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系統識別號 U0026-2807201419381300
論文名稱(中文) 藉由時域聚焦多光子激發顯微術之快速光學切片與超高解析度影像
論文名稱(英文) Fast Optical Sectioning and Super-Resolution Imaging by Temporal Focusing Multiphoton Excitation Microscopy
校院名稱 成功大學
系所名稱(中) 光電科學與工程學系
系所名稱(英) Department of Photonics
學年度 102
學期 2
出版年 103
研究生(中文) 鄭力中
研究生(英文) Li-Chung Cheng
學號 L78991021
學位類別 博士
語文別 英文
論文頁數 88頁
口試委員 指導教授-陳顯禎
口試委員-邱爾德
口試委員-董成淵
口試委員-傅永貴
口試委員-鄧熙聖
中文關鍵字 多光子吸收截面  時域聚焦  多光子激發顯微技術  布朗運動  多光子激發生物影像  非線性結構性照射顯微技術 
英文關鍵字 multiphoton absorption cross section  temporal focusing, multiphoton excitation microscopy  Brownian motions  multiphoton excited bioimage  nonlinear structured-illumination microscopy 
學科別分類
中文摘要 此論文首先介紹多光子激發螢光顯微術,其中包含雙光子、三光子以及四光子吸收截面的量化實驗,利用三種不同波長的光源來測量一些常見的螢光分子以及螢光蛋白,使用的光源波長分別為800 nm,1300 nm以及1680 nm,而且所測量出來的多光子吸收截面的數量級與量子力學的計算估計彼此吻合。再來主要為發展出了藉由時域聚焦的多光子激發顯微術(temporal focusing multiphoton excitation microscopy,TFMPEM),其可以提供高速的光學縱向切片影像;而TFMPEM中主要的元件有重複率(repetition rate) 10 kHz的飛秒雷射放大器(femtosecond laser amplifier)來提供的超瞬時脈衝強度(最大的脈衝能量在90 fs的脈衝寬度下為400 μJ/pulse)以及具有電致冷器和超高光粒子接收靈敏度的電子倍增感光耦合器(electron multiplying charge couple device)。目前可以得到幀速度(frame rate)高於100 Hz且激發面積大於200 × 100 μm2大小的多光子激發影像。同時我們利用此套高速光學切片影像系統即時觀測500 nm大小螢光球的布朗運動(Brownian motion),而此時系統的側向空間解析度為小於500 nm以及軸向激發體積深度為3.5 μm。更進一步,利用這套系統觀測雞腱的二倍頻訊號(second harmonic generation)影像,提供快速且具有軸向分辨率的生物影像於生物組織上。
為了改善系統的空間解析度,我們結合了此TFMPEM與非線性結構性照射顯微術(nonlinear structured illumination microscopy,NSIM)。在此改善系統當中,TFMPEM的繞射元件以及NSIM所需產生的結構性照射圖形是同時由一個高解析度的數位微型反射鏡元件(digital micromirror device)來完成,而且所產生的照射圖形也可以被完美的成像在樣品激發面上。藉由二階的非線性結構性照射顯微技術可以顯著的改善系統的側向以及縱向解析度,分別由397 nm至168 nm (改善2.4倍)以及由2.33 μm 至 1.22 μm (1.9倍)。更進一步我們觀測細胞骨架的微小管,其直徑大小約為25 nm,其改善影像中的微小管其側向距離接近側向解析度範圍可以被明顯的分辨出來。
英文摘要 In this thesis, first we report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. Then, a temporal focusing multiphoton excitation microscopy (TFMPEM) with fast optical sectioning has been developed. Key features of this microscope are the integrations of a 10 kHz repetition rate ultrafast amplifier featuring high instantaneous peak power (maximum 400 μJ/pulse at a 90 fs pulse width) and a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled camera into a temporal focusing microscope. This configuration can produce multiphoton images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm were observed in real-time with a lateral spatial resolution of less than 0.5 μm and an axial excitation volume depth of approximately 3.5 μm. Furthermore, the second harmonic images of chicken tendons demonstrate that the TFMPEM can provide high resolution axial sectioning for biotissues.
To improve the spatial resolution, we combined the TFMPEM with nonlinear structured-illumination microscopy (NSIM). The light diffraction of the TFMPEM and the excitation patterning of NSIM can be simultaneously and exactly implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TEMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4 folds) and from 2.33 μm to 1.22 μm (1.9 folds), respectively, in full width at the half maximum. Furthermore, the three-dimensional rendering image of a cytoskeleton cell with about 25 nm microtubules is improved and some close microtubules with the distance near the lateral resolution of 168 nm can be further distinguished.
論文目次 Abstract I
摘要 III
Acknowledgements V
Table of Content VII
List of Figures IX
List of Labels XIII
Abbreviations XIV
Chapter 1 Introduction 1
1.1 Introduction 1
1.2 Motivation 4
1.3 Outline 6
Chapter 2 Multiphoton Excitation Microscopy 7
2.1 Multiphoton excitation 9
2.2 Measurement of multiphoton absorption cross section 11
2.3 4PM of GFP labeled microglia in vivo 22
Chapter 3 Temporal Focusing Multiphoton Excitation Microscopy 26
3.1 Spatial focusing and temporal focusing 26
3.2 System setup 29
3.3 System performance 31
3.4 Brownian motions of fluorescent microbeads 35
3.5 Second harmonic generation imaging 39
Chapter 4 Spatial Resolution Improvements of Temporal Focusing
Multiphoton Excitation Microscopy 44
4.1 Temporal focusing and patterning via digital
micromirror devise 44
4.2 Reject background noise by HiLo 49
4.2.1 Theory 50
4.2.2 Experimental results 52
4.3 Super-resolution imaging with nonlinear structured
illumination 54
4.3.1 Structured illumination microscopy (SIM) 55
4.3.2 Nonlinear structured illumination microscopy (NSIM) 60
4.3.3 Experimental results 65
Chapter 5 Conclusions 75
References 77
Curriculum Vitae 85
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