拉曼光谱检测中荧光抑制方法的研究
|
摘要
拉曼光谱在化学、生物医学、材料、环保等领域有着非常广泛的应用。但是拉曼光谱检测经常受到荧光背景干扰,而且拉曼光强度远小于荧光强度以至于被荧光完全淹没,因此荧光抑制措施尤为重要。移频激发法(SERDS)是一种常用的荧光抑制方法,它采用两种波长相近的激发光源,获得两组光谱,将这两组光谱做差分,达到抑制荧光背景的目的。此方法具有抑制荧光能力强,适用范围广的优点。
本文采用波长为532nm,526.5nm的半导体泵浦固态激光器和全息窄带滤光片,设计并实现了一套价格相对便宜的SERDS拉曼光谱检测系统。本文设计并制作了光学密度高、带宽窄的全息滤光片,达到了滤除瑞利散射光的目的;对激发光源、激发光路、收集光路精心设计,提高信噪比。推导了解卷积模型从差值谱中复原拉曼光谱,详细分析了解卷积算法噪声大、负值、振荡产生的原因,为了克服解卷积算法的缺点,提出了多重能量约束迭代算法,并通过Matlab计算仿真,该算法信噪比高,收敛速度快。
利用自主设计的拉曼光谱系统,检测了常见的有机液体,例如甲醇、乙醇、丙酮等的拉曼光谱,拉曼位移均在实验误差范围内。对混有荧光物质阿维菌素的甲醇溶液进行检测,采用多重能量约束迭代算法,得到了质量较好的甲醇拉曼光谱,又成功检测出掺有荧光物质的乙腈溶液中乙腈的拉曼光谱,验证了SERDS方法能够有效的抑制荧光和多重能量约束迭代算法能够很好的复原拉曼光谱;最后对荧光很强的农药进行初步的尝试,虽没有得到预期的结果,但建立了相应的理论模型来分析主要原因,提出了今后的改进方向。
Raman spectroscopy is widely used in chemistry, biomedicine, materials, environmental protection and other areas. However, Raman spectroscopy is often hampered by strong fluorescence background that can easily bury the much weaker Raman signal. It’s necessary to take some proper fluorescence rejection measure in Raman detection. One efficient technique is the shifted-excitation Raman difference spectroscopy (SERDS), which incorporates two lasers to illuminate the sample alternatively. Two series of spectra are acquired to generate the difference spectrum in order to cancel out the fluorescence. SERDS can reject the fluorescence well, and it is applied to different samples.
This paper proposed a low cost SERDS system with 532 nm and 526.5 nm lasers and a home-made holographic notch filter. A high optical density and narrow bandwidth holographic filter was made in order to reject the Rayleigh scattering. The optical path of excitation and collecting were designed carefully. The deconvolution model of reconstructing Raman spectrum from difference spectrum was derived. The result reconstructed by deconvolution had three disadvantages, large noise, negative and oscillation. Then multi-energy constrained iterative algorithm was proposed to overcome these disadvantages. The algorithm was simulated to show the advantages of high signal-noise ratio and convergence rate.
Many common organic solvents such as methanol, alcohol and acetone were detected by this system. The measurement error of Raman shifts were within the experimental error. The Raman spectrum of methanol and acetonitrileare in strong fluorescence background were detected. Raman spectrum were well reconstructed by multi-energy constrained iterative algorithm. The results proved that the fluorescence can be effectively rejected by SERDS and Raman spectrum can be well reconstructed by the proposed algorithm. The fluorescent pesticide is also tried to detect, however, it is not successful. A theoretical model is built to analyze the main causes of the test failures.
本文采用波长为532nm,526.5nm的半导体泵浦固态激光器和全息窄带滤光片,设计并实现了一套价格相对便宜的SERDS拉曼光谱检测系统。本文设计并制作了光学密度高、带宽窄的全息滤光片,达到了滤除瑞利散射光的目的;对激发光源、激发光路、收集光路精心设计,提高信噪比。推导了解卷积模型从差值谱中复原拉曼光谱,详细分析了解卷积算法噪声大、负值、振荡产生的原因,为了克服解卷积算法的缺点,提出了多重能量约束迭代算法,并通过Matlab计算仿真,该算法信噪比高,收敛速度快。
利用自主设计的拉曼光谱系统,检测了常见的有机液体,例如甲醇、乙醇、丙酮等的拉曼光谱,拉曼位移均在实验误差范围内。对混有荧光物质阿维菌素的甲醇溶液进行检测,采用多重能量约束迭代算法,得到了质量较好的甲醇拉曼光谱,又成功检测出掺有荧光物质的乙腈溶液中乙腈的拉曼光谱,验证了SERDS方法能够有效的抑制荧光和多重能量约束迭代算法能够很好的复原拉曼光谱;最后对荧光很强的农药进行初步的尝试,虽没有得到预期的结果,但建立了相应的理论模型来分析主要原因,提出了今后的改进方向。
Raman spectroscopy is widely used in chemistry, biomedicine, materials, environmental protection and other areas. However, Raman spectroscopy is often hampered by strong fluorescence background that can easily bury the much weaker Raman signal. It’s necessary to take some proper fluorescence rejection measure in Raman detection. One efficient technique is the shifted-excitation Raman difference spectroscopy (SERDS), which incorporates two lasers to illuminate the sample alternatively. Two series of spectra are acquired to generate the difference spectrum in order to cancel out the fluorescence. SERDS can reject the fluorescence well, and it is applied to different samples.
This paper proposed a low cost SERDS system with 532 nm and 526.5 nm lasers and a home-made holographic notch filter. A high optical density and narrow bandwidth holographic filter was made in order to reject the Rayleigh scattering. The optical path of excitation and collecting were designed carefully. The deconvolution model of reconstructing Raman spectrum from difference spectrum was derived. The result reconstructed by deconvolution had three disadvantages, large noise, negative and oscillation. Then multi-energy constrained iterative algorithm was proposed to overcome these disadvantages. The algorithm was simulated to show the advantages of high signal-noise ratio and convergence rate.
Many common organic solvents such as methanol, alcohol and acetone were detected by this system. The measurement error of Raman shifts were within the experimental error. The Raman spectrum of methanol and acetonitrileare in strong fluorescence background were detected. Raman spectrum were well reconstructed by multi-energy constrained iterative algorithm. The results proved that the fluorescence can be effectively rejected by SERDS and Raman spectrum can be well reconstructed by the proposed algorithm. The fluorescent pesticide is also tried to detect, however, it is not successful. A theoretical model is built to analyze the main causes of the test failures.
引文
[1] Bauer C, Amram B, Agnely M. On-line monitoring of a latex emulsion polymerization by fiber-optic FT-raman spectroscopy[J]. Applied Spectroscopy, 2000, 54(4): 528-535.
[2] Wenz E, Buchholz V, Eichenauer H. Process for the production of graft Polymers [P]. US Patent: 10/281345, 2003, 10, 07.
[3] Otakar F, Jan J, Howell G M E. Raman spectroscopy as tool for the characterization of thio-polyaromatic hydrocarbons in organic minerals[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy. 2007, 68(4), 1065-1069
[4] Nicholas S, Catherine K, Neil S. Near-infrared Raman Spectroscopy for the epithelial of pre-cancer [J]. J. Raman Spectroscopy, 2002, 33: 564-573.
[5]胡军,胡继明.拉曼光谱在分析化学中的应用进展[J].分析化学, 2000, 28 (6) :764.
[6]梁鲁宁,林雷祥,董永宪等.拉曼光谱法鉴别常见炸药,刑事技术, 2003(5).
[7]成诚,于永爱. Raman光谱判别违禁品的算法结构研究.全国第十四届光散射技术学术年会会议论文集.
[8]杨群,王怡林,张鹏翔等.拉曼光谱对古青铜矛腐蚀情形的研究[J].光散射学报, 2001, 13 (1): 49 - 53.
[9]王荣,冯敏,王昌燧等.拉曼光谱在薛家岗古玉测试分析中的应用[J].光谱学与光谱分析, 2005, 25 (9) : 1422 - 1425.
[10]那娜,欧阳启名,乔玉青等.傅里叶变换红外光谱和近红外傅里叶变换拉曼光谱法无损鉴定中国字画[J].光谱学与光谱分析, 2004, 24(11) : 1327 - 1330.
[11] Eckhardt G, Wagner W. On the calculation of absolute Ramam scattering cross section from Raman scattering coefficients [J]. J. Molec. Spect. 1966, 19(4) : 409-411.
[12] Mosier-Boss P, Lieberman S, Newbery R. Fluorescence rejection in Ramanspectroscopy by shifted-spectra, edge detection, and FFT filtering techniques [J]. Applied Spectroscopy, 1995, 49(5): 630-637.
[13] Friedman J M, Hochstasser R M. The use of fluorescence quenchers in resonance Raman spectroscopy [J]. Chem. Phys. Lett, 1975, 33(2): 225-227.
[14] Macdonald A M, Wyeth P. On the use of photo bleaching to reduce fluorescence background in Raman spectroscopy to improve the reliability of pigment identification on painted textiles [J]. J. Raman Spectroscopy, 2006, 37(8): 830-835.
[15]姚启钧.光学教程[M].北京:高等教育出版社. 1981, 384-385.
[16] Hirschfeld T, Chase B. FT-Raman spectroscopy: development and justification [J]. Applied Spectroscopy, 1986, 40(2): 133-137.
[17] Arguello C A, Mendes G F, Leite R C. Simple technique to suppress spurious luminescence in Raman spectroscopy [J]. Applied Optics, 1974, 13(8): 1731-1732.
[18] Shreve A P, Cherepy N J, Mathies R A. Effective rejection of fluorescence interference in Raman spectroscopy using a shifted excitation difference technique [J]. Applied Spectroscopy, 1992, 46(4): 707-710.
[19] Stellman C M, Bucholtz F. Suppression of fluorescence interference via wavelength shift-keyed Raman spectroscopy using an argon ion laser and acousto-optic tunable filter [J]. Spectrochimica Acta Part A, 1998, 54(8): 1041-1047.
[20] McCain S T, Willett R M, Brady D J. Multi-excitation Raman spectroscopy technique for fluorescence rejection [J]. Applied Spectroscopy, 2008, 16(15): 10976-10988.
[21] Bright F V, Hieftje G M. A new technique for the elimination of fluorescence interference in Raman spectroscopy [J]. Applied Spectroscopy, 1986, 40(5): 583-586.
[22] Yaney P P. Reduction of fluorescence background in Raman spectra by the pulsed Raman technique [J]. J. Opt. Soc. Am, 1972, 62(11): 1927-1936.
[23] Tahara T, Hamaguchi H. Pico-second Raman spectroscopy using a streak camera [J]. Applied Spectroscopy, 1993, 47(4): 391-394.
[24] Cai T T, Zhang D, Amotz D B. Enhanced chemical classification of Raman imagesusing multi-resolution wavelet transformation [J]. Applied Spectroscopy, 2001, 55(9): 1124-1130.
[25] Lieber C A, Jansen A M. Automated method for subtraction of fluorescence from biological Raman spectra [J]. Applied Spectroscopy, 2003, 57(11): 1363-1367.
[26] Lavine B K, Davidson C E, Moores A J. Genetic algorithms for spectral pattern recognition [J]. Vibrational Spectroscopy, 2002, 28(1): 83–95.
[27] Matousek P, Towrie M, Parker A W. Fluorescence background suppression in Raman spectroscopy using combined Kerr gated and shifted excitation Raman difference techniques [J]. J. Raman Spectroscopy, 2002, 33(4): 238–242.
[28] Dennis A. Photo-bleaching and automatic baseline correction for Raman spectroscopy. [EB/OL]. PerkinElmer Life and Analytical Sciences, 2007, www. perkinelmer.com.
[29]王锭笙,李学铭.亚单分子层三聚氰胺的便携式拉曼检测[J].光散射学报, 2008, 20(4), 379-383.
[30]肖怡琳,张鹏翔,钱晓凡.几种农药的显微拉曼光谱和荧光光谱[J].光谱学与光谱分析, 2004, 24(5): 579-581.
[31] Martins M, Ribeiro D G. Shifted-excitation Raman difference spectroscopy for in vitro and in vivo biological samples analysis[J]. Biomedical optics express, 2010, 1(2): 617-626.
[32] Zhao J, Carrabba M. Automated fluorescence rejection using shifted excitation Raman difference spectroscopy[J], Applied Spectroscopy, 2002, 56(7), 834-844.
[33] Gonzalez R C. Digital image processing[M], Publishing house of electronics industry, beijing, 2002, 210-214.
[34] Tikhonov A N, Goncharsky A V, Stepanov V V, Yagola A G. Numerical Methods for the Solution of Ill-Posed Problems[M], Kluwer Academic Publishers, Amsterdam, 1995.
[35] Morhaˇc M , Matou?ek V. Complete positive deconvolution of spectrometric data[J], Digital Signal Processing, 2009, 19: 372-392.
[36]赵艳皎,吴建宏.便携式拉曼光谱仪技术的研究[J].苏州大学, 2002, 37-38.
[37]吴建宏,唐敏学.非均匀反射体全息窄带带阻滤光片[J].光学学报, 2000, 20(9): 1272-1276.
[38]沈杭城,潘建根.多通道快速光谱仪的波长定标[J].光学仪器, 2006, 28 (2): 52-55.
[39] Liou K N. An introduce to atmospheric radiation[M], Academic Press, US, 1980.
[2] Wenz E, Buchholz V, Eichenauer H. Process for the production of graft Polymers [P]. US Patent: 10/281345, 2003, 10, 07.
[3] Otakar F, Jan J, Howell G M E. Raman spectroscopy as tool for the characterization of thio-polyaromatic hydrocarbons in organic minerals[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy. 2007, 68(4), 1065-1069
[4] Nicholas S, Catherine K, Neil S. Near-infrared Raman Spectroscopy for the epithelial of pre-cancer [J]. J. Raman Spectroscopy, 2002, 33: 564-573.
[5]胡军,胡继明.拉曼光谱在分析化学中的应用进展[J].分析化学, 2000, 28 (6) :764.
[6]梁鲁宁,林雷祥,董永宪等.拉曼光谱法鉴别常见炸药,刑事技术, 2003(5).
[7]成诚,于永爱. Raman光谱判别违禁品的算法结构研究.全国第十四届光散射技术学术年会会议论文集.
[8]杨群,王怡林,张鹏翔等.拉曼光谱对古青铜矛腐蚀情形的研究[J].光散射学报, 2001, 13 (1): 49 - 53.
[9]王荣,冯敏,王昌燧等.拉曼光谱在薛家岗古玉测试分析中的应用[J].光谱学与光谱分析, 2005, 25 (9) : 1422 - 1425.
[10]那娜,欧阳启名,乔玉青等.傅里叶变换红外光谱和近红外傅里叶变换拉曼光谱法无损鉴定中国字画[J].光谱学与光谱分析, 2004, 24(11) : 1327 - 1330.
[11] Eckhardt G, Wagner W. On the calculation of absolute Ramam scattering cross section from Raman scattering coefficients [J]. J. Molec. Spect. 1966, 19(4) : 409-411.
[12] Mosier-Boss P, Lieberman S, Newbery R. Fluorescence rejection in Ramanspectroscopy by shifted-spectra, edge detection, and FFT filtering techniques [J]. Applied Spectroscopy, 1995, 49(5): 630-637.
[13] Friedman J M, Hochstasser R M. The use of fluorescence quenchers in resonance Raman spectroscopy [J]. Chem. Phys. Lett, 1975, 33(2): 225-227.
[14] Macdonald A M, Wyeth P. On the use of photo bleaching to reduce fluorescence background in Raman spectroscopy to improve the reliability of pigment identification on painted textiles [J]. J. Raman Spectroscopy, 2006, 37(8): 830-835.
[15]姚启钧.光学教程[M].北京:高等教育出版社. 1981, 384-385.
[16] Hirschfeld T, Chase B. FT-Raman spectroscopy: development and justification [J]. Applied Spectroscopy, 1986, 40(2): 133-137.
[17] Arguello C A, Mendes G F, Leite R C. Simple technique to suppress spurious luminescence in Raman spectroscopy [J]. Applied Optics, 1974, 13(8): 1731-1732.
[18] Shreve A P, Cherepy N J, Mathies R A. Effective rejection of fluorescence interference in Raman spectroscopy using a shifted excitation difference technique [J]. Applied Spectroscopy, 1992, 46(4): 707-710.
[19] Stellman C M, Bucholtz F. Suppression of fluorescence interference via wavelength shift-keyed Raman spectroscopy using an argon ion laser and acousto-optic tunable filter [J]. Spectrochimica Acta Part A, 1998, 54(8): 1041-1047.
[20] McCain S T, Willett R M, Brady D J. Multi-excitation Raman spectroscopy technique for fluorescence rejection [J]. Applied Spectroscopy, 2008, 16(15): 10976-10988.
[21] Bright F V, Hieftje G M. A new technique for the elimination of fluorescence interference in Raman spectroscopy [J]. Applied Spectroscopy, 1986, 40(5): 583-586.
[22] Yaney P P. Reduction of fluorescence background in Raman spectra by the pulsed Raman technique [J]. J. Opt. Soc. Am, 1972, 62(11): 1927-1936.
[23] Tahara T, Hamaguchi H. Pico-second Raman spectroscopy using a streak camera [J]. Applied Spectroscopy, 1993, 47(4): 391-394.
[24] Cai T T, Zhang D, Amotz D B. Enhanced chemical classification of Raman imagesusing multi-resolution wavelet transformation [J]. Applied Spectroscopy, 2001, 55(9): 1124-1130.
[25] Lieber C A, Jansen A M. Automated method for subtraction of fluorescence from biological Raman spectra [J]. Applied Spectroscopy, 2003, 57(11): 1363-1367.
[26] Lavine B K, Davidson C E, Moores A J. Genetic algorithms for spectral pattern recognition [J]. Vibrational Spectroscopy, 2002, 28(1): 83–95.
[27] Matousek P, Towrie M, Parker A W. Fluorescence background suppression in Raman spectroscopy using combined Kerr gated and shifted excitation Raman difference techniques [J]. J. Raman Spectroscopy, 2002, 33(4): 238–242.
[28] Dennis A. Photo-bleaching and automatic baseline correction for Raman spectroscopy. [EB/OL]. PerkinElmer Life and Analytical Sciences, 2007, www. perkinelmer.com.
[29]王锭笙,李学铭.亚单分子层三聚氰胺的便携式拉曼检测[J].光散射学报, 2008, 20(4), 379-383.
[30]肖怡琳,张鹏翔,钱晓凡.几种农药的显微拉曼光谱和荧光光谱[J].光谱学与光谱分析, 2004, 24(5): 579-581.
[31] Martins M, Ribeiro D G. Shifted-excitation Raman difference spectroscopy for in vitro and in vivo biological samples analysis[J]. Biomedical optics express, 2010, 1(2): 617-626.
[32] Zhao J, Carrabba M. Automated fluorescence rejection using shifted excitation Raman difference spectroscopy[J], Applied Spectroscopy, 2002, 56(7), 834-844.
[33] Gonzalez R C. Digital image processing[M], Publishing house of electronics industry, beijing, 2002, 210-214.
[34] Tikhonov A N, Goncharsky A V, Stepanov V V, Yagola A G. Numerical Methods for the Solution of Ill-Posed Problems[M], Kluwer Academic Publishers, Amsterdam, 1995.
[35] Morhaˇc M , Matou?ek V. Complete positive deconvolution of spectrometric data[J], Digital Signal Processing, 2009, 19: 372-392.
[36]赵艳皎,吴建宏.便携式拉曼光谱仪技术的研究[J].苏州大学, 2002, 37-38.
[37]吴建宏,唐敏学.非均匀反射体全息窄带带阻滤光片[J].光学学报, 2000, 20(9): 1272-1276.
[38]沈杭城,潘建根.多通道快速光谱仪的波长定标[J].光学仪器, 2006, 28 (2): 52-55.
[39] Liou K N. An introduce to atmospheric radiation[M], Academic Press, US, 1980.