生物醫學工程之發展日益健全，而分子影像為其中相當重要的環節之一，為了解決傳統顯微鏡只能擷取樣本輪廓資訊的限制，我們將超光譜影像技術融合至雙光子螢光顯微系統中，研發出雙光子螢光超光譜顯微術，而此技術結合了雙光子螢光激發機制之優點，具有光學切片、高縱向解析度與較低光破壞等特性，以及超光譜影像能獲得光譜維度資訊的優點，希望可將此系統用於活體研究中，並藉由所得到之空間與光譜資訊以增加顯微技術之應用層面，使得研究人員對於實驗樣本有更多了解，同時減少單單依賴樣本輪廓進行判定所造成的失誤機率。
本論文欲使用先前開發出之平行接收雙光子螢光超光譜顯微術進行實驗，並測試系統效率與解析度分析。除此之外，本論文最主要目的是試著將此套顯微系統實際應用至生物研究中，欲解決傳統顯微技術對於具有多重螢光來源樣本之限制，以光譜線性分離法提升光譜重疊現象的判斷能力。在生物研究中將分為兩部分進行，第一部分以所培養之動物細胞進行多螢光標定作為實驗樣本，進行光譜線性分離法之驗證，第二部分則利用老鼠皮膚組織進行實驗，將系統與分析法實際應用於醫療研究，試著將可能影響醫生影像判斷之二倍頻訊號與自發螢光濾除以驗證系統效能。

The development of biomedical engineering has gradually improved, and molecular imaging has been one of its most important techniques. To solve the limitation of a conventional microscope that merely detects the morphology of samples, we’ve combined hyperspectral imaging with two photon fluorescence microscopy and designed the setup of two photon fluorescence hyperspectral microscopy. This neoteric technique includes the advantages of both microscopies, providing deeper depth penetration, less photo damage to sample, higher axial resolution, and the addition of receiving spectral information of the targeted sample simultaneously. With these properties, it’s more suitable for biomedical research in vivo, it also provides more information of the samples to researchers, and reduces the misjudgment probabilities of researches that are dependent on the morphology.
Using the previously developed system, a non-de-scanned two-photon fluorescence hyperspectral microscope with parallel recording, experiments of cells and sectioning skin samples were carried out to test the efficiency of the system. Furthermore, the major aim is to try to actually apply this microscopy to biological researches and to overcome the limitation of multiple fluorescence labeling samples. Multispectral images might generate crosstalk and increase the difficulty of fluorescence discrimination. Therefore, we applied the applicably spectral analysis, linear unmixing, to improve the spectral discriminating ability.
In the biological research, there were two parts of experiments. In the first part, cultured cell lines were labeled with multiple fluorescence dyes and the hyperspectral images of these cells were analyzed to show the ability and correctness of linear unmixing. In the second part, mouse skin tissues with hair follicles being fluorescently labeled were used as samples. According to the experimental results and the spectral analyses, the fluorescence and second harmonic generation signals was easily separated. The unexpected signal sources, second harmonic generation, can be removed to avoid influencing doctor’s diagnoses. The two-photon hyperspectral imaging combined with linear unmixing has shown its ability to biomedical fields.

1. L. W. Jennifer, and J. H. Naomi, "Engineered Nanomaterials for Biophotonics Applications: Improving Sensing, Imaging, and Therapeutics," Annual Review of Biomedical Engineering, Vol 5, pp. 285-292, 2003.
2. P. Bonato, "Wearable Sensors/Systems and Their Impact on Biomedical Engineering," IEEE Engineering in Medicine and Biology Magazine, Vol 22, pp. 18-20, 2003.
3. C. H. Thomas, J. H. Collier, C. S. Sfeir, and K. E. Healy, "Engineering gene expression and protein synthesis by modulation of nuclear shape," PNAS, Vol 99, pp. 1972-1977, 2002.
4. L. J. T. Daniel, K. C. Antony, C. Julie, and T. Andrew, "Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging," Annals of Biomedical Engineering, Vol 34, pp. 23-38, 2006.
5. J. Enderle, and J. Bronzino, Introduction to biomedical engineering, Academic Press, 2011.
6. S. A. Wickline, and G. M. Lanza, "Nanotechnology for Molecular Imaging and Targeted Therapy," Circulation, Vol 107, pp. 1092-1095, 2003.
7. T. F. Massoud, and S. S. Gambhir, "Molecular imaging in living subjects: seeing fundamental biological processes in a new light," Genes & Development, Vol 17, pp. 545-580, 2003.
8. http://en.wikipedia.org/wiki/Wilhelm_R%C3%B6ntgen
9. R. Fitzgerald, "Phase-Sensitive X-Ray Imaging," Physics Today, Vol 53, pp. 23-26, 2000.
10. D. J. Brenner, and E. J. Hall, "Computed Tomography — An Increasing Source of Radiation Exposure," NEJM , Vol 357, pp. 2277-2284, 2007.
11. S. Ogawa, D. W. Tank, R. Menon, J. M. Ellermann, S. G. Kim, H. Merkle, and K. Ugurbil, "Intrinsic signal changes accompanying sensory stimulation: Functional brain mapping with magnetic resonance imaging," PNAS, Vol 89, pp. 5951-5955, 1992.
12. P. T. Callaghan, Principles of Nuclear Magnetic Resonance Microscopy, OUP, 1994.
13. http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance#Medicine
14. G. Turrell, and J. Corset, Raman Microscopy: Developments and Applications, Academic Press, 1996.
15. D. Zhang, P. Wang, M. N. Slipchenko, and J. X. Cheng, "Fast Vibrational Imaging of Single Cells and Tissues by Stimulated Raman Scattering Microscopy," J. Am. Chem. Soc., Vol 47, pp. 2282−2290, 2014.
16. J. W. Lichtman, and J. A. Conchello, "Fluorescence microscopy," Nature Methods, Vol 2, pp. 910–919, 2005.
17. P. Bastiaens, and A. Squire, "Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell," Trends Cell Biol., Vol 9, pp. 48–52, 1999.
18. V. Studer, J. Bobin, M. Chahid, H. S. Mousavi, E. Candes, and M. Dahan, "Compressive fluorescence microscopy for biological and hyperspectral imaging," PNAS, Vol 109, pp. E1679–E1687, 2011.
19. G. Placzek, The Rayleigh and Raman scattering, NTIS, 1962.
20. A. Zumbusch, G. R. Holtom, and X. S. Xie " Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering," APS, Vol 82, pp. 4142-4145, 1999.
21. W. M. Tolles, J. W. Nibler, J. R. Mcdonald, and A. B. Harvey, "A Review of the Theory and Application of Coherent Anti-Stokes Raman Spectroscopy (CARS)," Applied Spectroscopy, Vol 31, pp. 253–271, 1977.
22. P. R. Carey, "Raman Crystallography and Other Biochemical Applications of Raman Microscopy: Developments and Applications," Annu. Rev. Phys. Chem., Vol 57, pp. 527–554, 2006.
23. T. T. Le, S. Yue, and J. X. Cheng, "Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy," J Lipid Res., Vol 51, pp. 3091–3102, 2010.
24. A. R. Clapp, I. L. Medintz, and H. Mattoussi, "Förster Resonance Energy Transfer Investigations Using Quantum-Dot Fluorophores," Chemphyschem., Vol 7, pp. 47-57, 2006.
25. S. Ganesan, S. M. Ameer-beg, T. T. C. Ng, B. Vojnovic, and F. S. Wouters, "A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP," PNAS, Vol 103, pp. 4089–4094, 2006.
26. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, 1999.
27. E. B. Munster, and T. W. J. Gadella, "Fluorescence Lifetime Imaging Microscopy (FLIM)," Microscopy Techniques, Vol 95, pp. 143–175, 2005.
28. W. Denk, J. H. Strickler and W. W. Webb," Two-photon laser scanning fluorescence microscopy," Science, Vol 248, pp. 73–76, 1990.
29. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb," Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," PNAS, Vol 100, pp. 7075-7080, 2003.
30. http://www.evrogen.com/products/basicFPs.shtml
31. R. A. Schultz, T. Nielsen, J. R. Zavaleta, R. Ruch, R. Wyatt, and H. R. Garner, "Hyperspectral Imaging: A Novel Approach for Microscopic Analysis," Cytometry., Vol 43, pp. 239–247, 2001.
32. B. Kraus, M. Ziegler, and H. Wolff," Linear fluorescence unmixing in cell biological research," Modern Research and Educational Topics in Microscopy, pp. 863-872, 2007.
33. M. Buffington, and M. Gates," Advanced Imaging Techniques II: Using a Compound Microscope for Photographing Point-Mount Specimens," American Entomologist, Vol 54, pp. 222-224, 2008.
34. J. R. Swedlow, and M. Platani," Live Cell Imaging Using Wide-Field Microscopy and Deconvolution," Cell Structure and Function, Vol 27, pp. 335-341, 2002.
35. T. Horio, and H. Hotani," Visualization of the dynamic instability of individual microtubules by dark-field microscopy," Nature, Vol 321, pp. 605-607, 1986.
36. F. Zernike, "Phase Contrast, a New Method for the Microscopic Observation of Transparent Objects," Physica, Vol 9, pp. 686-698, 1942.
37. P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland," Two-photon excitation fluorescence microscopy," Annual Review of Biomedical Engineering, Vol.2, pp. 399-429, 2000.
38. K. D. Dorkenoo, H. Bulou, G. Taupier, A. Boeglin, and E. Sungur," Monitoring the Contractile Properties of Optically Patterned Liquid Crystal Based Elastomers," Advanced Elastomers - Technology, Properties and Applications, Chapter 2 , pp. 37-60, 2012.
39. T. Wilson," Confocal microscopy," Transactions of the American Microscopical Society, Vol 110, pp. 194-196, 1991.
40. S. Kim, T. Y. Ohulchanskyy, H. E. Pudavar, R. K. Pandey, and P. N. Prasad, " Organically Modified Silica Nanoparticles Co-encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy," J. Am. Chem. Soc., Vol 129, pp. 2669-2675, 2007.
41. http://www-psych.stanford.edu/~lera/psych115s/notes/lecture5/figures.html
42. E. K. Hege, D. O'Connell, W. Johnson, S. Basty, and E. L. Dereniak," Hyperspectral imaging for astronomy and space surviellance," Proc. SPIE, Vol 5159, pp. 380-391, 2003.
43. A. F. H. Goetz," Three decades of hyperspectral remote sensing of the Earth: A personal view," Remote Sensing of Environment, Vol 113, pp. S5-S16, 2009.
44. Z. P. Lee, K. L. Carder, C. D. Mobley, R. G. Steward, and J. S. Patch, "Hyperspectral remote sensing for shallow waters: 2. Deriving bottom depths and water properties by optimization, " Applied Optics, Vol 38 , pp. 3831-3843, 1999.
45. R. Zhang, Y. Ying, X. Rao, and J. Li," Quality and Safety Assessment of Food and Agricultural Products by Hyperspectral Fluorescence Imaging," Journal of the Science of Food and Agriculture, Vol 92, pp. 2397-2408, 2012.
46. N. Fairbairn, A. Christofidou, A. G. Kanaras, T. A. Newman, and O. L. Muskens," Hyperspectral darkfield microscopy of single hollow gold anoparticles for biomedical applications," Phys. Chem. Chem. Phys., Vol 15, pp. 4163-4168, 2013.
47. Y. Hiraoka, T. Shimi, and T. Haraguchi, "Multispectral imaging fluorescence microscopy for living cells," Cell Structure and Function, Vol 27, pp. 367-374, 2002.
48. D. M. Bonner, Further Developments in Scientific Optical Imaging, Springer, 2000.
49. N. Gat, "Imaging Spectroscopy Using Tunable Filters: A Review," Proc. SPIE, Vol 4056, pp. 50-64, 2000.
50. M. B. Sinclair, D. M. Haaland, J. A. Timlin, and H. D. T. Jones," Hyperspectral confocal microscope," Applied Optics, Vol 45, pp. 6283-6291, 2006.
51. F. R. Bertani, L. Ferrari, V. Mussi, E. Botti, A. Costanzo, and S. Selci," Living Matter Observations with a Novel Hyperspectral Supercontinuum Confocal Microscope for VIS to Near-IR Reflectance Spectroscopy," Sensors, Vol 13, pp. 14523-14542, 2013.
52. P. De Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. R. Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. J. Lever, P. Anand, M. A. A. Neil, and P. M. W. French, " Rapid hyperspectral fluorescence lifetime imaging," Microscopy Research and Technique, Vol 70, pp. 481-484, 2007.
53. A. F. Palonpon, J. Ando, H. Yamakoshi, K. Dodo, M. Sodeoka, S. Kawata, and K. Fujita, "Raman and SERS microscopy for molecular imaging of live cells," Nature Protocols, Vol 8, pp. 677–692, 2013.
54. L. Qingli, H. Xiaofu, W. Yiting, L. Hongying, X. Dongrong, and G. Fangmin, " Review of spectral imaging technology in biomedical engineering: achievements and challenges," Journal of Biomedical Optics, Vol 18, pp. 1-28, 2013.
55. M. E. Dickinson, G. Bearman, S. Tille, R. Lansford, and S. E. Fraser," Multi-Spectral Imaging and Linear Unmixing Add a Whole New Dimension to Laser Scanning Fluorescence Microscopy," BioTechniques, Vol 31, pp. 1272-1278, 2001.
56. R. Bro, and S. D. Jong," A Fast Non-negativity-constrained Least Squares Algorithm," Journal of Chemometrics, Vol 11, pp. 393-401, 1997.
57. X. Michalet, and S. Weiss," Using photon statistics to boost microscopy resolution," PNAS, Vol 103, pp. 4797-4798, 2006.
58. C. Y. Dong, K. Koenig, and P. So," Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium Using photon statistics to boost microscopy resolution," Journal of Biomedical Optics, Vol 8, pp. 450–459, 2003.
59. https://www.lifetechnologies.com/tw/zt/home/life-science/cell-analysis/labeling-chemi
stry/fluorescence-spectraviewer.html?ICID=svtool&UID=1142lip
60. M. Drobizhev, K. S. Makarov, G. Stobrawa, S. E. Tillo, T. E. Hughes, and A. Rebane," Two-photon absorption properties of fluorescent proteins," Nature Methods, Vol 8, pp. 393-399, 2011.
61. M. A. Albota, C. Xu, and W. W. Webb," Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm," Applied Optics, Vol 37, pp. 7352-7356, 1998.
62. F. Bestvater, E. Spiess, G. Stobrawa, M. Hacker, T. Feurer, T. Porwol, U. Berchner-Pfannschmidt, C. Wotzlaw, and H. Acker," Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging," Journal of Microscopy, Vol 208, pp. 108-115, 2002.
63. W. R. Zipfel, R. M. Williams, R. Christie, A. Y. Nikitin, B. T. Hyman, and W. W. Webb," Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," PNAS, Vol 100, pp. 7075-7080, 2003.
64. https://en.wikipedia.org/wiki/Quasi-phase-matching
65. R. LaComb, O. Nadiarnykh, S. S. Townsend, and P. J. Campagnola, " Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology," Optics Communications, Vol 281, pp. 1823-1832, 2008.