臺灣博碩士論文加值系統

雙光子顯微術因其優異的特性—包含自發性的光學斷層、較低的光漂白與光損害、較佳的影像對比度以及較長的穿透距離，近年來在生醫領域上，吸引不少學者的高度興趣。在此研究中，藉由觀測以780 nm波長激發、來自巨噬細胞 J774A.1的NAD(P)H自體螢光，得以瞭解螢光強度與細胞的代謝作用之關係。到目前為止，不僅證實了我們所觀測到的訊號的確來自細胞的代謝作用，同時指出LDL 的氧化對細胞所造成的傷害大於ox-LDL。
我們亦觀察了一些較簡單的系統以作為實驗模型，如蛋白質晶體及以YOYO-1染色的DNA纖維。將蛋白質晶體所發出的螢光，以極化分光鏡分為兩垂直方向之光束，並比較它們對雷射極化角度的強度變化，即可判斷我們所觀察的是否為單一晶體形式。此外，亦觀察到來自DNA纖維、因其結構之不對稱性而產生的二倍頻訊號。更進一步地，藉由分析螢光強度對雷射極化之，我們通常能夠分辨DNA纖維是較為延展伸長的，或較為聚集在一起。以上實驗說明了未來以雙光子顯微鏡分析染色質結構的可能性。
實驗結果闡明了雙光子顯微鏡是一有潛力的工具，可用於研究細胞之代謝作用，或可作為評價並量化生物分子之非線性光學反應之儀器。

Two-photon microscopy has attracted high interests on investigation of biomedicine recently, because of its several outstanding characteristics, including intrinsic optical section, lower photo-bleaching and photo damaging, higher contrast and longer penetrating distance. In this research, NAD(P)H autofluorescence of macrophages J774A.1 was observed by 780nm two-photon excitation, to understand the relationship between fluorescence intensity and cells’ metabolisms. So far, we have not only demonstrated that what we observed was from metabolism of cells, but also indicated that LDL oxidation causes more damage than ox-LDL does on macrophages.
Simpler systems used as models, such as protein crystals and YOYO-1 stained DNA fibers, were also observed. By separating fluorescence emitted from a protein crystal into two perpendicular polarization directions, and comparing their intensity response dependence to laser polarization, one can tell if what observed is a pure crystalline form. Besides, slight intrinsic Second Harmonic Generation (SHG) signal were collected from DNA fibers, due to the chirality of their structure. Moreover, we can usually tell DNA fibers are more extended or form aggregates, by analyzing the polarization dependence of fluorescence intensity. The above experiments indicate the possibility to analyze chromatin structure using a two-photon microscope in the future.
The experimental results bring out that two-photon microscopy is a potential tool to investigate metabolism of cells, and it can also be used as an adequate instrument to evaluate and to quantify the nonlinear responses from bio-molecules.

CHAPTER 1— INTRODUCTION 1
1.1 Review and Basic Principle of Optical Microscopy 1
1.2 Brief Review of Two-Photon Microscopy 5
1.2.1 History of Second Harmonic Generation Microscopy 5
1.2.2 History of Two-Photon Excitation Fluorescence (TPEF) Microscopy 6
1.3 Prospect of Two-Photon Microscopy 7
CHAPTER 2— PRINCIPLES OF TWO-PHOTON EXCITATION MICROSCOPY 10
2.1 Physics basis of TPEF microscopy 10
2.2 Optical Properties of TPEF microscopy 12
2.3 Physics basis of SHG microscopy 14
2.4 Optical Properties of SHG microscopy 16
2.5 Spectral Properties of TPEF and SHG 17
2.6 Intensity of TPEF and SHG 19
2.7 Description of a TPE microsocope Set Up 21
2.7.1 Light source 21
2.7.2 Scanning System and Beam Expander 22
2.7.3 Detecting System 23
CHAPTER 3—AUTOFLUORESCENCE AND METABOLISM OF MACROPHAGES 25
3.1 Introduction 25
3.2 TPE Microscope System in Nano Center, National Taiwan University 27
3.3 Materials and Methods 29
3.3.1 Cell Culture and Samples Preparation 29
3.3.2 Coating Condition of Culture Surfaces 30
3.3.3 Intensity Dependence of Laser Power and Glucose Concentration 30
3.3.4 Time Sequence Observation 30
3.3.5 Influence of Potassium Ions on Macrophages 30
3.3.6 Influences of Nature and Oxidized Low-Density Lipoprotein (LDL and ox-LDL) on Macrophages 31
3.4 Results and Discussion 31
3.4.1 Coating Condition of Culture Surface 31
3.4.2 Intensity Dependence of Laser Power and Glucose Concentration 33
3.4.3 Time Sequence Observation 35
3.4.4 Influence of Potassium Ions on Macrophages 36
3.4.5 Influences of Nature and Oxidized Low-Density Lipoprotein (LDL and ox-LDL) on Macrophages 37
CHAPTER 4— INTRINSIC NONLINEAR RESPONSE FROM BIO-MOLECULAR MODELS 41
4.1 Introduction 41
4.1.1 Protein Crystals 41
4.1.2 DNA Fibers and Chromatin 43
4.2 TPE Microscope System in Ecole Normale Supérieure (ENS) Cachan, France 44
4.2.1 Detection System 44
4.2.2 Scanning Method 45
4.2.3 Transmission Direction Detection Set Up 46
4.3 Materials and Methods 47
4.3.1 Calibration of Transmission Direction Detection 47
4.3.2 Observation of Protein Crystals 47
4.3.3 Observation of DNA Combing Fibers 48
4.3.3.1 Demonstration of SHG from DNA Combing Fibers 48
4.3.3.2 Analysis of TPEF from DNA Combing Fibers 48
4.3.3.3 Comparison of DNA Combing Fibers by TPEF and DIC Microscopes 49
4.3.4 Observation of DNA Manual Fibers 49
4.4 Results and Discussion 49
4.4.1 Calibration of Transmission Direction Detection 49
4.4.2 Observation of Protein Crystals 51
4.4.3 Observation of DNA Combing Fibers 52
4.4.3.1 Demonstration of SHG from DNA Combing Fibers 52
4.4.3.2 Analysis of TPEF from DNA Combing Fibers 53
4.4.3.3 Analysis of SHG from DNA Combing Fibers 60
4.4.3.4 Comparison of DNA Combing Fibers by TPEF and DIC Microscopes 62
4.4.4 Observation of DNA Manual Fibers 65
CHAPTER 5— CONCLUSION AND PROSPECTIVE 68
REFERENCE 71
APPENDIX 75

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