基態耗損結構照明三倍頻顯微術｜國立中央大學博碩士論文系統

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研究生：	洪瑞廷 Jui-Ting Hung
論文名稱：	基態耗損結構照明三倍頻顯微術 Ground State Depletion Structured Illumination Third Harmonic Generation Microscopy
指導教授：	陳思妤 Szu-Yu Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 光電科學與工程學系 Department of Optics and Photonics
論文出版年：	2019
畢業學年度：	108
語文別：	中文
論文頁數：	58
中文關鍵詞：	基態耗損、結構照明、三倍頻、顯微術
相關次數：	點閱：11 下載：0
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多光子顯微術由於激發過程是非線性，所以擁有良好的縱向及橫向解析度，且激發光波長較單光子顯微術長，因此樣本穿透深度也較深，其中倍頻訊號(如二倍頻及三倍頻)的激發過程中並無實際能階躍遷，滿足動量守恆，因此較不易產生光漂白及破壞樣本。
不同於螢光訊號，因為倍頻訊號屬於無實際能階躍遷，無法如螢光訊號般進行強度的調制，所以不適用螢光的超解析顯微術。1994年，S. W. Hell提出了基態耗損顯微術(Ground state depletion, GSD)這項技術，藉由降低基態電子數目，影響樣本的吸收，進而調制螢光的訊號強度。本論文利用倍頻訊號的一項特性：若樣本存在多光子吸收能階，將會增強倍頻訊號的強度，因此嘗試利用GSD來降低樣本的吸收，使倍頻的增益減弱，來達到調制倍頻訊號。
由於倍頻的激發需要極高的光強度，本論文將以二維點掃描顯微系統為架構來取得三倍頻影像，並導入耗損光(depletion light)，使之與激發光重合產生激態耗損，因三倍頻會滿足光子動量的方向，將以順向方式架設收光系統，使用濾波片將激發光及耗損光濾除，再使用光電倍增管接收三倍頻訊號。本研究以血紅素、黑色素為樣本進行基態耗損的測試，系統以1064 nm的飛秒雷射作為激發光，根據樣本的吸收光譜，以綠光連續波(Continuous wave, CW)雷射作為耗損光。為能進一步產生具有週期結構的耗損光，耗損光經過空間光調制器(Spatial Light Modulator, SLM)產生繞射，使兩道+1與1的繞射光在樣本的焦平面干涉，產生餘弦波的結構照明，如此將能以不同的耗損光強度產生週期性的三倍頻強度調制。

Due to nonlinear excitation, multi-photon microscopy could provide well performance at axial and lateral resolution. In addition, the wavelength of excitation light is longer than the wavelength of single-photon microscopy relatively. Hence, it has better penetration depth. Take harmonic generation for an instance, during the excitation process, there is no transition of real energy state. Complying with the conservation of momentum. Accordingly, cause less optical bleaching or damage to the interacted tissues.
Harmonic generation is different from fluorescence signal. There is no transition of real energy state. As a result, the super resolution microscopy of fluorescence is not
suitable for harmonic generation. Ground state depletion microscopy was published by S. W. Hell at 1994. By decreasing the number of electron of ground, affect the absorption of the sample. As a consequence, modulate the intensity of fluorescence. This essay apply a characteristic of harmonic generation: if there is real energy state of multi-photon absorption, it will enhance the intensity of harmonic generation. Therefore, we can decade the enhancement of harmonic generation by applying GSD. Finally, we can modulate the enhance the signal of harmonic generation.
Owing to the high intensity requirement of multi-photon. This essay will apply 2-D point scanning system to obtain THG image. Furthermore, involving the depletion light make the sample GSD。THG comply with the momentum of the insert photon. The system will set up as a forward system. THG will be detected by a photomultiplier tube, which is mounted with a UV filter.
This research consider melanin and hemoglobin as sample for GSD testing. Our system take 1064 nm femto-second laser as excitation laser. According to the absorption spectrum of sample, use 532 nm continuous wave laser as depletion light. In order to generate a structured illumination depletion light. Use Spatial Light Modulator (SLM) to diffract the light. Make use of the +1 and 1 order diffraction light to interfere at the focal plane of the sample. Consequently, generate the sinusoidal structured illumination. In this way, we can modulate the THG at different intensity of depletion light.

摘要…………………………………………………………………………………….i
Abstract………………………………………………………………………………..ii
目錄…………………………………………………………………………………...iv
圖索引…………………………………………………………………………....vi
第一章 緒論………………………………………..…………………………………1
    1.1 研究背景………………………………….…………………………………1
        1.1.1 光學顯微系統……………………………..…………………………1
        1.1.2 超解析顯微術………………………………………………..………2
    1.2 研究目的與動機…………………………………………………………….4
第二章 原理……………………………………………………………………..……6
    2.1 三倍頻訊號……………………………………………………………….…6
    2.2 基態耗損原理……………………………………………………………….8
    2.3 結構照明顯微術…………………………………………….…………..…11
2.3.1 廣域結構照明顯微術………………………………………………11
2.3.2 掃描式結構照明顯微術………………………………………...….13
2.3.3 耗損光結合掃描式系統之結構照明顯微術………………………14
第三章 系統設計與建構………………………………………………………..…..19
    3.1 掃描式三倍頻影像系統………………………………………………...…19
3.2 結構照明耗損光系統……………………………………………………...21
    3.2.1 結構照明耗損光建構……………………………………………....21
3.2.2 SLM控制參數與結構照明結果檢測………………………………25
3.2.3 結構耗損光之結果分析……………………………………………27
第四章 實驗結果與分析……………………………………………………………33
4.1 有機半導體材料Ir(piq)2(acac)……………………….…………………....33
    4.1.1 樣本備製……………………………………………………………33
        4.1.2 三倍頻影像及強度調制量測……………………………..………..34
4.2 人體血紅素Hemoglobin…………………………………………………..37
4.2.1 樣本備製……………………………………………………………38
4.2.2 三倍頻影像及強度調制量測………………………………………39
    4.3 黑色素Melanin…………………………………………………………….41
        4.3.1 樣本備製……………………………………………………………42
        4.3.2 三倍頻影像及強度調制量測……………………….……………43
第五章 結論…………………………………………………………………………46
參考文獻……………………………………………………………………………..47
圖索引
圖1-1 (a)繞射極限下PSF的強度分布；(b)相近可以解析的兩個點；(c)恰可解析
圖1-1的兩個點。…………………….…………………………………………….…2
圖1-2 STED系統架構：激發光與耗損光光路[9]……………………………….….3
圖1-3隨機光學重建顯微術取像過程與分子定位示意圖[14]……………………...3
圖1-4 (a)結構照明系統；(b)條紋照明樣本後所產生的莫瑞紋[15]………………..4
圖2-1 (a)玻璃縱向上三倍頻強度量測系統式意圖；(b) S-LAH64玻璃三倍頻強度
圖1-1變化，較弱的峰值訊號倍放大100倍以便呈現[23]……………………..….7
圖2-2 (a)三倍頻無實際能階轉換；(b)三倍頻有單光子的吸收能階；(c)三倍頻有
圖1-1雙光子的吸收能階；(d)三倍頻有三光子的吸收能階……………...………..8
圖2-3 Jablonski energy diagram [24]…………………………...…………….……….9
圖2-4 三個能階的在不同激發光下的電子數(黑線為基態、藍線為三重態、紅線
圖2-4為單重態)..……..………………………………………………….………….10
圖2-5 三倍頻有雙光子的吸收能階，且具有三重態T1…………………………...11
圖2-6 (a)條紋的強度分佈；(b)樣本的正空間；(c)廣域結構照明下的樣本；(d)廣
圖2-4域結構照明下收進CCD的影像；(e)條紋的頻率空間；(f)樣本的頻率空間；
圖2-4 (g)結構照明下被位移的頻率空間；(h)結構照明下CCD所取得之影像對應
圖2-4的頻率空間。…………………….…………………….………………….…..12
圖2-7 被解出來的三項未知數：(a) , (b) , (c)；(a)和(c)為位移的高頻資訊。……13
圖2-8 (a)餘弦波強度分佈的耗損光；(b)耗損光強度變化與樣本上基態電子數變
圖2-4化。.……………………………………………………………………..…….15
圖3-1 掃描式三倍頻影像系統示意圖；L：透鏡；L1：焦長75 mm；L2：焦長
圖3-1 250 mm；L6：焦長100 mm；Obj：物鏡；F1：390 nm low-pass filter。…19
圖3-2 掃描式三倍頻影像系統實際架構圖…………………………………….….20
圖3-3 系統二維掃描的電控訊號，x(t)為慢軸訊號，y(t)為快軸訊號………..….21
表3-1 為三倍頻PSF的半高全寬； 為2道耗損光能產生的干涉干涉條紋週期
圖3-4 系統二維掃描； 為3P-OTF的截止頻率； 為干涉條紋的頻率。……….22
圖3-4 使用高斯光束，在薄螢光樣本產生干涉條紋………………………….…...23
圖3-5 結構照明耗損光系統示意圖。L：透鏡；S：擋板；P：偏振片……...….24
圖3-6 結構照明耗損光系統實際架構圖…………………………………………..25
圖3-7 SLM之繞射角與雷射光間距示意圖……………………………………...…25
圖3-8 耗損光干涉示意圖……………………………….………………………….26
圖3-9 螢光樣本上的干涉條紋，SLM使用的週期為4白8黑，scale bar：10 µm；
圖3-10 (a)條紋週期為0；(b)條紋週期為2π/3；(c)條紋週期為4π/3。………...28
圖3-10 原始干涉條紋強度變化……………………………………………..……..28
圖3-11 強度校正後的的干涉條紋強度變化…………………………………..…..29
表3-2 三種強度曲線的擬合資料…………………………………………………..30
圖3-12 螢光樣本上的干涉條紋，SLM使用的週期為6白12黑，scale bar：10 
圖3-12 µm；(a)條紋週期為0；(b)條紋週期為2π/3；(c)條紋週期為4π/3。………30
圖3-13 強度校正後的的干涉條紋強度變化………………………………………31
表3-3 三種強度曲線的擬合資料…………………………………………………..32
圖4-1 Ir(piq)2(acac)吸收光譜與磷光光譜…………….…………………………….33
圖4-2 Ir(piq)2(acac)的白光顯微影像(a)和(b)為不同區域的影像；scale bar: 10 µm.34
圖4-3 Ir(piq)2(acac)結晶的白光顯微影像，scale bar：6.5 µm……………………35
圖4-4 有機材料在激發10秒內的強度變化………………………………………36
圖4-5 在不同強度的耗損光下，材料Ir(piq)2(acac)的三倍頻平均強度變化……36
圖4-6 材料Ir(piq)2(acac)在耗損光關閉後三倍頻的平均強度變化….…....……..36
圖4-7 帶氧與不帶氧血紅素吸收光譜[37] ………………………………………...38
圖4-8 聚合物小球的白光顯微影像影像，scale bar：10 µm……………………..39
圖4-9 聚合物小球的三倍頻影像；從最上層開始往下掃描，影像依序為(a)到(h)，圖4-9 每張高度差500 nm，scale bar：10 µm……………………………………40
圖4-10 血紅素coated聚合物小球 wide-field影像，scale bar：10 µm…...……41
圖4-11 三倍頻影像(a)有血紅素鍍膜的小球；(b)原始小球，scale bar：10 µm…41
圖4-12 黑色素吸收光譜[42]…………………………………………...…………..42
圖4-13 白光影像 (a)未處理的黑色素；(a)小分子狀態的黑色素；(b)放置一段時
圖4-13間凝聚後的黑色素；scale bar：10 µm ………………..…………………..43
圖4-14 黑色素樣本一的三倍頻影像(a)激發光剛打開；(b)激發光照射5秒後；(c)
圖4-14 激發後的黑色素白光影像，scale bar：10 µm…………………..………..43
圖4-15 黑色素樣本三的三倍頻影像：(a)激發光剛打開；(b)激發5秒後；(c)激發
圖4-15 10秒後，scale bar：10 µm…………...…………………………………….44
圖4-16 黑色素三倍頻訊號在10秒內的強度變化………………………………...44
圖4-17 黑色素樣本三在耗損光下的三倍頻影像：(a)激發光剛打開；(b)激發5秒
圖4-17 後；(c)激發10秒後，scale bar：10 µm……………………………………45
圖4-18 黑色素在耗損下的三倍頻訊號在10秒內的強度變化……………..……45

                                

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