱:選擇性雙光子螢光過飽和顯微術
論文名稱(外文):A Study on the Super-Resolution Capability by Using Selectively Over-Saturated Two-Photon Fluorescence Emission Microscopy
指導教授:孫啟光孫啟光引用關係
口試委員:高甫仁李翔傑
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:光電工程學研究所
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:2020
畢業學年度:108
語文別:中文
論文頁數:50
中文關鍵詞:超解析雙光子螢光顯微術飽和激發顯微術飽和雙光子激發螢光顯微術加強型綠螢光蛋白選擇性雙光子螢光過飽和顯微術
外文關鍵詞:super-resolutiontwo-photon microscopysaturation excitation microscopysaturated two-photon excitation microscopyenhanced green fluorescent proteinselectively over-saturated fluorescence emission in two-photon excitation
DOI:10.6342/NTU202000537
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光學顯微術被發明以來,由於其對於生物組織具有非侵入性,因此,被廣泛利用在活體生物組織的成像上。然而,其解析度受到物理上繞射極限的限制,光學顯微術的橫向解析度被限制在大於250 nm內,嚴重影響了人們對生物組織的認識。因此近幾十年來,有許多研究突破繞射極限的超解析技術被提出。然而,它們都受限於穿透深度(~20 μm)。
現今,穿透深度和解析度是光學成像系統觀察生物組織的兩個重要因素。其中,雙光子螢光顯微術,使用近紅外光作為光源;因此,雙光子螢光顯微術可應用在較厚的活體生物組織上;然而,紅外光在生物組織中的散射和吸收將使成像品質惡化;因此,雙光子螢光顯微術的空間解析度低於共軛焦顯微術。近年來,有一種稱為"飽和激發顯微術"的技術被提出,此技術可用於超解析影像上。此技術是基於時間調製,相對於在空間域提高解析度的超解析顯微術,飽和激發顯微術較不受生物組織中的散射和吸收的影響,因此可用於較厚的生物組織成像。透過結合雙光子螢光顯微術和飽和激發顯微術,即"飽和雙光子激發螢光顯微術"。透過此飽和雙光子激發螢光顯微術,並結合鼠腦透明化可獲得~2.4 mm的穿透深度和亞微米的超解析鼠腦影像。但此技術相較於繞射極限只提高了1.4倍的解析度。
為了突破此瓶頸,本篇論文我們架設了飽和雙光子激發螢光顯微術的系統。但有別於一般飽和激發顯微術、飽和雙光子激發螢光顯微術,是在低激發強度照射樣本;本實驗我們利用高雙光子激發強度去照射加強型綠螢光蛋白。我們發現,在高雙光子激發強度下,由於加強型綠螢光蛋白在樣本內濃度的不同,加強型綠螢光蛋白會選擇性的出現過飽和現象。在此條件下拍攝影像,影像的空間解析度相對於繞射極限可加強~4.2倍。我們稱此技術為"選擇性雙光子螢光過飽和顯微術"。在論文中,我們首先會說明選擇性雙光子螢光過飽和顯微術的機制和原理。並利用數值模擬,去計算加強型綠螢光蛋白在不同雙光子激發強度下,螢光在不同高階非線性項的響應。並從數學的角度去說明我們在模擬上所觀察到的過飽和現象。接著,我們利用所架設的選擇性雙光子螢光過飽和顯微鏡,去量測加強型綠螢光蛋白在不同雙光子激發強度下,螢光在不同高階非線性項的響應,以證實與理論相符。最後,我們將此技術直接應用在具有加強型綠螢光蛋白的離體鼠腦樣本上,並證明其可達到90 nm (與繞射極限相比好~4.2倍)的橫向解析度。討論其在不同激發強度下,影像所出現的現象與理論是否相同。最後討論其未來應用在超解析影像上的能力。
Since optical microscopy was invented, it has been widely used for imaging of living biological tissues because it is non-invasive to biological tissues. However, its resolution is limited by the physical diffraction limit, which seriously affects people to understand biological tissues; thus, in decades, many super-resolution techniques have been proposed to break the diffraction limit. However, they are all limited by the penetration depth (~ 20 μm).
Nowadays, penetration depth and resolution are two important factors in optical imaging to observe live biological specimens. Two-photon microscopy which uses near-infrared light as a source; therefore, it is a technique employed to imaging thick samples with less sample damage. However, by using long-wavelength light to excite fluorescence makes the spatial resolution of the two-photon microscopy lower than confocal microscopy. Moreover, scattering and absorption of light in biological tissues will also worsen the imaging quality; thus, to improve the spatial resolution of the two-photon microscopy is the main concern in technology development.
Recently, the other super-resolution fluorescence microscopy called saturation excitation (SAX) microscopy had been proposed, which is based on the temporal modulation; thus, it is less affected by the scattering effect of biological tissues compared with the above mentioned techniques. Through combining SAX and two-photon microscopy, saturated two-photon excitation (TP-SAX) microscopy can achieve 2.4 mm penetration depth with a submicron resolution in transparent mouse brain. However, the resolution only improve 1.4 times.
To further improve the spatial resolution of the TP-SAX microscopy, in this work, we built the TP-SAX microscope. However, instead of using low excitation intensity to excite the sample, we use high excitation intensity to excite the enhanced green fluorescent protein (eGFP). We found under high excitation intensity in two-photon excitation the eGFP sample selectively shows over saturation on fluorescence emission because of different concentrations, which we call selectively over-saturated fluorescence emission in two-photon excitation (2-SOFE). In this thesis, we firstly explain the mechanism and principle of 2-SOFE microscopy. The Numerical simulation is used to calculate the response of eGFP to different high-order harmonic frequencies under different two-photon excitation intensities. In addition, we explained the over-saturation phenomenon we have observed in the simulation under mathematics. Furthermore, we used a 2-SOFE microscope to measure the fluorescence response of eGFP solution under different two-photon excitation intensities to confirm that it is consistent with the theory. Next, we applied this technique directly to ex vivo mice brain samples with eGFP and demonstrated its lateral resolution can achieve ~ 90 nm. Finally, we discussed its future applications in super-resolution images.
誌謝 i
摘要 ii
Abstract iv
目錄 vii
圖目錄 ix
表目錄 xi
Chapter 1緒論 1
1.1 研究動機 1
1.2 現有超解析技術 1
1.3 飽和激發顯微術 3
1.4 反飽和激發顯微術 3
1.5 論文範圍 4
Chapter 2選擇性雙光子螢光過飽和顯微術 5
2.1 選擇性雙光子螢光過飽和顯微術的基本原理 5
2.2 過飽和顯微術的回顧 6
2.3 選擇性雙光子螢光過飽和顯微術理論模擬 12
2.4 模擬結果分析與討論 17
2.4.1 在數學上證明過飽和行為 17
2.4.2 理論解析度分析 19
Chapter 3 eGFP頻譜分析之方法與結果 25
3.1 實驗架構 25
3.2 研究前系統校正 27
3.2.1光路校正 27
3.2.2影像系統校正 27
3.3 頻譜分析結果 29
3.3.1 eGFP 樣本配置 29
3.3.2 eGFP頻譜分析過程與討論 29
Chapter 4 實驗結果與討論 32
4.1 2-SOFE影像分析 32
4.2 利用2-SOFE獲取超解析影像 36
4.3 進一步討論 40
4.3.1 螢光漂白 40
4.3.2 反飽和(reverse saturation)與過飽和(over-saturation)的比較 44
4.3.3 影像雜訊 44
Chapter 5 結論與未來展望 46
5.1 結論 46
5.2 未來展望 47
參考文獻 48
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