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研究生:陳怡哲
研究生(外文):Chen, Yi-Che
論文名稱:FocusClear®處理過後之透明生物顯微鏡影像的三維反疊積
論文名稱(外文):3D Deconvolution of FocusClear® Processed Transparent Biological Microscopic Images
指導教授:陳永昌陳永昌引用關係
指導教授(外文):Chen, Yung-Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:電機工程學系
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:62
中文關鍵詞:反疊積顯微鏡三維
外文關鍵詞:DeconvolutionMicroscopy3D
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自從顯微鏡發明以來,一直是醫學界和生物科技界重要的設備之一。對醫學界而言,能夠利用顯微鏡觀察病人的生理組織,有助於醫生更準確的判斷病情,減少錯誤判斷率。對生物科技而言,顯微鏡使能見微知著,讓科學家了解微觀世界的脈動,才能更詳細了解生物的運作,對現代科技的發展幫助很大。 顯微鏡的種類很多,解析度也各自不同,但最終目的不外乎希望能夠看到更清楚更細節的影像,當然價錢也是其中考量。本研究的目的是利用影像演算法以期增加顯微鏡的三維影像解析度。更進一步希望研發出低價卻高解析度的顯微鏡。 影響解析度的原因有很多,擷取影像時的背景光、感光器本身的雜訊、相機的畫素、甚至樣本自身來自不同焦距平面的模糊影像都會決定最後影像的品質。這份論文主要是針對共軛焦螢光顯微鏡,擷取生物組織的三維變焦影像(一系列二維影像堆疊),經由影像處理的三維反疊積(Deconvolution)後產生高解析度的三維生物影像。 在這整個演算法中,本論文著重於生物細胞組織經由光學顯微鏡擷取的影像,由於鏡頭的不完美而造成的模糊變型現象,利用盲反疊積(Blind Deconvolution)估計出的點擴散方程式(Point Spread Function)實行於整體三維影像中完成三維影像重建,減輕原本失焦所造成的模糊和偏光所產生的形變問題。
Since the invention of microscope, it has been one of the most important equipments in medical and biological technology communities. For medical applications, doctors investigate patients’tissues through microscopes, which help them better diagnose disease and decrease possibility of errors. For biologists, microscopes help them recognize things through micro world, understand how living creature works in detail. In summary, microscopy has great advantage to nowaday technology development. There are many kinds of microscopes, each with different resolutions, but eventually they are all in essence developed to observe images more clearly with more details. Price, of course, is one of the concerns. This research is intended to increase the resolution of 3D microscopy images by image processing algorithms. The factors affecting the captured resolution can be complex, including the backlight of the scene, noises of the sensor, pixel rate of the camera, and even the sample itself affects the final resolution due to defocus. This thesis is mainly focused on confocal fluorescence microscopy, capturing defocused 3D images (a series of 2D image stack), and then further image processing on the 3D image with 3D deconvolutionto to produce the 3D super-resolution sample. In the whole algorithm, this study focuses on processing the images of biological tissue from light microscopes. Since the imperfection of lenses results in blurriness and distortion of the original sample, Blind Deconvolution will be employed to estimate Point Spread Function on the raw 3D data for subsequent 3D reconstruction to alleviate these problems.
Abstract.....................ii
Table of Contents............iii
List of Figures..............vi
List of Tables...............ix

Chapter 1 Introduction.................1
1.1 Overview of 3D Microscopy....1
1.2 Motivation...................2
1.3 Thesis Organization..........3
Chapter 2 Related Works................4
2.1 3D Model.....................4
2.1.1 Image Formation..............4
2.1.2 Convolution Model............6
2.2 3D Reconstruction............7
2.2.1 Deconvolution................8
2.2.2 Blind Deconvolution..........9
Chapter 3 Microscopy...................10
3.1 Transmitted Light Bright-Field Microscopy...10
3.1.1 Transmitted Light............11
3.1.2 Bright Field.................12
3.1.3 Image Formation..............13
3.2 Fluorescent Microscopy.......13
3.2.1 Fluorescence.................13
3.2.2 Microscope Structure.........14
3.2.3 Image Formation..............14
3.2.4 Wide Field...................15
3.2.5 Advantages and Disadvantages.15
3.3 Confocal Microscopy..........16
3.3.1 Pinhole......................16
3.3.2 Raster Scan..................17
3.3.3 Photomultiplier..............17
3.3.4 Cone-like Illumination.......18
3.3.5 Conjugate Points.............18
3.3.6 Advantages and Disadvantages.19
Chapter 4 Deconvoluiton................20
4.1 Flowchart....................21
4.2 3D Image Stack...............21
4.3 User Cropped Region..........23
4.4 Blind Deconvolution..........24
4.4.1 Blind Deconvolution Flowchart...24
4.4.2 Initial Guess................26
4.4.3 Mathematical Model...........26
4.4.4 Point Spread Function Constraints...31
4.5 Point Spread Function........35
4.6 Deconvolution................35
4.7 Deconvoluted 3D Image Stack..35
Chapter 5 3D Super-Resolution..........36
5.1 Motivation...................36
5.2 System Flowchart.............38
5.3 Rotational Stage.............39
5.4 Image Stacks with Different Angles...40
5.5 Deconvolution................41
5.6 Registration.................42
5.7 Interpolation................43
Chapter 6 Experimental Results and Discussions...44
6.1 Experiment Setup.............44
6.2 Experimental Results.........48
6.2.1 FWHM (0.3, 0.6)..............50
6.2.2 FWHM (0.7, 1.4)..............53
6.2.3 FWHM (1.0, 2.0)..............56
6.3 Discussions..................60
Chapter 7 Conclusions and Future Works.61
7.1 Conclusions..................61
7.2 Future works.................62
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[2]T. F. Brooks and W. M. Humphreys, “A deconvolution approach for the mapping of acoustic sources (DAMAS) determined from phased microphone arrays,” Journal of Sound and Vibration, Volume 294, Issues 4-5, pp. 856-879, 2006.

[3]T.J. Holmes, “Acceleration of maximum-likelihood image restoration for fluorescence microscopy and other noncoherent imagery,” Journal of Optical Society of America A, Volume 8, Issue 6, pp. 893-907, 1991.

[4]T.J. Holmes and N. J. O’connor, “Blind deconvolution of 3D transmitted light brightfield micrographs,” Journal of Microscopy, Volume 200, Issue 2, pp. 114-127, 2000.

[5]T.J. Holmes, “Blind deconvolution of quantum-limited incoherent imagery: maximum-likelihood approach,” Journal of Optical Society of America A, Volume 9, Issue 7, pp. 1052-1061, 1992.

[6]D. Semwogerere and E. R. Weeks, “Confocal Microscopy,” Encyclopedia of Biomaterials and Biomedical Engineering (4th ed.), pp. 705-714, 2005.

[7]S. Kimura and C. Munakata, “Dependence of 3-D optical transfer functions on the pinhole radius in a fluorescent confocal optical microscope,” Applied Optics, Volume 29, Issue 20, pp. 3007-3011, 1990.

[8]M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Optics Letters, Volume 22, Issue 24, pp. 1905-1907, 1997.

[9]A. Erhardt, G. Zinser, D. Komitowski, and J. Bille, “Reconstructing 3-D light-microscopic images by digital image processing,” Applied Optics, Volume 24, Issue 2, pp. 194-200, 1985.

[10]N. Streibl, “Three-dimensional imaging by a microscope,” Journal of Optical Society of America A, Volume 2, Issue 2, pp. 121-127, 1985.

[11]http://www.martinmicroscope.com/MicroscopePages/TLbrightfield.htm

[12]http://microscopereview.blogspot.com/

[13]T.J. Holmes, S. Bhattacharyya, J. A. Cooper, D. Hanzel, V. Krishnamurthi, W. C. Lin, B. Roysam, D. H. Szarowski, and J. N. Turner, “Light Microscopic Images Reconstructed by Maximum Likelihood Deconvolution,” Handbook of Biological Confocal Microscopy (3rd ed.), Chapter 24, pp. 389-402, 2006.

[14]http://en.wikipedia.org/wiki/Poisson_distribution

[15]http://www.optinav.com/Iterative-Deconvolve-3D.htm

[16] http://www.amira.com/
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