反褶积课件_1

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1、Image-ProPremierBasicTrainingCourseDeconvolutionDiffractionOpticalsystemareimperfectatresolvingOpticalsystemsresolvelightfromaplanetoaplaneOutoffocusobjectsstillpresentinplaneoffocuswww.mediacy.comFocusingwww.mediacy.comFocusingExamplesofConvolutionPl

2、aneViewCapturedRealExamplesofConvolutionMaximumIntensityProjectionCapturedRealConvolutionConvolutiontheprocesswherebyinformationistransformedSystematictoopticalconditionsReversingtheeffectiscalleddeconvolutionPrinciplesofDeconvolutionIdentifypointsofli

3、ghtoriginMeasurepointspreadfunctionIdentifyoutoffocuslightmeasurementsReassignmeasuredlighttopointoforiginEffectofDeconvolutionIncreasedspatialresolutionIncreasedintensityresolutionNoiseremovalAcquisitionRequirementsNyquistspacingforacquisitione.g.100x

4、1.4NAOil550nm:XY<0.07um,Z<0.3umAcquireseveralplanesaboveandbelowtheplaneoffocusMatchlensandimmersionRIAvoidsaturation!DeconvolutionMethodsNearestNeighbourFast,easy,butnotveryaccurateInverseFilterCalculatefullvolume~PSF-1,applytodataIterativeDeconvoluti

5、on(MLE,others)Improveswithmoreiterations,reducesnoiseBlindIterativeDeconvolutionRefinesPSFaswellasthesampleDeconvolution,Confocal,orBoth?WFDeconvolutionPlusesMinimalphotobleaching(invivo,lowstabilitydyes)Fastacquisition(timeresolution,livesamples)Lowsy

6、stemcostWFDeconvolutionMinusesAbsorptionorRIchanges(lensingsamples)candistortreconstructionRequiresabove/belowslicesProcessingtime(canbedoneoff-line)Deconvolution,Confocal,orBoth?ConfocalPlusesCangodeepinabsorbing/distortingsamples(thicktissuesamples)S

7、ingleslicesrequireonlyasingleacquisitionFastertotaltimetoresultsConfocalMinusesPhotobleaching/phototoxicitySloweracquisitiontimesLowersignal/noiseratioHowdoIknowmydataisn’t“Photoshopped”?Deconvolvingknownstructures(beads,microtubules,etc)demonstratesth

8、atdeconvolutionaccuratelyrestoresthosestructures;withintheinformationlimitsofthemicroscopeIsdeconvolveddataquantitative?Note:fluorescenceimagingdoesnotprovideabsoluteintensitiesoutsidesinglemoleculeorinternalreference(ratio,FRET)imaging