拉曼散射 (Raman scattering) 顯微術是一項生物成像技術。不同於螢光顯微鏡需倚賴螢光標記標定檢測目標，拉曼散射的訊號是來自於樣本本身的化學鍵，因此可以在無標記 (Label-free) 的情況下取得具有高特徵性的影像。
　　拉曼散射顯微術當中的同調拉曼散射 (Coherent Raman scattering, CRS) 顯微術，可以在無標記的情況下獲得活體細胞的實時 (Real time) 影像。由於同調拉曼散射需要波長能夠調變的光源，我們首次利用多重薄片展頻 (Multiple-plate continuum, MPC) 技術所產生的超寬頻 (supercontinuum) 光源，搭建一套可以同時掃同調反斯托克斯散射 (Coherent anti-Stokes Raman scattering, CARS) 顯微術以及受激拉曼散射 (stimulated Raman scattering, SRS) 顯微術這兩種同調拉曼散射的影像系統。我們比較兩種技術的差別，並擷取兩種無標記果蠅腦的同調拉曼散射影像。

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　　Raman scattering microscopy is a biological imaging technique. Unlike fluorescence microscopy, which relies on fluorescent probes to label detection targets, Raman scattering signals stem from chemical bonds in the sample itself. Thus, it can obtain high-characteristic images without labeling (Label-free).
　　Coherent Raman scattering (CRS) in Raman scattering microscopy can conduct real-time live-cell imaging. Since coherent Raman scattering requires a tunable wavelength, we utilize a supercontinuum light source generated by a multiple-plates continuum (MPC) technique to provide arbitrarily tunable wavelengths. Also, we used this light source to build a system that can measure two coherent Raman images simultaneously that are Coherent anti-Stokes Raman scattering (CARS) microscope and Stimulated Raman scattering (SRS) microscope, respectively. We compared the differences between the two techniques and collect coherent Raman scattering images of Drosophila brains

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誌謝 1
摘要 2
Abstract 3
圖形目錄 6
表格目錄 7
第一章 簡介 8
第二章 理論 11
2.1 拉曼散射 11
2.2 自發拉曼散射 12
2.3 受激拉曼散射 13
2.4 同調反斯托克斯拉曼散射 14
第三章 實驗架構 18
3.1 多重薄片展頻 (Multiple-Plate Continuum) 19
3.2 濾波 (filtering) : 泵光及斯托克斯光波長選擇 22
3.3 訊號檢測技術 : 光電二極體、光電倍增管、電子訊號放大器及電子帶通濾波器 22
3.4 影像繪製方法 25
第四章 實驗結果 28
4.1 系統量測極限檢驗 28
4.1.1 時間延遲依賴性檢驗 28
4.1.2 強度依賴性檢驗 29
4.1.3 拉曼共振頻率檢驗 31
4.1.4 濃度依賴性檢驗 32
4.2系統空間解析度檢測 36
4.3 生物體拉曼影像 37
4.3.1 二維果蠅腦影像 37
4.3.2 三維果蠅腦立體影像 39
第五章 總結與未來工作 42
5.1 總結 42
5.2 未來工作 43
參考文獻 44

1. P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annual Review of Biomedical Engineering 2, 399-429 (2000).
2. M. J. Sanderson, I. Smith, I. Parker, and M. D. Bootman, "Fluorescence microscopy," Cold Spring Harbor Protocol (2014).
3. A. Ettinger and T. Wittmann, "Fluorescence live cell imaging," Methods Cell Biology 123, 77-94 (2014).
4. D. Zhang, M. N. Slipchenko, D. E. Leaird, A. M. Weiner, and J.-X. Cheng, "Spectrally modulated stimulated Raman scattering imaging with an angle-to-wavelength pulse shaper," Optics Express 21, 13864-13874 (2013).
5. X. Zhang, M. B. J. Roeffaers, S. Basu, J. R. Daniele, D. Fu, C. W. Freudiger, G. R. Holtom, and X. S. Xie, "Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy," ChemPhysChem 13, 1054-1059 (2012).
6. D. Fu, W. Yang, and X. S. Xie, "Label-free imaging of neurotransmitter acetylcholine at neuromuscular junctions with stimulated Raman scattering," Journal of the American Chemical Society 139, 583-586 (2017).
7. L. Wei, Z. Chen, L. Shi, R. Long, A. V. Anzalone, L. Zhang, F. Hu, R. Yuste, V. W. Cornish, and W. Min, "Super-multiplex vibrational imaging," Nature 544, 465-470 (2017).
8. L. Wei and W. Min, "Electronic preresonance stimulated Raman scattering microscopy," The Journal of Physical Chemistry Letters 9, 4294-4301 (2018).
9. S. Hong, T. Chen, Y. Zhu, A. Li, Y. Huang, and X. Chen, "Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules," Angewandte Chemie International Edition 53, 5827-5831 (2014).
10. L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C.-C. Lin, M. C. Wang, and W. Min, "Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering," Natutre Methods 11, 410-412 (2014).
11. X. S. Xie. Ji-Xin Cheng, Coherent Raman Scattering Microscopy, CRC press (2012).
12. C. S . Wang, "Theory of stimulated Raman scattering," Physical Review 182, 482-494 (1969).
13. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, "Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy," Science 322, 1857-1861 (2008).
14. L. Shi, H. Xiong, Y. Shen, R. Long, L. Wei, and W. Min, "Electronic resonant stimulated Raman scattering micro-spectroscopy," The Journal of Physical Chemistry B 122, 9218-9224 (2018).
15. C.-H. Lu, Y.-J. Tsou, H.-Y. Chen, B.-H. Chen, Y.-C. Cheng, S.-D. Yang, M.-C. Chen, C.-C. Hsu, and A. H. Kung, "Generation of intense supercontinuum in condensed media," Optica 1, 400-406 (2014).
16. Y.-C. Cheng, C.-H. Lu, Y.-Y. Lin, and A. H. Kung, "Supercontinuum generation in a multi-plate medium," Optics Express 24, 7224-7231 (2016).
17. T. Nagy, P. Simon, and L. Veisz, "High-energy few-cycle pulses: post-compression techniques," Advances in Physics: X 6, 1845795 (2021).
18. T.-a. Ishibashi and H.-o. Hamaguchi, "Partially coherent anti-Stokes Raman scattering (PCARS)," Chemical Physics Letters 175, 543-547 (1990).
19. X. Wang, A. Zhang, M. Zhi, A. V. Sokolov, and G. R. Welch, "Glucose concentration measured by the hybrid coherent anti-Stokes Raman-scattering technique," Physical Review A 81, 013813 (2010).
20. D. Fu, G. Holtom, C. Freudiger, X. Zhang, and X. S. Xie, "Hyperspectral imaging with stimulated Raman scattering by chirped femtosecond lasers," The Journal of Physical Chemistry B 117, 4634-4640 (2013).
21. Y. Bi, C. Yang, Y. Chen, S. Yan, G. Yang, Y. Wu, G. Zhang, and P. Wang, "Near-resonance enhanced label-free stimulated Raman scattering microscopy with spatial resolution near 130 nm," Light: Science & Applications 7, 81 (2018).
22. P. Wang, M. N. Slipchenko, J. Mitchell, C. Yang, E. O. Potma, X. Xu, and J.-X. Cheng, "Far-field imaging of non-fluorescent species with subdiffraction resolution," Nature Photonics 7, 449-453 (2013).
23. A. Volkmer, "Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy," Journal of Physics D: Applied Physics 38, R59-R81 (2005).
24. F. M. Kamga and M. G. Sceats, "Pulse-sequenced coherent anti-Stokes Raman scattering spectroscopy: a method for suppression of the nonresonant background," Optics Letters 5, 126-128 (1980).