一种多波段光源的显微多光谱成像系统研究
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摘要
复合染色方法可以解决传统宫颈癌联合筛查无法在同一张涂片上实现的问题。针对复合染色带来的DNA吸光度干扰和色彩还原失真等问题,本文建立了一套多波段光源的显微光谱成像系统.组合光源的设计兼顾彩色图像的还原和吸光度模型的建立,采用多元线性回归法建立多光谱剥离模型,利用该模型从混叠的多光谱图像中剥离出福尔根染料的真实吸光度用于DNA定量分析,同时合成符合医生诊断习惯和要求的伪彩色图像.实验结果表明:选用的四个波段的光源的带宽为12nm/12nm/14nm/19nm,是符合要求的窄带单色光;以显微测微尺为标尺,以原光源测量参数为标准,测得组合光源的光斑偏移量最大不超过3个刻度,其平行度和同轴度满足成像系统要求;合成的伪彩色图像颜色明亮饱满、背景干净,不会对阅片医生诊断造成干扰和障碍;吸光度剥离模型可以成功将福尔根染料真实吸光度剥离出,能够准确计算DNA含量.选用小鼠肝脏涂片作为测量标本,测量计算的DI值符合肝细胞多倍体性的特点;选择宫颈阳性样本进行对比实验,两种方法测得的二倍体细胞和四倍体细胞DI值相吻合.本系统具有较高的准确性和可靠性.
Compound staining method can solve the problem that traditional combination screening of cervical cancer can not be carried out on the same smear.In view of the problems of DNA absorbance interference and color restoration distortion caused by compound staining,a microscopic spectral imaging system with multi-band illuminants was established in this paper.The design of composite illuminants gave consideration to both the restoration of color image and the establishment of absorbance model,multi-spectral unmixing model were constructed by multiple linear regression method,and by using this modle Feulgen′s real absorbance were unmixed from the mixing muti-spectral images for DNA quantitative analysis,and pseudo color images were also composed to meet the doctors′requirements and habits at the same time.Experimental results show that:the Full Width at Half Maximum(FWHM)of the chosed four-band illuminants were 12 nm/12 nm/14 nm/19 nm,and they were satisfactory narrowband monochromatic lights;The measured maximum offset of spot does not exceed 3 scale by using a microscope micrometer as a ruler and the original illuminant as a standard,so parallelism and coaxiality of the composite illuminants met the requirements of imaging system;The synthesis pseudo color images were bright and full,and have clean background,they will not cause interference and obstacles to doctor′s diagnosis.The absorbance unmixing model can successfully unmixing the real absorbance of feulgen and accurately calculate the content of DNA:The liver smear of mice was used as the measuring specimen and the calculated DNA index(DI)values accorded with the polyploidy of liver cells;In contrast experiments using a cervical positive sample,the DI values of the diploid and tetraploid cells calculated by the two methods were almost equal,therefore,this method has higher accuracy and reliability.
Compound staining method can solve the problem that traditional combination screening of cervical cancer can not be carried out on the same smear.In view of the problems of DNA absorbance interference and color restoration distortion caused by compound staining,a microscopic spectral imaging system with multi-band illuminants was established in this paper.The design of composite illuminants gave consideration to both the restoration of color image and the establishment of absorbance model,multi-spectral unmixing model were constructed by multiple linear regression method,and by using this modle Feulgen′s real absorbance were unmixed from the mixing muti-spectral images for DNA quantitative analysis,and pseudo color images were also composed to meet the doctors′requirements and habits at the same time.Experimental results show that:the Full Width at Half Maximum(FWHM)of the chosed four-band illuminants were 12 nm/12 nm/14 nm/19 nm,and they were satisfactory narrowband monochromatic lights;The measured maximum offset of spot does not exceed 3 scale by using a microscope micrometer as a ruler and the original illuminant as a standard,so parallelism and coaxiality of the composite illuminants met the requirements of imaging system;The synthesis pseudo color images were bright and full,and have clean background,they will not cause interference and obstacles to doctor′s diagnosis.The absorbance unmixing model can successfully unmixing the real absorbance of feulgen and accurately calculate the content of DNA:The liver smear of mice was used as the measuring specimen and the calculated DNA index(DI)values accorded with the polyploidy of liver cells;In contrast experiments using a cervical positive sample,the DI values of the diploid and tetraploid cells calculated by the two methods were almost equal,therefore,this method has higher accuracy and reliability.
引文
[1]ZHU Fang-pei,REN Qing-ling.The new progress of cervical cancer and precancerous lesions in early screening[J].Modern Oncology,2016,24(1):149-152.朱方培,任青玲.宫颈癌及癌前病变早期筛查的新进展[J].现代肿瘤医学,2016,24(1):149-152.
[2]REMMERBACH T W,WEIDENBACH H,POMJANSKI N,et al.Cytologic and DNA-cytometric early diagnosis of oral cancer[J].Anal Cell Pathol,2001,22(4):211-221.
[3]KANG Yi-qing,OUYANG Xin-hua,HE Dan,et al.Super early screening of cervical lesions clinical analysis of cervical cell DNA[J].Medical Innovation of China,2013,10(9):134-135.康一青,欧阳新华,何丹,等.宫颈细胞DNA超早期筛查宫颈病变的临床分析[J].中国医学创新,2013,10(9):134-135.
[4]ZHOU Hong,LUO Xin.Advantages of cells DNA ploidy analysis of cervical lesions in large population countries prior screening[J].Chinese Journal of Family Planning&Gynecotokology,2016,8(9):11-15.周宏,罗新.细胞DNA倍体分析技术在人口大国宫颈病变先行筛查中的优势[J].中国计划生育和妇产科,2016,8(9):11-15.
[5]TAN Jing-lang,CHEN Qiong,QIAO Sha-sha,et al.Clinical application of DNA quantitative analysis combined with liquid based cytology in cervical cancer screening[J].Hebei Medicine,2016,22(2):261-264.谭景浪,陈琼,乔莎莎,等.细胞DNA定量分析联合液基细胞学在宫颈癌筛查中的临床应用研究[J].河北医学,2016,22(2):261-264.
[6]王睿.DNA定量细胞学联合液基细胞学在宫颈病变中的应用价值[D].西宁:青海大学研究生院,2015.
[7]孙小蓉,汪键.DNA定量细胞学[M].第2版.武汉:湖北科学技术出版社,2011.
[8]WU Zheng,ZENG Li-bo,WU Qiong-shui.Study of cervical exfoliated cell′s DNA quantitative analysis based on multispectral imaging technology[J].Spectroscopy and Spectral Analysis,2016,36(2):496-501.
[9]陆婉珍.现代近红外光谱分析技术[M].第2版.北京:中国石化出版社,2007.
[10]WANG Xin-quan,HUANG Min,GAO Xiao-hui,et al.Potable multispectral imager based on LCTF[J].Acta Photonica Sinica,2010,39(1):71-75.王新全,黄旻,高晓惠,等.基于液晶可调谐滤光片的便携式多光谱成像仪[J].光子学报,2010,39(1):71-75.
[11]WANG Yi-zhe,ZHANG Feng-shan.A new kind of optical thin-film component-multi-channel band-pass filter[J].Infrared,2003(1):15-20.
[12]LIAO Cheng-sheng,WU Zheng,ZENG Li-bo,et al.Real-time multi-spectral imaging system based on multi-channel narrow-band filter and color image sensor[J].Acta Photonica Sinica,2017,46(4):0411001.廖乘胜,吴正,曾立波,等.基于多通道窄带滤光片和彩色图像传感器的实时多光谱成像系统[J].光子学报,2017,46(4):0411001.
[13]ZUO Fen,CHEN Lei,XU Chun-sheng.Simultaneous phase-shifting interferometry based on two-dimension grating[J].Acta Optica Sinica,2007,27(4):663-667.左芬,陈磊,徐春生.基于二维光栅分光的同步移相干涉测量技术[J].光学学报,2007,27(4):663-667.
[14]WANG Jia-hua,WU Zheng,WU Qiong-shui,et al.The study on combined screening method for TBS and cervical cancer cell′s DNA quantitative analysis based on multi-spectral imaging technology[J].Spectroscopy and Spectral Analysis,2017,37(2):545-550.
[15]ZHANG Hao,YAO Xu-guo,MAO Xue-qin,et al.Measurement of rice neek blast severity by using multi-spectral imagine[J].Hunan Agricultural Sciences,2009(1):65-68.
[16]WEI Qing-zhu,XIA Chao-yong,HUANG Zhong-xin,et al.The application of liver cell smear in evaluating the precise and accuracy of image analyzer[J].Chinese Journal of Pathology,2004,33(6):580-581.魏清柱,夏潮涌,黄中新,等.肝细胞涂片在评估图像分析仪的精确性和准确性中的应用[J].中华病理学杂志,2004,33(6):580-581.
[2]REMMERBACH T W,WEIDENBACH H,POMJANSKI N,et al.Cytologic and DNA-cytometric early diagnosis of oral cancer[J].Anal Cell Pathol,2001,22(4):211-221.
[3]KANG Yi-qing,OUYANG Xin-hua,HE Dan,et al.Super early screening of cervical lesions clinical analysis of cervical cell DNA[J].Medical Innovation of China,2013,10(9):134-135.康一青,欧阳新华,何丹,等.宫颈细胞DNA超早期筛查宫颈病变的临床分析[J].中国医学创新,2013,10(9):134-135.
[4]ZHOU Hong,LUO Xin.Advantages of cells DNA ploidy analysis of cervical lesions in large population countries prior screening[J].Chinese Journal of Family Planning&Gynecotokology,2016,8(9):11-15.周宏,罗新.细胞DNA倍体分析技术在人口大国宫颈病变先行筛查中的优势[J].中国计划生育和妇产科,2016,8(9):11-15.
[5]TAN Jing-lang,CHEN Qiong,QIAO Sha-sha,et al.Clinical application of DNA quantitative analysis combined with liquid based cytology in cervical cancer screening[J].Hebei Medicine,2016,22(2):261-264.谭景浪,陈琼,乔莎莎,等.细胞DNA定量分析联合液基细胞学在宫颈癌筛查中的临床应用研究[J].河北医学,2016,22(2):261-264.
[6]王睿.DNA定量细胞学联合液基细胞学在宫颈病变中的应用价值[D].西宁:青海大学研究生院,2015.
[7]孙小蓉,汪键.DNA定量细胞学[M].第2版.武汉:湖北科学技术出版社,2011.
[8]WU Zheng,ZENG Li-bo,WU Qiong-shui.Study of cervical exfoliated cell′s DNA quantitative analysis based on multispectral imaging technology[J].Spectroscopy and Spectral Analysis,2016,36(2):496-501.
[9]陆婉珍.现代近红外光谱分析技术[M].第2版.北京:中国石化出版社,2007.
[10]WANG Xin-quan,HUANG Min,GAO Xiao-hui,et al.Potable multispectral imager based on LCTF[J].Acta Photonica Sinica,2010,39(1):71-75.王新全,黄旻,高晓惠,等.基于液晶可调谐滤光片的便携式多光谱成像仪[J].光子学报,2010,39(1):71-75.
[11]WANG Yi-zhe,ZHANG Feng-shan.A new kind of optical thin-film component-multi-channel band-pass filter[J].Infrared,2003(1):15-20.
[12]LIAO Cheng-sheng,WU Zheng,ZENG Li-bo,et al.Real-time multi-spectral imaging system based on multi-channel narrow-band filter and color image sensor[J].Acta Photonica Sinica,2017,46(4):0411001.廖乘胜,吴正,曾立波,等.基于多通道窄带滤光片和彩色图像传感器的实时多光谱成像系统[J].光子学报,2017,46(4):0411001.
[13]ZUO Fen,CHEN Lei,XU Chun-sheng.Simultaneous phase-shifting interferometry based on two-dimension grating[J].Acta Optica Sinica,2007,27(4):663-667.左芬,陈磊,徐春生.基于二维光栅分光的同步移相干涉测量技术[J].光学学报,2007,27(4):663-667.
[14]WANG Jia-hua,WU Zheng,WU Qiong-shui,et al.The study on combined screening method for TBS and cervical cancer cell′s DNA quantitative analysis based on multi-spectral imaging technology[J].Spectroscopy and Spectral Analysis,2017,37(2):545-550.
[15]ZHANG Hao,YAO Xu-guo,MAO Xue-qin,et al.Measurement of rice neek blast severity by using multi-spectral imagine[J].Hunan Agricultural Sciences,2009(1):65-68.
[16]WEI Qing-zhu,XIA Chao-yong,HUANG Zhong-xin,et al.The application of liver cell smear in evaluating the precise and accuracy of image analyzer[J].Chinese Journal of Pathology,2004,33(6):580-581.魏清柱,夏潮涌,黄中新,等.肝细胞涂片在评估图像分析仪的精确性和准确性中的应用[J].中华病理学杂志,2004,33(6):580-581.