激光共聚焦显微技术在共定位应用中的常见问题
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摘要
荧光共定位分析是当今生物显微成像中一个极为常见的技术。本文从以下4个方面详细介绍了使用Zeiss LSM880型激光扫描共聚焦显微镜(confocal laser scanning microscope,CLSM)进行共定位研究时常见的问题及解决方法:1.如何用共聚焦进行共定位实验;2.如何进行共定位分析;3.如何评价共定位;4.如何判定共定位结果。在共定位研究中,只要按照共定位实验的基本要求拍摄图像,并按照上述方法进行分析,便可得到准确可靠的共定位分析结果。
The colocalization of fluorescence is a common technique in the biological imaging.In order to get true results of colocalization,we describe the following four aspects in detail,which are common problems of colocalization by Zeiss LSM880(confocal laser scanning microscope,CLSM),as well as the solutions.First,how to use CLSM for colocalization experiments.Second,how to conduct colocalization analysis.Third,how to evaluate colocalization.Fourth,how to determine the colocalization results.In the colocalization study,we can get the accurate and reliable colocalization analysis results if we take the images strictly following the basic requirements of the colocalization experiment and analyse them according to the above methods.
The colocalization of fluorescence is a common technique in the biological imaging.In order to get true results of colocalization,we describe the following four aspects in detail,which are common problems of colocalization by Zeiss LSM880(confocal laser scanning microscope,CLSM),as well as the solutions.First,how to use CLSM for colocalization experiments.Second,how to conduct colocalization analysis.Third,how to evaluate colocalization.Fourth,how to determine the colocalization results.In the colocalization study,we can get the accurate and reliable colocalization analysis results if we take the images strictly following the basic requirements of the colocalization experiment and analyse them according to the above methods.
引文
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[13]姚远,冯姝婷,赵鸿梅,李艳.雌激素受体alpha和肿瘤转移相关因子1在胃癌细胞中的表达和共定位[J].吉林大学学报(医学版),2013,39(1):5-7.Yao Y,Feng S T,Zhao H M,Li Y.Expressions and colocalization of ERαand MTA1in gastric cancer cells[J].Journal of Jilin University(Medicine Edition),2013,39(1):5-7.
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[2]李琳,刘德旺,田野.激光共聚焦显微镜技术在中药研究中的应用[J].中成药,2017,39(5):1038-1041.Li L,Liu D W,Tian Y.Future prospects of confocal laser scanning microscopy in taditional chinese medicine[J].Chinese Tmditional Patent Medicine,2017,39(5):1038-1041.
[3]李叶,黄华平,林培群,崔艳梅,李勤奋,郑勇奇.激光扫描共聚焦显微镜的基本原理及其使用技巧[J].电子显微学报,2015,34(2):169-176.Li Y,Huang H,Lin P Q,Cui Y M,Li Q F,Zheng Y Q.The fundamentals and techniques in laser scanning confocal microscopy[J].Journal of Chinese Electron Microscopy Society,2015,34(2):169-176.
[4]岳磊,张垚,马卓.激光扫描共聚焦显微镜实验技术与应用[J].哈尔滨商业大学学报(自然科学版),2015,31(3):263-266.Yue L,Zhang Y,Ma Z.Technique and application skills of laser scanning confocal microscopy[J].Journal of Harbin University of Commerce(Natural Sciences Edition),2015,31(3):263-266.
[5]王静,李辉.激光扫描共聚焦显微镜在脑部医学成像中的应用[J].激光杂志,2016,37(10):102-105.Wang J,Li H.Laser scanning confocal microscope used in medical imaging of the brain[J].Laser Journal,2016,37(10):102-105.
[6]段妍,关苑君,蓝秀健,张选红,吴珏珩.ZeissLSM710激光扫描共聚焦显微镜的使用和管理[J].实验室研究与探索,2012,31(8):228-230,239.Duan Y,Guan Y J,Lan X J,Zhang X H,Wu J H.Use and management of laser scanning confocal microscopy Zeiss LSM 710[J].Research and Exploration in Laboratory,2012,31(8):228-230,239.
[7]许佳玲,王晓红,罗剑文,龙钊,陈云凤,张以顺.蔡司LSM 7DUO NLO激光共聚焦显微镜的应用和管理[J].实验室研究与探索,2017,36(6):281-285.Xu J L,Wang X H,Luo J W,Long Z,Chen Y F,Zhang YS.Application and administration of laser scanning confocal microscopy Zeiss LSM 7DUO NLO[J].Research and Exploration in Laboratory,2017,36(6):281-285.
[8]孙学俊,闫喜中,郝赤.激光共聚焦扫描显微镜技术简介及其应用[J].山西农业大学学报(自然科学版),2016,36(1):1-14.Sun X J,Yan X Z,Hao C.Confocal laser scanning microscopy,ageneral guideline and its applications[J].Journal of Shanxi Agricultural University(Natural Sciences Edition),2016,36(1):1-14.
[9]Pastorek L,Sobol M,Hozák P.Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns[J].Histochemistry and Cell Biology,2016,146:391-406.
[10]Ellen C.Technical Review:colocalization of antibodies using confocal microscopy[J].The Anatomical Record,2014,297:183-187.
[11]何薇,耿慧武,左恒,阮操,潘林鑫,范礼斌,刘晓颖.FK506结合蛋白25及其突变体与CLIC1蛋白在真核细胞内共定位的研究[J].安徽医科大学学报,2016,51(10):1391-1395.He W,Gen H W,Zuo H,Ruan C,Pan L X,Fan L B,Liu X Y.The co-localization of FKBP25and its deletion mutants with CLIC1[J].Acta Universitatis Medicinalis Anhui,2016,51(10):1391-1395.
[12]于海波,李红玲,张新富,肖义,方沛菊,吕春娇,侯伟.基于Nile Red的线粒体靶向性增强型硫化氢荧光探针及其细胞成像[J].化学学报,2015,73:450-456.Yu H B,Li H L,Zhang X F,Xiao Y,Fang P J,Lv C J,Hou W.A mitochondrial-targetable and turn-on fluorescent probe based on nile red and monitoring for H2S in living cells[J].Acta Chimica Sinica,2015,73:450-456.
[13]姚远,冯姝婷,赵鸿梅,李艳.雌激素受体alpha和肿瘤转移相关因子1在胃癌细胞中的表达和共定位[J].吉林大学学报(医学版),2013,39(1):5-7.Yao Y,Feng S T,Zhao H M,Li Y.Expressions and colocalization of ERαand MTA1in gastric cancer cells[J].Journal of Jilin University(Medicine Edition),2013,39(1):5-7.
[14]王玥.LSM 710/780brief manual[R].Zeiss,2015:35-37.Wang Y.LSM 710/780brief manual[R].Zeiss,2015:35-37.
[15]Wu Y,Zinchuk V,Zinchuk O.Critical evaluation of quantitative colocalization analysis in confocal fluorescence microscopy[J].Interdisciplinary Sciences-Computational Life Sciences,2012,4:27-37.
[16]Zinchuk V,Zinchuk O,Okada T.Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images:pushing pixels to explore biological phenomena[J].Acta Histochemica Et Cytochemica,2007,40(4):101-111.
[17]Zinchuk V,Zinchuk O.Recent advances in quantitative colocalization analysis:focus on neuroscience[J].Progress in Histochemistry and Cytochemistry,2009,44:125-172.