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研究生： 王筑萱
Chu-hsuan Wang
論文名稱： Calibrating z distance for confocal microscope
指導教授： 林耿慧
Keng-hui Lin
口試委員:
學位類別： 碩士
Master
系所名稱： 理學院 - 生物物理研究所
Graduate Institute of Biophysics
論文出版年： 2014
畢業學年度： 102
語文別： 英文
論文頁數： 66
中文關鍵詞： 共軛焦顯微鏡 、z軸校正 、折射率不匹配
外文關鍵詞： confocal microscope, z calibration, index mismatch
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    • 共軛焦顯微鏡已被廣泛運用於建立三維模型中，它藉由在不同垂直方向的位置拍攝一系列堆疊的二維影像來建立模型，但是尚未有基於影像特徵來精確測量三維的距離的方法，要藉由影像來確認垂直方向的移動距離是更為困難。而且當鏡頭的介質與樣品的浸泡液體折射率不相同時，在拍攝影像時會遇到許多問題，像是焦平面移動、像差、吸收與散射，當我們拍攝較深的影像時這些現象會造成影像的變形和亮度的降低。
    　　在這篇論文中，主要考慮折射率對三維成像的影響，並把重點放在z方向上距離的校正，我們採用一個簡單的裝置，將一個二維週期排列的螢光圖形固定在與垂直夾角60度的平面上，再藉由比較原始圖形與z軸投影圖形來計算x、y與z之間的變換係數。接著有系統地變化不同折射率的浸泡溶液並找出z的變換係數、亮度下降與折射率匹配之間的關係。並在結尾探討樣品與浸製液體折射率不匹配的問題。

    Confocal microscopes have been widely used to produce a 3D image by acquiring stacks of 2D images. Yet to have accurate 3D distance measurement between features based on images, it is necessary to calibrate pixel-to-micron conversation, which is especially difficult along the optical axis (z direction). When there is a refractive index difference between immersion medium of a sample and an objective, there are problems such as focus shift, aberrations, absorption and scattering which contribute to dramatic reduction in signal intensity as the focus gets deeper into the sample. In this thesis, I will look into the effects of refractive index on 3D imaging with the emphasis on z distance calibration. We employ a simple device which consists of a 2D periodic and fluorescent pattern mounted at 60 degrees with respect to z direction. We can easily compare the interval distance between the original and z-projected image to compute x, y, and z conversion factors and also intensity patterns. We systematic change the immersion medium index and find the relationship between z-conversion factor, intensity drop, and index-mismatch.

    Abstract i 中文摘要 ii 致謝 iii Contents iv List of Figures vi Chpater 1. Introduction 1 1.1 Three-dimensional Confocal Microscopy 1 1.1.1 The principle of a confocal microscope 1 1.1.2 Numerical aperture 3 1.1.3 Point Spread Function 3 1.2 Focus Shift 5 1.3 Spherical Aberration 7 Chpater 2. Experiments 8 2.1 The mounting device for z calibration 8 2.2 Preparation of calibration sample 10 2.3 refractive index medium preparation 13 2.4 Point Spread Function Measurement 14 Chpater 3. Results and Discussions 16 Z calibration factor 16 Z calibration by staining glass 20 Point spread function measurement and other effect of index mismatch 24 Chpater 4. Conclusion and Outlook 36 Reference 37 Appendix 39 Accuracy of refractometer 39 Fast Fourier Transform and fitting gauss curve code 40 Looking for FFT mask size code 44 Show fitting result on image code 46 Multi-direction gauss fit 47 Light path from sphere inner 53 Effective focal length estimate 56

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