基于双光子共振的生物组织的非线性效应
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摘要
近年来,许多应用于生物医学领域的显微技术广泛地运用了双光子激发荧光和光学二次谐波作为信号源。但至今为止,仍然有许多飞秒激光与生物组织体相互作用的非线性效应有待探索。利用钛宝石飞秒激光器作为光源的Zeiss LSM510 META共焦扫描显微镜,我们对两种不同的生物样品进行非线性复合成像,并得到不同波长下双光子荧光和二次谐波信号强度随激发功率变化的关系曲线。基于实验结果,发现双光子荧光和二次谐波强度随飞秒激光激发功率的增长并不符合简单的平方关系。为了研究这种非线性现象的起因,提出二次谐波产生机制的一种新观点,并建立了生物组织瞬态双光子吸收的方程组,通过求解和数值计算,最终得到的理论曲线与实验结果基本一致,证明了该理论用来解释生物组织中的非线性效应确实具有其合理性。这项工作对研究光与生物组织相互作用的非线性理论提供了有价值的结果。
Recently, most nonlinear microscopic techniques used for biological study have relied on the combination of two-photon excited fluorescence (TPEF) and second harmonic generation(SHG). However, certain non-linear effects in biological tissue excited by femtosecond (fs) laser have not been fully explored. Utilizing Ti: sapphire femtosecond laser equipped with Zeiss LSM 510 META laser scanning microscopy, we obtain non-linear compound imaging from two different specimens and the relationship curve between normalized TPEF/SHG intensity and excitation power. Based on experimental results, we find that the intensity of TPEF and SHG varying with excitation power dose not exactly obey a simple quadratic relationship. To systematically investigate the cause of such non-linear phenomenon, we develop a new viewpoint on the mechanism of SHG, and establish the equations of transient two-photon absorption theory. By using numerical calculation, the results show that theoretical curve exhibited a similar trend coinciding with experimental data. Moreover, theory together with experimental result has a capability to interpret nonlinear effect. As a result, this study may provide some possibly valuable conclusion on further investigating nonlinear theory in biological tissues.
Recently, most nonlinear microscopic techniques used for biological study have relied on the combination of two-photon excited fluorescence (TPEF) and second harmonic generation(SHG). However, certain non-linear effects in biological tissue excited by femtosecond (fs) laser have not been fully explored. Utilizing Ti: sapphire femtosecond laser equipped with Zeiss LSM 510 META laser scanning microscopy, we obtain non-linear compound imaging from two different specimens and the relationship curve between normalized TPEF/SHG intensity and excitation power. Based on experimental results, we find that the intensity of TPEF and SHG varying with excitation power dose not exactly obey a simple quadratic relationship. To systematically investigate the cause of such non-linear phenomenon, we develop a new viewpoint on the mechanism of SHG, and establish the equations of transient two-photon absorption theory. By using numerical calculation, the results show that theoretical curve exhibited a similar trend coinciding with experimental data. Moreover, theory together with experimental result has a capability to interpret nonlinear effect. As a result, this study may provide some possibly valuable conclusion on further investigating nonlinear theory in biological tissues.
引文
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2. P. T. C. So, C. Y. Dong, B. R. Masters, and K. M. Berland, "Two-photon excitation fluorescence microscopy," Annu. Rev. Biomed. 2000,2, 399-429.
3. V. E. Centonze and J. G. White, "Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging," Biophys. J. 1998, 75,2015-2024.
4. F. Helmchen and W. Denk, "Deep tissue two-photon microscopy," Nat. Methods. 2005, 2, 932-940.
5. W.R.Zipfel, R.M.Williams, et al. "Nonlinear magic: multiphoton microscopy in the biosciences", Nature Biotechnol. 2003, 21, 1369-1377.
6. P.J. Campagnola and L.M. Loew, "Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms," Nature Biotechnol. 2003,21,1356-1360.
7. W.R. Zipfel, R.M. Williams, et al. "Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation," Proc. Natl. Acad. Sci. USA. 2003,100, 7075-7080.
8. A. Zoumi, A. Yeh, and BJ. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. USA. 2002, 99,11014-11019.
9. A. Zoumi, et al. "Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy," Biophysical Journal. 2004, 87, 2778-2786.
10. E. Brown, et al. "Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation," Nat. Med. 2003,9, 796-800.
11. P. J. Campagnola, M.D. Wei, A. Lewis, and L.M. Loew, "High-resolution nonlinear optical imaging of live cells by second harmonic generation," Biophys. J. 1999, 77, 3341-3349.
12. S. Lin, R. J. Wu, H. Tan, et al. "Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy," Opt. Lett. 2005, 30, 2275-2277.
13. R. Richards-Kortum and E. Sevick-Muraca, "Quantitative optical spectroscopy for tissue diagnosis," Annu. Rev. Phys. Chem. 1996, 47, 555-606.
14. I. Georgakoudi, B.C. Jacobson, et al. "NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes," Cancer Res.2002, 62, 682-687.
15. M.C. Skala, J.M. Squirrell, et al. "Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous," Cancer. Res.2005, 65, 1180-1186.
16. Levene M J, Dombeck D A, et al. "In vivo multiphoton microscopy of deep brain tissue," J. Neurophysiol. 2004, 91 1908-1912.
17. W. Denk, J. H. Strickler, and W. W. Webb. "Two-photon laser scanning fluorescence microscopy," Science. 1990, 248.73-76.
18. M. Goppert-Mayer, "Uber elementarakte mit zwei quantensprungen," Ann. Phys. 1931,5,273-294.
19. W. Kaiser, C.G.B. Garrett, "Two-photon excitation in CaF_2:Eu~(2+)," Phys. Rev. Lett. 1961, 7, 229-231.
20. D.M. Friedrich, V.M. McClain, "Two-photon molecular electronic spectroscopy," Annu. Rev. Phys. Chem. 1980, 31, 559-577.
21. F. Bestvater, E. Spiess, G. Stobrawa, et al. "Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging," J. Microsc. 2002, 208, 108-115.
22. Yasushi Hiraoka, Tokuko Haraguchi, "Fluorescence imaging of mammalian living cells", Chromosome Research, 1996, Vol 4 ,199,173-176.
23. E. Niggli and M. Egger, "Applications of multi-photon microscopy in cell physiology," Front. Biosci. 2004, 9, 1598-1610.
24. Shuangmu Zhuo, Jianxin Chen, "Multimode nonlinear optical imaging of the dermis in ex vivo human skin based on the combination of multichannel mode and Lambda mode" OPTICS EXPRESS.2006, Vol. 14, No. 17.7810-7820.
25. B.R. Masters, P.T.C. So, and E. Graton, "Multiphoton excitation fluorescence Microscopy and spectroscopy of in vivo human skin," Biophys. J. 1997, 72, 2405-2412.
26. Shuangmu Zhuo, Jianxin Chen, "The layered-resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy", Phys. Med. Biol. 2007, 52, 4967-4980
27. Shuangmu Zhuo, Jianxin Chen, "Sequential Multitrack Nonlinear Ex vivo Imaging of Esophageal Stroma Based on Backscattered Second-harmonic Generation and Two-photon Autofluorescence", Scanning, 2007, Vol.29, 219-224.
28. D.W. Piston, B.R. Masters, and WW. Webb, "Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy," J. Microsc.1995,178,20-32.
29. P.T.C. So, H. Kim, and I .E. Kochevar, "Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures,'' Opt. Exp. 1998, 3,339-351.
30. B.D. Bennet, T. Jeton, G. Ying, et al. "Quantitative subcellular imaging of glucose metabolism with in intact pancreatic islets," J. Biol. Chem. 1996,271,3647-3655.
31. Pierre Collinet, Marie Delemer, "Fluorescence diagnosis of cervical squamous intraepithelial lesions: A clinical feasability study", Photodiagnosis and Photodynamic Therapy, 2007,4, 112—116.
32. Chun-Chin Wang, Feng-Chieh Li, "Utilizing nonlinear optical microscopy to investigate the development of early cancer in nude mice in vivo,"Proc. of SPIE-OSA Biomedical Optics, SPIE,2007, Vol. 6630, 66300Y
33. Bhawalkar JD, Kumar ND, Zhao CF, "Two-photon photodynamic therapy." J Clin Laser Med. Surg. 1997, 15(5):201-4.
34. E. Werkmeister, H. Kerdjoudj, "Multiphoton microscopy for blood vessel imaging: new non-invasive tools," Clinical Hemorheology and Microcirculation, 2007, 3777-3788.
35. Levene M J, et al. "In vivo multiphoton microscopy of deep brain tissue", J. Neurophysiol. 2004, 91 1908-1912.
36. P. Theer, M.T. Hasan, and W. Denk, "Two-photon imaging to a depth of 1000 microns in living brains by use of a Ti:A1203 regenerative amplifier," Opt. Lett. 2003, 28, 1022-1024.
37. Bloembergen N. Nonlinear Optics. W.A.Benjamin,Inc.,1977
38. Shen Y.R. The principles of Nonlinear Optics. John Wliey&Sons, Inc, 1984
39. Boyd R.W. Nolinear Optics. Academic Press, 1992
40. P.A. Franken, A.E. Hill, C.W. Peters, G. Weinreich, "Generation of optical harmonics," Phys. Rev. Lett. 1961,7, 118-119.
41. Fine S, Hansen W P. "Optical second harmonic generation in biological systems." Appl Opt, 1971, 10(10):2350-2353.
42. J. Gannaway, CJ.R Sheppard, "Second harmonic imaging in the scanning optical microscope," Opt.Quantum. Electron. 1978,10. 435-439.
43. Freund, M. Deutsch, A. Sprecher. "Connective tissue polarity." Biophys. J, 1986, 50:693-712.
44. Campagnola P J, Andrew C M, Mark T, et al. "Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. " Biophys J, 2002, 81(1): 493-508.
45. Patrick Stoller, Peter M. Celliers, et al. "Imaging collagen orientation using polarization-modulated second harmonic generation." Proc. SPIE, 2002, 4620:157-165.
46. P. Stoller, B.-M Kim, A. M. Rubenchik, et al. "Measurement of the second order non-linear susceptibility of collagen using polarization modulation and phase-sensitive detection." Proc. SPIE, 2001, 4276:11-16.
47. Williams R M, Zipfel W R, Watt W W. "Interpreting Second-Harmonic Generation Images of Collagen I Fibrils." Biophysical Journal, 2005,88:1377-1386.
48. Francois Legare, Christian, Bjorn R Olsen, "The Role of Backscattering in SHG Tissue Imaging," Biophysical Journal, 2007, 93, 4; ProQuest Medical Library, 1312-1320
49. Theodossis A Theodossiou, Christopher Thrasivoulou, et al. "Second Harmonic Generation Confocal Microscopy of Collagen Type I from Rat Tendon Cryosections, " Biophysical Journal, 2006, 91, 12; pg. 4665
50.Patrick Stoller,Karen M.Reiser,et al."Polarization-Modulated Second Harmonic Generation in Collagen." Biophysical Journal,2002,82(6):3330-3342.
51.Paul J.Campagnola,Heather A.Clark,William A.Mohler,et al."Second-harmonic imaging microscopy of living cells." Journal of Biomedical Optics,2001,6(3):277-286.
52.Kim B M,Eichler J,Dasilva L B."Frequency Doubling of Ultrashort Laser Pulses in Biological Tissues." Appl Opt,1999,38(34):7145-7150.
53.Tang Zhilie,Xing Da,"Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy," Science in China Ser.G Physics and Astronomy,2004,Vol.47,No.1,8-16
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