一種新穎的方法來顯示細胞內粒線體的動態反應

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林芬緣(Fen-Yuan Lin) 查詢紙本館藏

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光電科學研究所碩士在職專班

論文名稱

一種新穎的方法來顯示細胞內粒線體的動態反應
(Novel Approach in Displaying Dynamic Variation within Living Mitochondria)

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摘要(中)

“眼見為憑”在細胞生物學的研究領域上，依然是個真理。目前，縱然有許多數值方法已被開發出來，作為擷取訊息的有利工具，但多數粒線體的相關研究，則仍然仰賴目視觀察。相對地，利用影像處理來同步顯示活細胞內部訊息萬變的動態反應的方法，截至目前依然附之闕如。
這篇論文報告了如何運用影像處理，來快速、同步地顯示粒線體由於氧化還原、膜電位以及運動性能等等的改變，所誘發的一連串動態反應。不同類型的運用則印證了這個方法的可適性：轉殖roGFP2-mito氧化還原綠螢光蛋白的PC12細胞，搭配全反射螢光顯微鏡；標定了TMRE螢光的PTC細胞，則使用雷射誘發螢光顯微鏡來偵測內膜電位變化的反應等等。
為了將重要訊息(經常是難以清楚地被肉眼所感測與辨識的)從背景雜訊中抽離出來，並且“顯示”粒線體內部的動態變化，一種嶄新的影像處理方法於焉產生；隨之，則運用數值方法來分析並建構不同參數之間的關連性。簡言之，這套方法不僅僅可以全面性偵測動態反應的瞬時變化(小至100 ms) ，同時也在影片中明確顯示出反應所發生的位置，這除了有益於擴展我們的“眼界”，同時也將提升細胞甚或胞器的研究，使之更臻於完善。

摘要(英)

“Seeing is believing” is true even in cell biology research. The most of researches treated of mitochondrial problems by visual inspection, whenas several mathematical calculations were developed for contributing detailed knowledge. However, in contrast, a simultaneous imaging method for soon displaying dynamic change was still left unoccupied to present significance in living cells.
This report presents a rapid and simultaneous approach with imaging/displaying dynamic changes in mitochondrial redox and potential as well. A variety of utilizations were applied in the following: Pheochromocytoma (PC12) cells expressed mitochondrial targeted redox-sensitive green fluorescent protein sensor (roGFP2-mito) under total internal reflection fluorescence microscopy (TIRFM), and primary culture of papillary thyroid carcinoma (PTC) stained with mitochondrial membrane potential indicator, tatramethylrhodamine ethyl ester (TMRE) observed by laser induced epi-fluorescence microscopy (epi-FM).
In order to extract important knowledge that may not be clearly visible to the human eye from background and displayed promptly in sub-mitochondrion in the meanwhile, a novel image processing algorithm should be required; likewise numerical analysis in constructing relevance between numerous parameters was processed getting on. As a result, may not only intensity change but happening location displayed instantly in 100 ms could be demonstrated throughout in living cells in sequences. Obviously, it will be useful to advance our research or expend our knowledge in cellular level or below.

關鍵字(中)

★ 粒線體
★ 影像處理
★ 細胞

關鍵字(英)

★ mitochondrion
★ image processing
★ cell

論文目次

List of Figure......................................................................................... vii
List of Table.................................................. ........................................ x
Table of Abbreviation ............................................................................ xi
Chapter 1 Introduction
1-1 Background .................................................................................... 1
1-2 Mitochondrion ………………………........….…...……...…….... 2
1-2-1 Functions and Morphology………………………………... 3
1-2-2 Mitochondrial Membrane Potential …………………….… 5
1-2-3 Redox (Oxidation/Reduction Reaction) …………..……… 6
1-2-4 Basic Principle of Mitochondrial Metabolism ………….… 7
1-3 Fluorescent Probes for Mitochondrial Status
1-3-1 roGFP2-mito: Indictor of Redox ………….……………… 10
1-3-2 TMRE: Measurements of Inner Membrane Potential …… 12
1-4 Equipments
1-4-1 Epi-Fluorescence Microscopy ……………………………14
1-4-2 Total Internal Reflection Fluorescence Microscopy
(TIRFM) ………………………………………….…….. 16
1-5 Cell Culture ...…….......................................................................18
1-6 Experiment Flow Chart .…………….......................................... 19
Chapter 2 Image Processing
2-1 Introduction ………..………….................................................... 21
2-2 Scheme and Steps ………..…………........................................... 22
2-3 Imaging Mitochondrial Redox State in PC12 cells ……..…….... 28
2-4 Imaging Mitochondrial Membrane Potential in PTC cells .…...... 30
vi
Chapter 3 Quantitative Analysis
3-1 Data Functions and Optimal Descriptors ……………….............. 32
3-2 Scheme and Steps ………..…………........................................... 34
Chapter 4 Result
4-1 Mitochondrial Redox in PC12 cells:
Effect of Taxol Treatment on Mitochondria
4-1-1 Importance of Image Processing ……………………….… 37
4-1-2 Knowledge from Quantitative Analysis ………………….. 42
4-2 Mitochondrial Membrane Potential in PTC cells
Effect of Clodronate on Mitochondria……………………………47
Chapter 5 Discussion
5-1 Significance of Functional Displaying and Identification ……… 53
5-2 Potential of Noise Mask ……………….…………………….…. 54
5-3 Dual-Channel Fluorescence for Functional Tracking …………... 54
5-4 Effect of Taxol Treatment on Mitochondrial Organization …….. 55
Chapter 6 Conclusion ………………..................................................... 57
Reference ................................................................................................ 58
Appendix A ……………....…………..................................................... 60
Appendix B ……………....….…........................................................... 66

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指導教授

張榮森、楊德明
(J.S. Chang、D.M. Yang)

審核日期

2007-7-21

推文