荧光漂白恢复技术PPT

荧光漂白恢复技术（fluorescencerecoveryafterphotobleaching，FRAP）09生物季刚裴家平Page2FRAP关键技术简介FRAP是用来测定活细胞的动力学参数。FRAP是上世纪70年代开创的利用荧光标记法研究活体细胞中各类分子迁移特性的技术。当前生命科学研究已进入分子水平，应用多种手段已获得大量关于细胞内分子的定性知识，基本了解了各细胞器的分子组成，找到许多在细胞生命周期中发挥重要作用的蛋白质，确认了许多重要蛋白质之间相互作用的存在并初步描绘出一些信号通路。但细胞的各种生物学功能和现象都是借助相关分子实现的，当前的研究重点和难点就集中于解答分子实现各种功能的机制。这就需要掌握活细胞生理状态下分子运动的各种信息，近年来发展的一些技术手段为获得有关重要信息提供了有力的工具，FRAP技术就是其中一种重要方法。Page3FRAP原理InFRAP,aregionofthecellisexposedtohigh-intensitylaserlight,causingthefluorophoreswithinthatregiontoirreversiblylosetheirabilitytofluoresce.Recoveryoffluorescenceinthatregionyieldsinformationaboutmoleculardiffusionandbindinginthecell.Page4FRAP技术的基本要求选择合适的荧光探针具备精确可控的激光激发和荧光检测设备激光扫描共聚焦显微镜Page5FRAP的三个阶段用尽可能弱的激光扫描全细胞使用强激光在短时间内扫描漂白区域用尽可能弱的激光扫描全细胞漂白前漂白漂白后恢复Page6注：漂白前和漂白后恢复都用尽可能弱的激光扫描全细胞，目的是得到扫描图像而不引起荧光淬灭，漂白阶段则使用强激光在短时间内扫描漂白区域，目的是使区域内的荧光全部淬灭。在整个过程中，监测漂白区域在各时间段的荧光强度变化并绘制曲线，从恢复曲线及其数据就可以得到关于分子迁移速率、动态分子比例等信息。Page7Page8FRAP实验注意事项做FRAP实验还应当注意以下几点，否则就易得到错误结果。（1）注意实验温度的控制。（2）漂白区域大小的选择和荧光恢复检测时间的长短要根据具体情况而定。（3）激发光的波长和强度应不会使细胞严重损伤；尽量减少漂白前和漂白后的荧光淬灭。Page9FRAP技术的不足之处第一,它只能检测膜蛋白的群体移动,而不能观察单个蛋白的移动。其次,它不能证明膜蛋白在移动时是否受局部条件的限制。Page10FRAP的应用Page11荧光漂白恢复测定巨噬细胞Fc受体的荧光恢复率和运动分数（山东大学硕士学位论文《荧光漂白恢复测定巨噬细胞Fc受体的流动性》，李建业，2005.5）采用Alexa488标记的羊抗鼠IgG间接标记巨噬细胞Fc受体，用激光共聚焦系统的强脉冲激光漂白，弱强度激光扫描，光电倍增管(PMT)检测，Fluoview软件记录数据并分析处理。Page12漂白区域的荧光强度随时间的变化示于图2.11。Page13漂白以后恢复过程荧光强度的变化示于图2.13。Page14漂白区域在漂白前后的荧光强度随时间的变化示于图2.14。说明：细胞膜具有流动性，膜表面上Fc受体能够迁移。Page15ProteinDiffusioninMammalianCellCytoplasm（PLoSONE|August2011|Volume6|Issue8|e22962）Methods1.CellcultureNordenlaboratoryfelinekidney(NLFK)andHeLacellsweregrowninDulbecco’smodifiedEaglemedium(DMEM)supplementedwith10%fetalbovineserum(Gibco,Paisley,UK)at37℃inthepresenceof5%CO2.2.FRAPexperimentsTheFRAPexperimentswereperformedonalaserscanningconfocalmicroscopeFV1000withanIX-81microscopeframe(Olympus,Tokyo,Japan)usinganOlympusUPLSAPO606(NA=1.2)waterimmersionobjective.Thesamplestagewasheatedto37uCpriortheexperiments.Toimagethecellgeometry,aconfocalstackwasacquiredbeforeandaftertheFRAPexperiment.Thevoxelsizewasadjustedto(200nm)3or(150nm)3.Thepinholesizewasadjustedto1Airyunit.The514nmlaserlinewasusedforEYFPexcitationandtheemittedfluorescencewasdetectedusinga530–600nmbandpassfilter.Imagingwasperformedwithalaserintensityof0.1–2Forbleachingacircular(r=1.85mmand2.83mm)regionofinterest(ROI)wasdefinedinthemiddleofthecytoplasm.AsbleachingtimesinFRAPareusuallyratherlargecomparedtothetimescalesofthemeasureddiffusionprocesses,theregionofthecell,whichisactuallybleached,isusuallylargerthanthedefinedROI.Thesizeoftheactuallybleachedregionanditsintensitydistributionweremeasuredbybleachingfixedcells(Fig.1).ImageJ[32]wasthenusedtoconstructanaverageshapeandintensityprofileofthatregion.Page16ThedurationofthebleachprocesswasmeasuredbyperformingFRAPexperimentsinwhich10imageswerecollectedbeforethebleachpulseand1afterthepulse.Thebleachtimewasextractedbymeasuringthetimewhentheframesimmediatelybeforeandafterthebleachpulseweretaken.Theaverageimagingtimeofoneframewassubtracted,andthedurationofthebleachprocesswasplottedasafunctionofiterations(bleachingtime),yieldingalinearslope.IntheLSCMused(OlympusFV1000),theshortestpossiblebleachprocedure(1iteration)lasted36mswithanadditionalrelayof18msbeforethenextimagescan,amountingto54msfortheentireprocess.Toachieveenoughbleachingforthedataanalysis,10iterationswereperformed(Fig.2),andthelaserintensitywassetto100%byusinganacousto-opticaltunablefilter.3.ImageprocessingTherawimagesoftheconfocalmicroscopewereconvertedto8-bitgreyscaleimages.Onlylinearadjustmentsoftheimagebrightnessandcontrastwereperformed,avoidingsaturation.Thegray-scaleimageswerecoloredwithanappropriatelook-uptableandconvertedtoRGBimages.4.ConventionalFRAPanalysisPage17Figure1.FRAPexperimentinanNLFKcellstablyexpressingEYFP.(a)Theaverage(n=10)bleachprofilemeasuredonfixedcellsexpressingEYFP.Scalebar2mm.(b)Fluorescencedistributionbeforethebleachpulseandthepositionofthecircularbleacharea(diameter20pixels,FWHM3.7mm).Subsequentimagesshowthefluorescencedistributionimmediately(t=0ms),and250msand1safterthebleachpulse.Scalebar10mm.(c)Themeasuredrecoverycurve(Axelrodnormalization)andafitbythefree-diffusionmodelofSoumpasis.doi:10.1371/journal.pone.0022962.g001Page18FRAPanalysisWeperformedFRAPexperimentsonEYFP-expressingNLFKandHeLacells.WhenthemeasuredrecoverydatawereanalyzedbytheSoumpasismethod,wefoundacytoplasmdiffusioncoefficientofD~0:75+0:3mm2/s(n=8)fortheNLFKcellsandD~1:83+0:28mm2/s(n=13)fortheHeLacells.Thedifferenceintheliquidphaseproperties(‘viscosity’)ofthesecellsisstatisticallysignificant(pv0:05),whichindicatesthattheyhavedifferentmacromolecularconcentrations.Theaveragecytoplasmdiffusioncoefficients,SDcpT,were15:5+2:7mm2/sfortheNLFKand20:6+5:0mm2/sfortheHeLacells,beingthusquitesimilar.Inbothcelllinesthecytoplasmdiffusioncoefficient,Dcp(r),variedsignificantly(Fig.7c).Page19Theemphasiswashereonthecytoplasm,butthemethodautomaticallyproduceddiffusioncoefficientsforthenucleus.Thenucleosoldiffusioncoefficients,Dnsol,werefoundtobe28:5+16:3mm2