臺灣博碩士論文加值系統

近幾年來，外生螢光染劑已被廣泛的應用在生物影像技術之上。在各類螢光染劑中，綠螢光蛋白 (GFP) 因其本身具有高穩定性與高發光效率的優勢，因此，常被應用於非侵入性分子與細胞生物間的標定。同時，並可用來監控基因的表現、蛋白質分布以及蛋白質之間的交互作用。在本篇論文中，我們提出一套變溫螢光量測系統，用來量測在不同溫度下樣品螢光特性之改變，而後我們提出一套演算法來分析樣品於不同溫度下螢光分佈的情形，進而探討樣品之熱效應。
光同調斷層攝影術 (OCT) 的研究大多著重於系統的改良，一般可分為系統的解析力與成像速度的改良。在此，我們主要針對成像技術改良上提出一套全反射式快速掃描的光延遲線系統，用來取代傳統速度較慢的步進馬達。本系統最大的特色就是系統本身結構緊密、穩定、容易製造並在掃描的過程中可降低光損耗。我們將此光延遲系統應用於 OCT 中，當系統內的掃描鏡轉動 ± 9.6°時，經由計算可達到樣品的掃描深度約為 2.9 mm 。而後透過實驗來驗證系統的可行性。
未來我們希望能將此兩套系統進行整合，並利用 OCT 來量測材料內部物質受熱效應影響之變化，並針對不同樣品內部之光學性質進行探討。

Recently, the exogenous fluorescent agent has been applied to bio-imaging techniques widely. In different fluorescent agents, the green fluorescent agent has high-stability and high-luminescence efficiency superior in itself, thus, it usually used to the lable of noninvasive
molecular and cell biology. It also use to monitor the gene phenomenon, rotein distribution, and protein interaction at the same time.
In the thesis, we offer a spectrum analyzing system to use to analysis the fluorescent property changing of the sample in different temperature, and then, we also offer a algorithm to analysis the phenomenon of the fluorescent spectrum distributions. Furthermore, we will treat the thermal effect of the samples.
The more research of optical coherence tomography (OCT) is mainly the systems improvement. One is the resolution of the systems, and the other is imaging speed improvement. In the thesis, we focus on improve the imaging speed and provide a real time all reflective optical delay line system to alternate the traditional stepper motor. The best distinguishing of this system is closed, stable, easily fabricated, and in scanning process, it can lower the light loss. We applied this system to the OCT, when the scanning mirror of the system tuned ± 9.6°, the scanning depth of the sample will arrived 2.9 mm by calculated. Then we will identify the system capability by the experiment.
In the future, we hope to combine with these two system, and use OCT to analyze the changing with thermal effect inside structure, and treat to inside optical properties.

目錄
中文摘要.....................................................................Ⅰ
Abstract.....................................................................Ⅱ
致謝.........................................................................Ⅲ
目錄.........................................................................Ⅴ
圖索引.......................................................................Ⅹ
表索引......................................................................XIV
第一章 緒論...................................................................1
1.1 前言......................................................................1
1.2 研究動機..................................................................1
1.2.1 光造影技術..............................................................1
1.2.2 螢光標定技術............................................................3
1.2.3 生醫造影技術整合........................................................5
1.3 論文架構..................................................................5
第二章 變溫光譜量測系統.......................................................7
2.1 背景知識..................................................................7
2.1.1 溫度標示與量測..........................................................7
2.1.2 席貝克效應..............................................................8
2.1.3 熱電偶原理..............................................................9
2.1.4 熱電製冷片原理.........................................................10
2.1.5 物質的光譜.............................................................12
2.1.5.1 吸收光譜.............................................................12
2.1.5.2 放射光譜.............................................................13
2.1.5.3 螢光光譜.............................................................13
2.2 儀器簡介.................................................................14
2.2.1 溫度控制器.............................................................14
2.2.2 T-type 熱電偶..........................................................16
2.2.3 串列轉換器.............................................................17
2.2.4 多層熱電致冷片.........................................................19
2.2.5 光譜分析儀.............................................................20
2.2.6 Nd-YAG Laser...........................................................21
第三章 變溫光譜量測系統架設與量測............................................22
3.1 前言.....................................................................22
3.2 樣品來源與製備...........................................................22
3.3 量測系統架設.............................................................24
3.4 資料處理.................................................................26
3.4.1 溫度控制器驅動.........................................................26
3.4.2 溫度控制儀控程式撰寫...................................................26
3.4.2.1 訊號分析程式.........................................................27
3.4.2.2 溫度控制程式.........................................................28
3.4.2.3 光譜分析程式.........................................................30
3.5 溫度變化量測結果分析.....................................................31
3.5.1 綠螢光蛋白發光原理.....................................................31
3.5.2 綠螢光蛋白變溫光譜量測.................................................32
3.5.3 綠螢光蛋白變溫光譜量測結果分析.........................................36
3.5.4 Jablonski diagram......................................................38
3.6 未來展望.................................................................39
第四章 全反射式光延遲線系統..................................................41
4.1 背景知識.................................................................41
4.1.1 干涉原理...............................................................41
4.1.2 麥克森干涉儀...........................................................41
4.2 光同調斷層攝影術.........................................................44
4.2.1 理論推導...............................................................46
4.2.2 解析能力...............................................................50
4.2.2.1 軸向解析度...........................................................50
4.2.2.2 橫向解析度...........................................................52
4.3 全反射式光延遲線.........................................................53
4.3.1 光延遲線簡介...........................................................53
4.3.2 光延遲線系統...........................................................54
4.3.3 全反射式新型光延遲線系統...............................................57
4.3.4 演算法.................................................................59
4.3.5 模擬分析...............................................................65
第五章 全反射式光延遲線系統架設與量測結果....................................67
5.1 前言.....................................................................67
5.2 儀器介紹.................................................................67
5.2.1 超亮二極體.............................................................67
5.2.2 掃描鏡.................................................................68
5.2.3 光偵測器...............................................................68
5.2.4 帶通濾波器.............................................................69
5.3 量測系統架設.............................................................70
5.4 資料處理.................................................................72
5.4.1 訊號分析程式...........................................................72
5.4.2 掃描程式...............................................................74
5.4.3 繪圖程式...............................................................75
5.5 實驗結果分析.............................................................75
5.5.1 單頻光源測試...........................................................75
5.5.2 寬頻光源測試...........................................................77
5.5.3 蓋玻片量測.............................................................79
第六章 結論與未來展望........................................................82
6.1 成果與結論...............................................................82
6.2 未來展望.................................................................83
第七章 參考文獻..............................................................84
圖索引
第一章 緒論...................................................................1
圖1.1、各類螢光蛋白吸收與放射光譜圖。.........................................3
圖1.2、綠螢光蛋白 (GFP) 結構圖。..............................................4
圖1.3、綠螢光蛋白之發光機團與結構式。.........................................5
第二章 變溫光譜量測系統.......................................................7
圖2.1、席貝可效應。...........................................................9
圖2.2、基本熱電偶電路。......................................................10
圖2.3、熱電致冷片剖面圖。....................................................11
圖2.4、熱電致冷片工作原理示意圖。............................................11
圖2.5、溫度控制器。(b) 外觀；(a)、(c) 內部構造圖。...........................15
圖2.6、接點示意圖。..........................................................15
圖2.7、串列轉換器外觀。......................................................17
圖2.8、連接方式簡圖。........................................................18
圖2.9、多層熱電致冷片。......................................................19
圖2.10、USB2000 光譜分析儀內部構造。.........................................20
第三章 變溫光譜量測系統架設與量測結果........................................22
圖3.1、變溫光譜量測平台。....................................................22
圖3.2、基因轉殖方法。........................................................23
圖3.3、經由基因轉殖後之擬尺蠖幼蟲。..........................................23
圖3.4、變溫螢光量測系統架設圖。..............................................24
圖3.5、RLCPro 控制面板。(a) 操控面板，(b) 參數設定面板。.....................26
圖3.6、不同溫區下輸出訊號之字串。(a)-25℃，(b)-5℃，(c)+5℃與(d) +25℃.......28
圖3.7、溫度控制程式操作介面圖。..............................................29
圖3.8、光譜分析程式操作介面圖................................................31
圖3.9、螢光發光原理示意圖。..................................................32
圖3.10、不同溫度下螢光光譜歸一化分佈情形。...................................34
圖3.11、螢光光譜經歸一化分佈情形。...........................................35
圖3.12、-10℃、-2℃、2℃和10℃ 螢光光譜分佈圖。..............................36
圖3.13、峰值強度與溫度變化關係圖。...........................................37
圖3.14、Jablonski 螢光能階示意圖。...........................................38
第四章 全反射式光延遲線系統..................................................41
圖4.1、光干涉示意圖。(a) 建設性干涉，(b) 破壞性干涉。........................41
圖4.2、麥克森干涉儀。........................................................42
圖4.3、模擬單頻光之干涉訊號。................................................43
圖4.4、OCT 基本架構。........................................................44
圖4.5、反射鏡之干涉與解調後訊號。(a) 模擬之干涉訊號，(b) 解調後訊號。........49
圖4.6、多層界面之干涉與解調後訊號。(a) 模擬之干涉訊號，(b) 解調後訊號。......50
圖4.7、高斯光束之傳遞。......................................................52
圖 4.8、快速掃描光延遲線 (RSODL) 系統。......................................56
圖4.9、光延遲線系統。(a) 結構圖，(b)、(c) 實體圖。...........................58
圖4.10、全反射式光延遲線系統之示意圖。(a) 俯視圖，(b) 側視圖。...............59
圖4.11、全反射式光延遲線系統數學符號示意圖。(a) 俯視圖，(b) 側視圖。.........60
圖4.12、全反射式光延遲線系統光路徑示意圖。...................................60
圖4.13、全反射式光延遲線系統光路徑說明圖-1。.................................64
圖4.14、全反射式光延遲線系統光路徑說明圖-2。.................................65
圖4.15、掃描鏡於不同角度下光程變化關係圖。...................................66
第五章 全反射式光延遲線系統架設與量測結果....................................67
圖5.1、超亮二極體 (superlumdiodes，SLD) 光源頻譜。...........................67
圖5.2、M3ST’掃描鏡針對不同波長入射光之反射率。..............................68
圖5.3、P-N 二極體空乏區生成示意圖。..........................................69
圖5.4、通濾波器示意圖。(a)高通濾波器，(b)低通濾波器。........................70
圖5.5、帶通濾波器示意圖......................................................70
圖5.6、全反射式光延遲線系統架設。............................................71
圖5.7、干涉訊號校正程式介面圖。..............................................73
圖5.8、掃描程式介面圖。......................................................74
圖5.9、繪圖程式介面圖。......................................................75
圖5.10、掃描鏡於不同角度下單頻光源光強度變化圖。(a) 參考光束強度變化，(b)干涉訊號強度變化。.................................................................76
圖5.11、掃描鏡於不同角度下光程變化之實驗值與理論值比較圖。...................77
圖5.12、掃描鏡於不同角度下寬頻光源光強度變化圖。(a) 反射鏡干涉訊號，(b)一片載玻片干涉訊號，(a) 二片載玻片干涉訊號。.........................................79
圖5.13、蓋玻片干涉訊號。.....................................................80
圖5.14、蓋玻片干涉訊號經由Hiber transform 解調。.............................80
圖5.15、一片蓋玻片二維影像。.................................................81
圖5.16、多片蓋玻片二維影像。.................................................81
表索引
第一章 緒論...................................................................1
表1.1、各式造影技術比較。.....................................................2
第二章 變溫光譜量測系統.......................................................7
表2.1、接觸式與非接觸式溫度感測器比較。.......................................8
表2.2、不同形式之熱電偶可量測溫度範圍。......................................16
表2.3、PC/RS232 與 ICM4 連接方法。...........................................18
表2.4、T48 與 ICM4 連接方法。................................................18
表2.5、多層熱電致冷片規格。..................................................19
表2.6、USB2000 光譜分析儀內部構造說明。......................................21
第四章 全反射式新型光延遲線系統..............................................41
表4.1、常見之光延遲線系統。..................................................54

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