流式细胞原理讲座.ppt

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1、FlowCytometryBectonDickinsonAsiaLimitedCompanyFlowCytometryBasicPrincipleBasicPrincipleofFlowCytometerConceptionCombinedSystemBasicPrincipleWhatCanaFlowCytometerTellusaboutaCell?ReviewWhatisFlowCytometer?Atechnologythatsimultaneouslymeasuresmultiplecharacteristicsofsinglecellsatarapidrat

2、e.AModernCytometerNeedsaCombinedSystemof:FluidicsTointroduceandfocusthecellsforinterrogationOpticsTogenerateandcollectthelightsignalsElectronicsToconverttheopticalsignalstoproportionalelectronicsignalsanddigitizethemforcomputeranalysisComputerBasicPrincipleFlowCellInjectorTipFluorescence

3、signalsFocusedlaserbeamSheathfluidForwardAngleLightScatterFALSSensorLaser90DegreeLightScatterFALSSensor90LSSensorLaserDotplotLaserFluorescenceDetectorsFluorescenceFALSSensorFluorescencedetector(PMT3,PMT4etc.)488nmlaser+-FluorescenceActivatedCellSortingChargedPlatesSinglecellssortedintote

4、sttubesFALSSensorFluorescencedetectorOpticalFiltersDichroicFilter/Mirrorat45degReflectedlightTransmittedLightLightSourceStandardBandPassFiltersTransmittedLightWhiteLightSource630nmBandPassFilter620-640nmLightStandardLongPassFiltersTransmittedLightLightSource520nmLongPassFilter>520nmLight

5、TransmittedLightLightSource575nmShortPassFilter<575nmLightStandardShortPassFiltersPMTPMTPMTPMTDichroicFiltersBandpassFiltersFlowCytometryOpticsLaser1234FlowcellEthidiumPEcis-ParinaricacidTexasRedPE-TRConj.PIFITC600nm300nm500nm700nm400nm457350514610632488CommonLaserLinePropidiumIodide400n

6、m500nm600nm700nmPIDNAExcitationEmisson300nm400nm500nm600nm700nmFluorescein(FITC)400nm500nm600nm700nmWavelengthProteinExcitationEmisson300nm400nm500nm600nm700nmExcitationEmisson300nm400nm500nm600nm700nmPhycoerytherin(PE)ProteinAllophycocyanin(APC)Protein632.5nm(HeNe)ExcitationEmisson300nm

7、400nm500nm600nm700nmBasicsofFlowCytometryCellsinsuspensionflowinsingle-filethroughanilluminatedvolumewheretheyscatterlightandemitfluorescencethatiscollected,filteredandconvertedtodigitalvaluesthatarestoredonacomputerFluidicsOpticsElectronicsWhatCanaFlowCytometerTellUsAbou