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系統識別號 U0026-2209201709572100
論文名稱(中文) 藉由結構照明技術來消除時域聚焦多光子激發螢光影像之背景雜訊
論文名稱(英文) Background Noise Cancellation of Temporal Focusing-based Multiphoton Excited Fluorescence Images by Structured Illumination
校院名稱 成功大學
系所名稱(中) 工程科學系
系所名稱(英) Department of Engineering Science
學年度 106
學期 1
出版年 106
研究生(中文) 陳冠瑋
研究生(英文) Guan-Wei Chen
學號 n96041494
學位類別 碩士
語文別 中文
論文頁數 47頁
口試委員 指導教授-陳顯禎
口試委員-林俊佑
口試委員-張家源
中文關鍵字 時域聚焦多光子激發  軸向激發  結構照明  HiLo  希爾伯特轉換 
英文關鍵字 Widefield  temporal focusing multiphoton excitation microscope  axial excitation confinement  structured illumination  HiLo  Hilbert transform 
學科別分類
中文摘要 本論文主要透過模擬來討論時域聚焦雙光子激發影像藉由適當的演算法將具有二維結構圖案的結構照明影像和均勻照明影像做還原得到一張具有更好的光學切片能力之影像,並討論結構照明之弦波頻率與調製方程式的關係與對於演算法還原效果的影響以及演算法之優缺點。結構照明中使用的二維圖形可以透過解調變HiLo和變異數HiLo來做還原,但還原出來的影像品質卻因為軸向激發能力的提升而變差,因為演算法本身就是靠著兩張影像的差異性來判斷是否為焦平面,提升軸向激發侷限固然可以減少部分離焦訊號,但卻造成兩張影像之間的背景訊號差異變大,使得原來被演算法判定為背景雜訊的部分被視為焦平面上的訊號而保留了下來,導致最後的品質變差。反而是被結構照明所使用的條紋頻率對於演算法消除離焦訊號的能力扮演著至關重要的角色,當頻率越高時還原的影像品質越好。其原因可歸於系統收光時會把離焦的高頻訊號給濾除,使得該部分的訊息並沒有隨著焦平面一樣受到結構照明調變至高頻然後透過演算法分離。在藉由模擬拍攝螺旋狀分佈長方形樣本的影像中,隨著結構照明的條紋頻率由0.63 μm-1提高到1.26 μm-1,在透過演算法把離焦背景消除後,殘留的離焦訊號也由離焦距離約0.5 ~ 0.75 μm減少到0.25 ~ 0.5 μm之間就會幾乎消失。同時以柱狀分佈USAF為樣本,在條紋頻率皆為1.26 μm-1的情況下以解調變HiLo、變異數HiLo、希爾伯特轉換的方式將離焦訊號消除以及未經過任何處理之均勻照明影像與原始資料做互相關得到係數分別為0.96、0.94、0.95、0.81。比起未經處理過的影像,經過演算法處理的影像與原始資料的相似度有了明顯的提升。其中解調變HiLo以及希爾伯特轉換消除離焦訊號的能力皆很優秀,但希爾伯特轉換在雙光子的情況下需要精準的相位而解調變HiLo則是需要額外的參數調整,變異數HiLo的優點是可以使用任意圖形的結構照明影像來還原,但效果相較另外兩者較差。
英文摘要 It has close relationship between axial confinement excitation (ACE) in temporal focusing multiphoton excitation microscopy (TFMPEM) and the distribution of the beam in the back focal aperture. When the distribution of beam is more close to flat-top, the effective numerical aperture is larger and the ACE can thus be improved. Our lab combines the different orientation of sinusoidal patterns to fill the back focal aperture to achieve the ACE from 3 μm to 1.5 μm.
The utilizing of two-dimensional patterns in structured illumination can be reconstructed by single sideband demodulation based HiLo and local variation based HiLo. We find that the key to eliminate the defocused background in the algorithm is not ACE but the frequency of the stripe. The higher frequency of pattern we use, the better image we obtain. It is because that the system is the essence of low-pass filter. The farther away from the focal plane, the higher frequency signal will be rejected. Therefore, the pattern will exist only near focal plane when we illuminate high frequency stripe on sample. According to the above statement, the higher frequency of the sinusoidal pattern we used, the easier the defocused signal will be eliminated instead of being viewed as an in focused information and preserving. Moreover, the cross correlation of the image reconstructed by single sideband demodulation based HiLo, local variation based HiLo, Hilbert transform and the image without processing by algorithm between raw data is 0.96, 0.94, 0.95, and 0.81. The similarity of raw data and the image is enhance significantly after the process of algorithm, and it can be viewed as the basis for the ability of eliminating the out of focus information.
論文目次 摘要 I
Extended Abstract III
誌謝 VI
第一章 序論 4
1-1 前言 4
1-2 文獻回顧 5
1-3 研究動機與方法 7
1-4 論文架構 8
第二章 超快雷射激發和時域聚焦之機制與成像模擬 9
2-1 多光子激發 9
2-2 空間聚焦與時域聚焦 10
2-3 時域聚焦多光子激發顯微術之系統與影像模擬架構 12
2-3-1 數值孔徑與三維點擴散方程式之關係 15
2-3-2 結構照明之圖案設計 18
2-3-3 時域聚焦雙光子激發成像模擬 19
第三章 消除離焦訊號演算法之研究與分析 22
3-1理論架構 22
3-1-1 單邊帶解調變HiLo演算法 22
3-1-2 區域變異數HiLo演算法 26
3-1-3 希爾伯特轉換演算法 27
3-2 結構照明之圖案頻率對於演算法效果之影響 28
3-3 二維結構照明之影像還原 31
3-4 軸向激發對於影像還原效果之影響 35
第四章 結論與未來展望 42
參考文獻 45




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[20] A. Vaziri and C. V. Shank, “Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing,” Opt. Express 18, 19645-19655(2010).
10.1002/jbio.201600287
[4] M. A. A. Neil, R. Juˇskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905-1907 (1997).
[5] C. Ventalon and J. Mertz, “Quasi-confocal fluorescence sectioning with dynamic speckle illumination,” Opt. Lett. 30, 3350-3352 (2005).
[6] C. Ventalon and J. Mertz, “Dynamic speckle illumination microscopy with translated versus randomized speckle patterns,” Opt. Express 14, 7198-7209 (2006).
[7] D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33, 1819-1821 (2008).
[8] S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14, 030502 (2009).
[9] J. Kim and J. Mertz, “Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection,” J. Biomed. Opt. 15, 016027 (2010).
[10] D. Lim, T. N. Ford, K. K. Chu, and J. Mertz, “Optically sectioned in vivo imaging with speckle illumination HiLo microscopy,” J. Biomed. Opt. 16, 016014 (2011).
[11] T. N. Ford, D. Lim and J. Mertz, “Fast optically sectioned fluorescence HiLo endomicroscopy,” J. Biomed. Opt. 17, 021105 (2012).
[12] X. Zhou, M. Lei, D. Dan, B. Yao, J. Qian, S. Yan, Y. Yang, J. Min, T. Peng, T. Ye and G. Chen, “Double-Exposure Optical Sectioning Structured Illumination Microscopy Based on Hilbert Transform Reconstruction,” PLoS One 10, 1-9 (2015).
[13] K. Patorski, M. Trusiak, and T. Tkaczyk, “Optically-sectioned two-shot structured illumination microscopy with Hilbert-Huang processing,” Opt. Express 22, 9517-9527 (2014).
[14] Z. R. Hoffman and C. A. DiMarzio, “Single-image structured illumination using Hilbert transform demodulation,” J. Biomed. Opt. 22, 056011 (2017).
[15] K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express 4, 2396-2410 (2013).
[16] D. Kim, Ultrafast Optical Pulse Manipulation in Three Dimensional-Resolved Microscopic Imaging and Microfabrication, PH.D. Thesis, MIT (2009).
[17] 鄭力中,廣視域多光子激發顯微術之開發與應用,國立成功大學光電科學與工程研究所碩士論文 (2010)。
[18] J. W. Goodman, Introduction to Fourier Optics, Roberts & Company (2005).
[19] L. C. Cheng, C. H. Lien, Y. D. Sie, Y. Y. Hu, C. Y. Lin, F. C. Chien, C. Xu, C. Y. Dong, and S.-J. Chen, “Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device,” Biomed. Opt. Express 5, 2526-2536 (2014).
[20] A. Vaziri and C. V. Shank, “Ultrafast widefield optical sectioning microscopy by multifocal temporal focusing,” Opt. Express 18, 19645-19655(2010).
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