local field fluorescence microscopy imaging cellular signals in intact hearts 2017 yuriana aguilar-sanchez资料.pdf

该【local field fluorescence microscopy imaging cellular signals in intact hearts 2017 yuriana aguilar-sanchez资料 】是由【四婆子】上传分享，文档一共【13】页，该文档可以免费在线阅读，需要了解更多关于【local field fluorescence microscopy imaging cellular signals in intact hearts 2017 yuriana aguilar-sanchez资料 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..JournalofVisualizedExperimentsVideoArticleLocalFieldFluorescenceMicroscopy:ImagingCellularSignalsinIntactHeartsYurianaAguilar-Sanchez*1,DiegoFainstein*2,3,RafaelMejia-Alvarez4,,UniversityofCalifornia,Merced2CentrodeInvestigacionesCardiovasculares,UniversidaddelaPlataandConicet3FacultaddeIngenieria,UniversidadNacionaldeEntreRios4DepartmentofPhysiology,MidwesternUniversity5SchoolofEngineering,UniversityofCalifornia,Merced*TheseauthorscontributedequallyCorrespondenceto:ArielL.******@:https://eo/55202DOI:doi::CellularBiology,Issue121,Intactheart,fluorescence,pulsedlocalfield,calcium,actionpotential,Rhod-2,di-8-ANEPPSDatePublished:3/8/2017Citation:Aguilar-Sanchez,Y.,Fainstein,D.,Mejia-Alvarez,R.,Escobar,:.(121),e55202,doi:(2017).AbstractIntheheart,,,wepresentthespectroscopictechniqueoflocalfieldfluorescencemicroscopy(LFFM)[Ca2+],intra-sarcoplasmicreticulum[Ca2+](PLFFM)useslaserlightpulseswhilecontinuouswaveLFFM(CLFFM),light-emittingdiodes(LEDs)-coherentarrangementiscalledpulsedLEDfluorescencemicroscopy(PLEDFM).VideoLinkponentofthisarticlecanbefoundathttps://eo/55202/'scontractionisinitiatedbyanincreaseinintracellular[Ca2+].TherelationshipbetweenelectricalexcitabilityandchangesinintracellularCa2+releasehasbeenhistoricallystudiedinenzymaticallydissociatedcells1,,ellsareelectrically,metabolicallyandmechanicallycoupled3,,themyocytesarenotonlyphysicallyuncoupled,,despitetheenormousadvantagesthathaveemergedfromthestudyofisolatedcellsundervoltageclampconditions,6,7,,wedescribetheadvancesobtainedintheknowledgeofcardiacphysiologybytheuseoflocalfieldfluorescencemicroscopy2+(LFFM)+,intrasarcoplasmicreticulum(SR),10,electrocardiograms9,electricalactionpotentials(APs),urrentrecordingsandflashphotolysisofcagedcompounds4,,,11,12,13,14havebeenpublishedbyourgroupusingLFFMtechniques,(Figure1),theopticalresolutiondependsonthediameterandthenumericalaperture(NA),(S/N),?2017JournalofVisualizedExperimentsMarch2017|121|e55202|Page1of13:..JournalofVisualizedExperimentsInordertoexcitetheexogenousfluorescentindicatorsloadedinthetissue,,12(PLFFM)employedalow-costpicosecondlaser(Figure1a,PLFFM).,+.Unfortunately,thetemporaljitteringofthepulsesandvariationsinamplitudefrompulse-to-(CW)lasersareusuallyusedasthemainilluminationsourceinLFFM(Figure1b,CLFFM).,multipledyeshavingd2+ifferentspectralpropertiesca2+nbeexcitedtoperformmeasurementsofavarietyofphysiologicalvariables,forexample,Rhod-a,MagFluo4forintra-SRCaanddi-8-,othertypesoflightsourcescanbeusedincludinglight-emittingdiodes(LEDs).Inthiscase,theexcitationlightsourceconsistedofanInGaNLED(Figure1c,PLEDFM).InLEDs,---8-ANEPPSandforCa2+transientsrecordedusingFluo-4orMag-Fluo-4,weusedanLEDthathasatypicalpeakemissionat485nm(blue)andahalfwidthof20nm(Figure1d).ForCa2+transientsrecordedwithRhod-2,theLEDhadatypicalpeakemissionat540nm(green)andahalfwidthof35nm(Figure1d).,-,,themaindrawbackofusingLEDsisthattheiremissionprofileshaveangulardisplacements(±15°)fromthemainaxis,,,,theemittedlightisfocusedwithanobjectiveontoaphotodetector(Figure1).:atransimpedanceamplifierhavingaresistivefeedbackelement(Figure1e)orbyanintegratortoconvertthecurrentintoavoltage(Figure1f).Usingthefirstapproach,,(*).Apeaktrackingalgorithmwasimplementedtodetectthepeak12(red)andthebase(green)+(Rhod-2),theoutputvoltageoftheintegratorisafunctionofthecurrentandcapacitivefeedback(Figures2d,2e,and2f).Figure2fshowstwoconsecutiveintegrationcycles:,althoughmorelaborious,providesalargerS/-,anyA/,,independently,+usedtomeasuretransmuralelectricalorintracellular[Ca]levelswithminor-,(-201).ExperimentsmissionoftheVenezuelanInstituteforScientificResearch(IVIC).Copyright?2017JournalofVisualizedExperimentsMarch2017|121|e55202|Page2of13:..:140NaCl,,2CaCl2,1MgCl2,,10glucose,-up11,%O2usingasubmergedplastic"airstone"""-(seeFigure4)thatallowsretro-,theheart'(,injectwith20unitsor200μL):,-absorbablesurgicalsuture,tietheaortaontothehorizontalLangendorffappa