不同的生物样本和切片,在进行拉曼探测时,会因为聚焦位置的差异及系统自身的原因导致同一光谱多次检测得到的信号强度不同,难以作定量分析。因此,需要找到一种可行性强的内标或外标的方法,从而有利于比较分析不同强度的拉曼带,实现定量检测。本文以犬类髋关节软骨切片为例,在偏振与非偏振情况下,研究标准正态变换(SNV)及多元散射校正(MSC)等常用方法对拉曼光谱的处理效果,并探究伊红染色剂(Eosin)作为一种新的内标的实际使用效果。结果发现:在非偏振条件下,MSC处理效果更理想;在偏振条件下,以伊红的特征峰1501 cm-1作为拉曼内标效果更理想。本研究证明了伊红的拉曼光谱不具有各向异性,有利于实现生物组织偏振拉曼光谱测量的归一化,且操作简单,结果更可靠。本文的结果为生物样本的拉曼光谱定量分析提供了一种可行的新方法。
Abstract
The Raman intensity of different biological samples or slices at the same wavenumber would be probably different when repeatedly detected at the same location,which was due to fine variation of focused position and the system deviation and made quantitative analysis much hard.Consequently,it was necessary to get a more feasible approach of defining internal or external standard for quantitative detection,which would be helpful for comparing Raman spectra captured under different conditions directly.Under the condition of polarization and depolarization,the Raman spectra of canine hip articular cartilage slices were detected and processed by standard normal variate (SNV) and multi-scattering correction (MSC) methods.In addition,eosin used as a novel internal standard to preprocess Raman signal was discussed and explored.It was found that the MSC method was most suited for the depolarization condition,as well as 1501 cm-1 band of eosin as internal standard for the polarization condition.The Raman band of eosin didn′t show anisotropy with polarization angel,which was effective to use as internal standard for the normalization of Raman spectra of biological tissue.Further,the internal standard method based on eosin brought more simple operation and more accurate results than MSC and SNV.Therefore,this study provided a promising novel method for the quantitative analysis of biological samples based on Raman spectroscopy.
关键词
定量分析 /
拉曼光谱 /
伊红染色剂 /
MSC /
SNV /
软骨
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Key words
Quantitative analysis /
Raman spectroscopy /
Eosin /
MSC /
SNV /
Cartilage
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参考文献
[1] Vo-Dinh T,Yan F,Wabuyele M B.Surface-enhanced Raman scattering for biomedical diagnostics and molecular imaging.Surface-Enhanced Raman Scattering:Physics and Applications 2006,103,409-426.
[2] 周殿凤,柯惟中,衡航等. 紫外辐射对牛血清白蛋白影响的拉曼光谱分析[J].光散射学报,2006,18 (3) :241-247 (ZHOU Dianfeng,KE Weizhong,HENG Hang et al.Raman spectroscopic study on the influence of UV radiation on BSA[J].The Journal of Light Scattering,2006,18(3):241-247)
[3] Kneipp J,Witting B,Bohr H,et al.Surface-enhanced Raman scattering:a new optical probe in molecular biophysics and biomedicine.Theoretical Chemistry Accounts 2010,125(3-6),319-327.
[4] 褚小立,袁洪福,陆婉珍.近红外分析中光谱预处理及波长选择方法进展与应用[J].化学进展,2004,16(4):528.
[5] Seasholtz,Mary Beth,Kowalski B.The parsimoy principle applied to multivariate calibration.Analytica Chimica Acta 277.2(1993):165-177.
[6] Gao Hao,Wang Xiao,Shang L W,et al.Design and Application of Small NIR-Raman Spectrometer Based on Dichroic and Transmission Collimating.SPECTROSCOPY AND SPECTRAL ANALYSIS,2018,38(06):1933-1937.
[7] Gao Hao,Zhai M Y,Shang L W,et al.Articular Cartilage Optical Clearing Research by Raman Spectroscopy.SPECTROSCOPY AND SPECTRAL ANALYSIS,2018,38(08):2425-2429.
[8] VMclean D I,Zeng H,Lui H,et al.Toward instrument-independent quantitative measurement of fluorescence intensity in fiber-optic spectrometer systems[J].Applied Optics,2007,46(29):7132-7140.
[9] Isaksson T,Naes T.The Effect of Multiplicative Scatter Correction (MSC) and Linearity Improvement in NIR Spectroscopy[J].Applied Spectroscopy,1988,42(7):1273-1284.
[10] Chen J,Iyo C,Terada F,et al.Effect of multiplicative scatter correction on wavelength selection for near infrared calibration to determine fat content in raw milk[J].J Near Infrared Spectrosc,2002,10(1):301-307.
[11] 芦永军,曲艳玲,宋敏.多元散射校正算法用于近红外相关光谱的处理研究[C].全国近红外光谱学术会议.2006.
[12] Wen T C,Ming T L,Han N L,et al.Micro-Raman spectroscopy used to identify and grade human skin pilomatrixoma[J].Microscopy research technique,2005,64(2):75-79.
[13] Eoghan ó Faoláin,Mary B,Hunterb,Joe M.Byrne,et al.A study examining the effects of tissue processing on human tissue sections using vibrational spectroscopy[J].Vibrational Spectroscopy,2005,38(1-2):121-127.
[14] Richard Ellis,Ellen Green,C.Peter Winlove.Structural Analysis of Glycosaminoglycans and Proteoglycans by Means of Raman Microspectrometry [J].Connective Tissue Research,2009,50(1):29-36.
[15] 吴正洁,黄耀熊,王成,等.多种拉曼光谱归一化法对乙醇定量分析的研究[J].光谱学与光谱分析,2010,30(4):971-974.
[16] 李梁,邵磊,霍剑青等.拉曼光谱退偏度测量实验的设计及其应用的研究[J].物理与工程,2008,18(3):65-69.
[17] Kazanci M,Roschger P,Paschalis E P,et al.Bone osteonal tissues by Raman spectral mapping:Orientation-composition[J].Journal of Structural Biology,2006,156(3):489-496.
[18] Kazanci M,Wagner H D,Manjubala N I,et al.Raman imaging of two orthogonal planes within cortical bone[J].Bone,2007,41(3):456-461.
[19] Raghavan M,Sahar N D,Wilson R H,et al.Quantitative polarized Raman spectroscopy in highly turbid bone tissue.[J].Journal of Biomedical Optics,2010,15(3):037001.
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脚注
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基金
江苏省六大人才高峰计划(SWYY-034);国家自然科学基金(61378087);南京航空航天大学研究生创新基地(实验室)开放基金(kfjj20180309)
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