宽光谱长工作距弱荧光信号检测显微物镜设计
|
摘要
针对癌细胞突变基因诱导荧光信号弱、光谱覆盖范围宽、现有显微镜不能检测等局限,本文设计了光谱波段为450~800nm、数值孔径为0.95的荧光显微物镜。物镜采用++-结构,因宽光谱、大数值孔径像差校正难度大、透镜片数多、装调困难,前组设置成敏感组分,承载物镜装调的调校功能,承担90%以上光焦度;中间组为弱光焦度组分,用于校正大数值孔径下的二级光谱,显著降低了二级光谱校正元件的加工难度;后组为负光焦度组分,用于平像场和增大物镜的工作距离。物镜的设计参数为:总长58mm、工作距0.21mm、视场0.625mm、倍率40×、数值孔径0.95,结果表明:其像质接近衍射极限,畸变小于0.2%,满足多种癌细胞突变基因的弱信号生物监测设计要求。
Because the fluorescence signal induced by a cancer cell is weak and has a wide spectral range,along with the detection limits of existing microscopes,this paper proposes fluorescence microscope objectives with a spectral range of 450—800 nm and a numerical aperture of 0.95.The objective used"+ +-"structure because the correction of aberration,caused by a wide spectrum and high numerical aperture,is difficult.The objective must include a set number of lenses and the alignment process is difficult;the front group,considering more than 90%focal power,is set as the sensitive component,which is useful for adjusting the alignment process;the middle group is the weak focal component,which is used to correct the secondary spectrum caused by the large numerical aperture,so that the difficulty in manufacturing the apochromatic elements is reduced significantly.The posterior group is the negative component,which is useful for flattening the image field andincreasing the working distance of the objective.The objective parameters are as follow:the total length is 58 mm,the working distance is 0.21 mm,the magnification is 40×,and the full field is0.625 mm.The results show that the image quality nearly reaches the diffraction limit and the distortion is less than 0.2%,meeting the surveilling requirements of the weak fluorescence signal from cancer cells.
Because the fluorescence signal induced by a cancer cell is weak and has a wide spectral range,along with the detection limits of existing microscopes,this paper proposes fluorescence microscope objectives with a spectral range of 450—800 nm and a numerical aperture of 0.95.The objective used"+ +-"structure because the correction of aberration,caused by a wide spectrum and high numerical aperture,is difficult.The objective must include a set number of lenses and the alignment process is difficult;the front group,considering more than 90%focal power,is set as the sensitive component,which is useful for adjusting the alignment process;the middle group is the weak focal component,which is used to correct the secondary spectrum caused by the large numerical aperture,so that the difficulty in manufacturing the apochromatic elements is reduced significantly.The posterior group is the negative component,which is useful for flattening the image field andincreasing the working distance of the objective.The objective parameters are as follow:the total length is 58 mm,the working distance is 0.21 mm,the magnification is 40×,and the full field is0.625 mm.The results show that the image quality nearly reaches the diffraction limit and the distortion is less than 0.2%,meeting the surveilling requirements of the weak fluorescence signal from cancer cells.
引文
[1]LI S Y.The human cancer genome:laboratory analysis and clinical application[J].Laboratory Medicine,2014,29(5):414-434.
[2]高志博,杨锦.肿瘤精准免疫治疗与基因检测[J].生物产业技术,2017(2):27-33.GAO ZH B,YANG J.Genetic testing for precision cancer immunotherapy[J].Biotechnology&Business,2017(2):27-33.(in Chinese)
[3]LAI Z Y,QIN Q,YU B,et al..Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma[J].Chinese Medical Journal,2014,127(12):2337-2341.
[4]陈孙霞,王晓庆,徐晓恩,等.共培养体系中高红色荧光蛋白(RFP)标记结肠癌细胞的检测技术[J].复旦学报(医学版),2014,41(5):602-609.CHEN S X,WANG X Q,XU X,et al..The technology of detecting high red fluorescence protein(RFP)labeled colon cancer cells in a co-culture system[J].Fudan Univ.J.Med.Sci.,2014,41(5):602-609.(in Chinese)
[5]胡笳,熊蕾,郑玉萍,等.脉络膜转移癌的荧光素和吲哚菁绿血管造影的影像分析[J].国际眼科杂志,2017,17(3):529-531.HU J,XIONG L,ZHENG Y P,et al..FFA and ICGA imaging analysis of choroidal metastatic carcinoma[J].International Eye Science,2017;17(3):529-531.(in Chinese)
[6]何国培.荧光光谱医学成像诊断技术研究[D].哈尔滨:哈尔滨工业大学,2007.HE G P.Fluorescence Spectrum Imaging Techniques for Medical[D].Harbin:Harbin Institute of Technology,2007.(in Chinese)
[7]FEJLMANN T,REINHEIMER S,GENG C,et al..cPAS-based sequencing on the BGISEQ-500to explore small non-coding RNAs[J].Clinical Epigenetics,2016,8(1):123.
[8]FROLOV D N,VINOGRADOVA O,FROLOV V,et al..Optical design of objectives-Lcf planfluor pol for 27 mm observation in the polarizing microscope[C].Freeform Optics,2017:JTu5A.14.
[9]徐明飞,庞武斌,徐象如,等.高数值孔径投影光刻物镜的光学设计[J].光学精密工程,2015,24(4):740-746.XU M F,PANG W B,XU X R.Optical design of high-numerical aperture lithographic lenses[J].Opt.Precision Eng.,2015,24(4):740-746.(in Chinese)
[10]ROBEN S,BRAKHANE S,ALT W,et al..High numerical aperture(NA=0.92)objective lens for imaging and addressing of cold atoms[J].Optics Letters,2017,42(6):1043-1046.
[11]郁道银,谈恒英.工程光学[M].北京:机械工业出版社,2011:112-115.YU D Y,TAN H Y.Engineering Optics[M].Beijing:China Machine Press,2011:112-115.(in Chinese)
[12]王东海.氟化钙晶体化学机械抛光工艺研究[D].长春:长春理工大学,2009.WANG D H.Study on Chemical Mechanical Polishing of Calcium Fluoride Single Crystal[D].Changchun:Changchun University of Science and Technology,2009.(in Chinese)
[13]DE ALBUQUERQUE B F,SASIAN J,DE SOUSA L,et al..Method of glass selection for color correction in optical system design[J].Optics Express,2012,20(13):13592-13611.
[14]International standard ISO 19012-1-2013,Microscopesdesignation of microscope objectives[S].2013.
[15]ZHOU E Y,LIU L H,LIU Y,et al..Design of high NA flat-field microscope objective for near infrared[J].Infrared and Laser Engineering,2017,46(7):718006.
[16]薛金来,巩岩,李佃蒙.N.A.0.75平场复消色差显微物镜光学设计[J].中国光学,2015,8(6):957-963.XUE J L,GONG Y,LI D M.Optical design of the N.A.0.75plan-apochromatic microscope objective[J].Chinese Optics,2015,8(6):957-963.(in Chinese)
[17]萧瑟新,高兴宇,肖华鹏.一种无CaF240倍平场复消色差金相显微物镜:中国,201420867956.7[P].2014.XIAO S X,GAO X Y,XIAO H P.A flat-apochromatic metallographic microscope objective without CaF2:China,201420867956.7[P].2014.(in Chinese)
[2]高志博,杨锦.肿瘤精准免疫治疗与基因检测[J].生物产业技术,2017(2):27-33.GAO ZH B,YANG J.Genetic testing for precision cancer immunotherapy[J].Biotechnology&Business,2017(2):27-33.(in Chinese)
[3]LAI Z Y,QIN Q,YU B,et al..Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma[J].Chinese Medical Journal,2014,127(12):2337-2341.
[4]陈孙霞,王晓庆,徐晓恩,等.共培养体系中高红色荧光蛋白(RFP)标记结肠癌细胞的检测技术[J].复旦学报(医学版),2014,41(5):602-609.CHEN S X,WANG X Q,XU X,et al..The technology of detecting high red fluorescence protein(RFP)labeled colon cancer cells in a co-culture system[J].Fudan Univ.J.Med.Sci.,2014,41(5):602-609.(in Chinese)
[5]胡笳,熊蕾,郑玉萍,等.脉络膜转移癌的荧光素和吲哚菁绿血管造影的影像分析[J].国际眼科杂志,2017,17(3):529-531.HU J,XIONG L,ZHENG Y P,et al..FFA and ICGA imaging analysis of choroidal metastatic carcinoma[J].International Eye Science,2017;17(3):529-531.(in Chinese)
[6]何国培.荧光光谱医学成像诊断技术研究[D].哈尔滨:哈尔滨工业大学,2007.HE G P.Fluorescence Spectrum Imaging Techniques for Medical[D].Harbin:Harbin Institute of Technology,2007.(in Chinese)
[7]FEJLMANN T,REINHEIMER S,GENG C,et al..cPAS-based sequencing on the BGISEQ-500to explore small non-coding RNAs[J].Clinical Epigenetics,2016,8(1):123.
[8]FROLOV D N,VINOGRADOVA O,FROLOV V,et al..Optical design of objectives-Lcf planfluor pol for 27 mm observation in the polarizing microscope[C].Freeform Optics,2017:JTu5A.14.
[9]徐明飞,庞武斌,徐象如,等.高数值孔径投影光刻物镜的光学设计[J].光学精密工程,2015,24(4):740-746.XU M F,PANG W B,XU X R.Optical design of high-numerical aperture lithographic lenses[J].Opt.Precision Eng.,2015,24(4):740-746.(in Chinese)
[10]ROBEN S,BRAKHANE S,ALT W,et al..High numerical aperture(NA=0.92)objective lens for imaging and addressing of cold atoms[J].Optics Letters,2017,42(6):1043-1046.
[11]郁道银,谈恒英.工程光学[M].北京:机械工业出版社,2011:112-115.YU D Y,TAN H Y.Engineering Optics[M].Beijing:China Machine Press,2011:112-115.(in Chinese)
[12]王东海.氟化钙晶体化学机械抛光工艺研究[D].长春:长春理工大学,2009.WANG D H.Study on Chemical Mechanical Polishing of Calcium Fluoride Single Crystal[D].Changchun:Changchun University of Science and Technology,2009.(in Chinese)
[13]DE ALBUQUERQUE B F,SASIAN J,DE SOUSA L,et al..Method of glass selection for color correction in optical system design[J].Optics Express,2012,20(13):13592-13611.
[14]International standard ISO 19012-1-2013,Microscopesdesignation of microscope objectives[S].2013.
[15]ZHOU E Y,LIU L H,LIU Y,et al..Design of high NA flat-field microscope objective for near infrared[J].Infrared and Laser Engineering,2017,46(7):718006.
[16]薛金来,巩岩,李佃蒙.N.A.0.75平场复消色差显微物镜光学设计[J].中国光学,2015,8(6):957-963.XUE J L,GONG Y,LI D M.Optical design of the N.A.0.75plan-apochromatic microscope objective[J].Chinese Optics,2015,8(6):957-963.(in Chinese)
[17]萧瑟新,高兴宇,肖华鹏.一种无CaF240倍平场复消色差金相显微物镜:中国,201420867956.7[P].2014.XIAO S X,GAO X Y,XIAO H P.A flat-apochromatic metallographic microscope objective without CaF2:China,201420867956.7[P].2014.(in Chinese)