基于图像拼接的高通量数字PCR荧光基因芯片读取系统的设计
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摘要
针对高通量数字聚合酶链式反应荧光基因芯片检测的需求,提出了一种基于荧光显微光学技术的基因芯片检测系统。系统以无限远荧光显微系统为框架,通过制冷CCD一次完成较大视场的成像,顺序移动基因芯片得到全部图像,通过图像拼接完成检测,切换二向色镜组实现检测不同荧光通道的目的。光学系统分辨率可达16.3μm、曝光时间500ms,目前只需要拼接35次,即可在1min内完成对28mm×16mm的基因芯片内两万多荧光通道的检测,极大的提高了检测效率。
In face of the requirement of the detection of high throughput digital polymerase chain reaction(dPCR)fluorescence gene chip,a new optical detection system based on fluorescence microscopy is proposed.The design of this system is depending on an afocal fluorescence microscopy system.It can complete the imaging of the larger field of view through refrigerating CCD.All the images can be got by moving the gene chip in sequence,and the complete image can be obtained through image splicing.By changing the dichroic mirror and filter,the detection system can detect different kinds of fluorescence gene chip.The optical system has a resolution of 20 mm,an exposure time of 500 ms and 35 times of splicing.It can complete the detection of the gene chip with more than 20000 channel within 1minutes,and the area of the gene chip is 28mm×18mm.This system greatly reduces the detection time.
In face of the requirement of the detection of high throughput digital polymerase chain reaction(dPCR)fluorescence gene chip,a new optical detection system based on fluorescence microscopy is proposed.The design of this system is depending on an afocal fluorescence microscopy system.It can complete the imaging of the larger field of view through refrigerating CCD.All the images can be got by moving the gene chip in sequence,and the complete image can be obtained through image splicing.By changing the dichroic mirror and filter,the detection system can detect different kinds of fluorescence gene chip.The optical system has a resolution of 20 mm,an exposure time of 500 ms and 35 times of splicing.It can complete the detection of the gene chip with more than 20000 channel within 1minutes,and the area of the gene chip is 28mm×18mm.This system greatly reduces the detection time.
引文
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[4]Belgrader P,Tanner S C,Regan J F,et al.Droplet digital PCR measurement of HER2copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue[J].Clinical Chemistry,2013,59(6):991—994.
[5]Kumaresan P,Yang C J,Cronier S A,et al.High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets[J].Analytical Chemistry,2008,80(10):3522—3529.
[6]Barrett A N,McDonnell T C,Chan K C,et al.Digital PCR analysis of maternal plasma for noninvasive detection of sickle cell anemia[J].Clinical Chemistry,2012,58(6):1026—1032.
[7]Corbisier P,Bhat S,Partis L,et al.Absolute quantification of genetically modified MON810 maize(Zea mays L)by digital polymerase chain reaction[J].Analytical and Bioanalytical Chemistry,2010,396(6):2143—2150.
[8]Tewhey R,Warner J B,Nakano M,et al.Microdroplet-based PCR amplification for large scale targeted sequencing[J].Nat Biotechnol,2009,27(11):1025—1031.
[9]Denk W,Piston D W,Webb W W.Multi-Photon molecular excitation in laser-scanning microscopy[J].Handbook of Biological Confocal Microscopy,2006:535—549.
[10]赵维谦,任利利,盛忠,等.激光共焦显微光束的偏转扫描[J].光学精密工程,2016,24(6):1257—1263.Zhao Weiqian,Ren Lili,Sheng Zhong,et al.Beam deflection scanning for laser confocal microscopy[J].Optics and Precision Engineering,2016,24(6):1257—1263.
[11]Yin C,Glaser A K,Leigh S Y,et al.Miniature in vivo MEMSbased line-scanned dual-axis confocal microscope for point-ofcare pathology[J].Biomedical Optics Express,2016,7(2):251—263.
[12]B9hnke M,Masters B R.Confocal microscopy of the cornea[J].Progress in Retinal and Eye Research,1999,18(5):553—628.
[13]王忠立,刘佳音,贾云得.基于CCD与CMOS的图像传感技术[J].光学技术,2003,29(3):361—364.Wang Zhongli,Lei Jiayin,Jia Yunde.Image sensing technology based on CCD and CMOS[J].Optical Technique,2003,29(3):361—364.
[14]郁道银,谈恒英.工程光学[M].北京:机械工业出版社,2011:123—125.Yu Daoyin,Tan Hengying.Engineering optics[M].Beijing:China Machine Press,2011:123—125.
[15]崔泽实,郭德伦.荧光显微镜的功能配置及应用要点[J].医疗装备,2004,17(09):4—6.Cui Zeshi,Guo Delun.Functional configuration and application points of fluorescence microscope[J].Medical Equipment,2004,17(09):4—6.
[16]李湘宁,贾宏志,张荣福,等.工程光学[M].北京:科学出版社,2011:165—166.Li Xiangning,Jia Hongzhi,Zhang Rongfu,et al.Engineering Optics[M].Beijing:Science Press,2011:165—166.