Camera pixel size and photon collection - Andor Technology

Figure 2: Large pixel sizes are used in EMCCD and CCD cameras to gain advantage of the superior photon collection efficiency and improved probability of photon ... PartoftheOxfordInstrumentsGroup Expand Collapse OxfordInstruments Applications Products News Events Contact en cn jp Investors Careers LifeScience CamerasEMCCDCamerassCMOSCamerasLowNoiseCCDCamerasSuper-ResolutionCamerasMulti-WavelengthImagingEx-DemoCameras ConfocalMicroscopyDragonflyConfocalMicroscopesBenchtopConfocalMicroscopesRevolutionUpgradesandWindows10MicroscopySolutionsPhotostimulationSolutionsIntegratedLaserEngineCryostatsforMicroscopy ControlSoftwareSolisPackagesSoftwareDevelopmentKitImageAnalysisSoftwareImarisPackagesImarisFreeTrialKomet MarketsCancerCellBiologyDevelopmentalBiologyNeuroscienceSingleMoleculeVirology   PhysicalScience CamerassCMOSCamerasEMCCDCamerasLargeCCDCamerasIntensifiedandGatedCamerasX-RayandNeutronCamerasSpectroscopyCamerasEx-DemoCameras SpectroscopySpectrographSolutionsDetectorsforUVtoNIRandSWIRSpectroscopyAccessoriesOpticalCryostatsOpticalCryostatsforSpectroscopyOpticalCryostatsforMicroscopy ControlSoftwareSolisPackagesSoftwareDevelopmentKit MarketsAdvancedMaterialsAstronomyBio-ScienceChemistryFluidMechanicsMetals,Alloys,Composites&CeramicsQuantumOpticsTomography   OEM PortfolioOEMRelationshipswithAndorScientificCamerasSpectroscopySolutionsSoftwareOpticalCryostatsMicroscopySolutions   Learning LEARNINGCENTREUpcomingWebinarsOn-DemandWebinarsMicroscopyTrainingCourseCaseStudiesApplicationNotesTechnicalArticles TOOLS AstronomyCalculatorCameraWindowSelectorHeliumCostCalculatorResolutionCalculatorSignaltoNoiseCalculatorThirdPartySoftwareCompatibility   Support ServiceandSupportSupportRequestRequestanewRMAFindaSupportCentreServiceProductsLiveAssist DownloadsSoftwareDownloadsDriversSoftware/UserManualsQuick-startGuidesCodeSnippets   News Events Contact eCommerce LearningCentre Asset WhenBiggerPixelsRule-PixelSizeandPhotonCollectionEfficiency Oneofthetrendsthatcanbeseeninmicroscopyinrecentyearshasbeentheinterestinimagingsensorswithlargerfieldsofviewandsmallerpixelsizestomaintainimagedetailwhenworkingatlowermagnificationssuchas20x.Butaresmallerpixelsalwaysbetter,oraretherestillconditionsinwhichlargerpixelsareadvantageous?Inthisarticleweconsiderpixelsizeandwhetherlargerpixelsizesarestilluseful,andifso,wheremaythisapply. TheRoleofthePixel Theroleofapixelinasensoristocollectphotonswithinasubunitoftheimagingareaandsubsequentlyconvertthesetoanelectricalsignal.Bydigitizingthesesignals,animagecanberecreatedfromthevaluesreceivedateachpixel.Themoreefficientatgatheringlightandconvertingthislighttoanelectricalsignal,themoresensitivethedetectorcanbe.ThishasbeenakeydriverofCCDandCMOSsensortechnologieswithprocessessuchasback-illuminationanduseofmicrolensbeingexamplesofwaystoboosttheefficiencyofthesedevices.Thelatestsensorsavailablehavequantumefficienciesreachingupto95%i.e.95%ofincomingphotonsareconvertedintoelectronsinthephotosensitiveregionofthesiliconcomparedtodevicesalittleover10yearsagobeinglimitedtoaround60%peakQE. Figure1:Modernsensorformatsallowforhighsensitivitybyacombinationofhighphotoncollectionefficiencyandconversionandlownoiseelectronics.Thisillustrationshowsaback-illuminatedsensorinwhichthecircuitryisbeneaththephotosensitiveregionandthusdoesnotblocklightfromreachingtheregioninwhichphotonsmaybeconvertedtoelectrons. Whateffectsdoespixelsizehaveonimaging? Developmentsinsensordesignandfabricationhasenabledevermoresmallerpixelstobepackedintosensors.Whilemorepixelscanbebetter,therearetrade-offssothepixelsizemustbebalancedtosuittheneedsoftheapplication.Therearethreemainaspectsofhowpixelsizemayimpactimaginginadditiontoadvancesinothersensortechnologyenhancementssuchasback-illumination.Thesearesummarizedintable1: SensorParameter ImageParameter Description PhotonCollectionEfficiency Signaltonoise Themoresensitiveasensoris,themorelowlevelinformationcanbeobtained.Thiscanbethedifferencebetweendetectingasignalandnot.Asensorwithahighphotoncollectionabilitywillhelpcollectmoresignalwhichwillboostthesignalagainstthenoiseofthesensoritself. Imagesampling“Resolution” ImageDetail ResolutionistheabilitytodistinguishtwoobjectsfromtheirAirypattern.TheresolutionisstrictlydefinedbywhatispossiblethroughthemicroscopeviatheRayleighequation:0.61xWavelength/objectiveNA.TheimagingdetectorshouldhavesufficientsamplingoftheimagetomeetNyquistcriteriaandmaintainthatresolutionandthustheimagedetail. Welldepth DynamicRange Dynamicrangeistherangebetweenthelowestandhighestlevelsofsignalwithinoneimage.Dynamicrangeofthesensorcanbederivedfromthewelldepthofthesensordividedbythenoisefloor.Thisassumesthatthesensorsupportingelectronicdesignisoptimisedtohandlethepotentialdynamicrangeanddosoinalinearrelationshipi.e.bit-depthandamplifierdesigns Table1:Sensorparameterslinkedtopixelsizeandrelationshiptotheimage Matchingpixelsizestomicroscopefieldofviewandmaintainingresolutionisdiscussedmorecomprehensivelyinthearticle:OptimizingfieldofViewandResolutionforMicroscopy.Inthefollowingsectionswewillfocusonpixelsizeandphotoncollectionefficiency. PixelsizeandPhotonCollectionEfficiency Intable2wecanseethepixelsizesofarangeofcurrentcommerciallyavailablehighperformanceimagingsensorsthatmaybeusedformicroscopy: SensorTechnology Camera Pixelsize(µm) PixelArea(µm2) Differenceinpixelphotoncollectionarea CMOS Back-illuminatedCMOS 4.6 21.2 1x FrontilluminatedsCMOSe.g.Zyla4.2PLUS 6.5 42.25 2x Back-illuminatedsCMOS e.g.Sona-6 6.5 42.25 2x Back-illuminatedsCMOSe.g.Sona-11 11 121 5.7x EMCCD Back-illuminatedEMCCDe.g.iXon888 13 169 8x Back-illuminatedEMCCDe.g.iXon897 16 256 12x CCD Back-illuminatedCCDe.g.iKon-M 13 169 8x Back-illuminatedCCDe.g.iKon-L 13.5 182.25 8.6 Table2:Thephotoncollectionareasforeachpixelforarangeofdifferentcameramodels Ingeneral,CMOSsensorshavesmallerpixelsizesthanEMCCDandCCDsensors.TheCMOSsensorarchitecturealsoallowshigherspeedsandlargersensorsizesthanCCDbaseddesigns.ThesequalitiessuitgeneralfluorescenceimagingapplicationsallowinggoodimagedetailatcommonmagnificationsforcellbiologystudiesandhavemadesCMOSthedominantdetectortechnologyformanymicroscopists.DespitetheriseofsCMOSdetectors,EMCCDandCCDdetectorshaveremainedasthebestoptionforsomeofthemostchallengingimagingapplicationsandthislookssettocontinueforsometimetocome:•EMCCDcamerasuseElectronMultiplicationtoeffectivelyeliminatereadoutnoise.Thisuniquefeaturemakesthissensortechnologyidealformakingprecisequantitativemeasurementsdowntoevenasinglephotonsuchassinglemoleculeimaging,butalsoforlive-cellconfocalimagingandsuperresolutionsystems.•DeepcooledCCDcamerasfeatureordersofmagnitudelessdarkcurrentthanCMOSdesignsmakingthemperfectlysuitedforlongexposureluminescenceexperimentsthataredarkcurrentlimitedandtheslowerreadoutofCCDisnotafactor. ThelargepixelsizesthattheseEMCCDandCCDcamerashavealsoplaysanimportantfactorinwhytheyremainsuitablefortheseimagingscenarios.Whenphotoncollectionisthepriority,alargerpixelactseffectivelyasalargercatchmentareaforphotons-feedinginmoresignaltothesensor.ThisiswhythelatestEMCCDsensordesignsfeaturepixelsizesintherangeof13-16µmwhicharenativelysuitedtophotoncollectionathighmagnificatione.g.100x.NotethatEMCCDcameraswithsmallerpixelsizesareavailable,buttheyhavenotgainedanymarkettractionastheydonotprovideapplicationbenefitsoverlargepixelEMCCD,ortosCMOScameraoptions.Inthefollowingillustration,aniXonUltra888EMCCDcamerahasa4xhighernativepixelareathanatypicalsCMOScameraandan8xlargerpixelareathantheCMOSsensor. Figure2:LargepixelsizesareusedinEMCCDandCCDcamerastogainadvantageofthesuperiorphotoncollectionefficiencyandimprovedprobabilityofphotonbeingdetected.Thisexampleillustratestherelativesizesof13µm,6.5and4.6µmpixel. WhataboutthelargerpixelsCMOSsensorformats? Therearesomeback-illuminatedsCMOScamerasavailablewithapixelsizeof11µmpixel-whichisarelativelylargepixelwhencomparedtotypicalCMOScameras.Thesecameramodels,suchastheSona-11sCMOSseries,cantakeadvantageofthelightgatheringpower3-foldhigherthanthe6.5µmpixelsizetypicallyfoundinsCMOScameras. Forsomeapplicationssuchassinglemoleculedetection,andforhighmagnification,thelargerpixelsCMOScamerasliketheSona-11canoffersomebenefits: HighersensitivityoverthesmallerpixelsCMOScamerasandfieldofview Higherspeedsoveralargerfieldofview,andincreasedimagesamplingcomparedtotheEMCCDcameras. However,forthemorechallengingsinglemoleculeexperiments,thelargerpixelsizeandhighQEisnotalwaysenough.ElectronmultiplicationprocessuniquetoEMCCDsensorsboostthesignalmanyfoldpriortoreadoutsothesignalisfarabovethenoisefloorofthecamerareadoutelectronicsandinpracticeproventooperateatphotonlevelsbelowwhatispossiblefromanyCMOStechnology SignaltoNoiseComparisons Herewecompare2cameraswithallfactorsthesame(QE,readnoise,darkcurrent)otherthantheirpixelsizeandcomparetheeffectofpixelsizeonrelativethesignaltonoiseperformanceaslightofagivenilluminationfallsoneachsensor. Camera PixelSize(µm) PixelArea(µm) Signal(photons) Noise 1 6.5 42.25 10 2 2 13 169 40 2 Table3:SimplifiedSignaltoNoisecomparisontoillustratetheincreaseinthesignalcomponentprovidedbypixelsize. Inthesimplestcomparison,camera2wouldlooktoprovideasignificantlyhighersignaltonoiseratiosincethesignalcomponenthasincreasedforeachpixel.Toconsidersignaltonoiseforimagingcamerasweneedtousethefollowingsignaltonoiseequationtomodelthedifferentsignalandnoisecomponentsinvolved: Usingthisinformationwecanplotthesignaltonoiseagainstthenumberofphotons,accountingforilluminationintensityoverthesensorbyexpressingphotonsonanareabasis–inthisexamplewewillexpressper13µm2sensorarea: Figure2:Acomparisontheeffectofpixelsizeonsignaltonoiseperformanceof2sensorswithallfactorsthesameotherthanpixelsizewithincidentlightasphotonsnormalisedoverper13µm. Fromthiswecanseethatthelargerpixeldoesindeedallowforahighersignaltonoise-some~2.5xhigherat10photonsper13µm2.Thiswilltranslatetothefollowingbenefits: Improvedsensitivity–havingalowerlimitofdetection Higherconfidenceindataatlowsignallevels Reduceexposureswhenlightismoreplentifulwhichisbeneficialtomanylivecellstudies. Wecanthenapplythistoreal-worldcameraexamples: Figure3:Camerasdevelopedforsensitivityandhighestpossiblesignaltonoisefeaturelargepixelsizes.Illuminationcorrectedforpixelsizebynormalizingper13µmarea. Inthecomparisonillustratedinfigure3wecanseethatthecameraswithlargerpixelsprovidethebettersignaltonoisefiguressuchastheiXonUltra888andSona-11modelswhichalignswiththeirintendedapplications,thatprioritizesensitivityanddetection,overspatialresolutionatlowlightlevels.TheotherCMOSmodelswithsmallerpixelsdonotprovideashighsignaltonoiseratioseventhoughtheyhavelownoiselevels.Theywillprovideimprovedspatialresolution. CanyouusepixelbinningtoincreaseSignaltoNoisePerformanceofsmallpixelCMOSsensors? Smallerpixelscanbebinned–wherebythesignalinanumberofpixelsmaybepooledtogethertoincreasetheoverallsignallevel.Acommonexamplewouldbe2x2binningwhichwouldgroupthesignalfor4pixelstogetherasshowninfigure3.ThisworkseffectivelyforCCDinboostingspeedandsensitivityastheserialnatureofCCDallowseachpixelvaluetobeaddedbeforeareadnoiseisadded.Binningalsoworksespeciallywelloutsideofimagingforspectroscopymeasurementsasverticalbinningmaybeappliedwithoutconcernoflosingresolutionintheyaxis. Figure4:2x2binningappliedtosensortoboostphotoncollectionacross4pixelsandthesignaltonoiseratioattheexpenseofspatialresolution. ThearchitectureofCMOShoweverisdifferent.Eachrowhastobereadoutsequentiallyonarow-by-rowbasisbeforebinning-meaningthatfortheCMOSsensorsinusetodaybinningwillnotdeliverthebenefitstosignaltonoise(andspeed)thatitdoesforCCD.DigitalbinningforsCMOSstillcombineshevaluesfor4pixelsinthecaseof2x2binning,butthereadnoisealsoincreases2-foldsincethe2pixelrowsarereadout. Figure5:WhilebinningdoesnotworkaswellforCMOSasCCD,itstillallowsaboostinsignaltonoisecomparedtowithoutattheexpenseofspatialresolution. Forthesmaller4.6µmpixelsoftheCMOSsensor3x3binningwouldberequiredtoobtainthesamephotoncollectionareaasa13µmpixel.3x3binningwouldthusincreasereadnoiseinthissensortypebyafactorof3. Whataboutopticalmatching? Ideallythesensorsizeshouldfitwithintheuniformregionavailabletothemicroscopeandthepixelsizewouldmatchtheobjectiveandmagnificationusedforimaging.However,thisissometimesnotthecasefordifferentcombinationofsensors,microscopesandapplicationrequirements.Additionalpostobjectivemagnificationmaybeusedtoavoidvignettingeffects,orexpandthefieldofviewtocoverthatofalargersensorarea.TheotherapplicationofadditionalmagnificationistohelpmeetoptimalsamplingoftheimagetomeetorexceedNyquistcriteriae.g.addinga2xlenswithreducethepixelsizebyx2andhelpimprovespatialresolution,butreducethefieldofview.Thus,whileuseful,thereareanumberofconsiderationsofusingadditionallensesintheopticalsystem.Thistopicisdiscussedfurtherinthearticle:OptimizingfieldofViewandResolutionforMicroscopy. LargestPixelsmeanLargerWellDepthsforHighDynamicRange Thefinalaspectofimagingperformancethatislinkedtopixelsizethatcanbebeneficialisthewelldepthofthesensorandhowthiscanbeusedtoallowforhighdynamicranges.Whenimplementedcorrectly,largerpixelsoftenallowlargersignalhandingcapacitiesthanispossibleusingsmallerpixels. SensorTechnology Camera Pixelsize(µm) WellDepth(e-) CMOS Back-illuminatedCMOS 4.6 7,000 FrontilluminatedsCMOSZyla4.2PLUS 6.5 30,000 Back-illuminatedsCMOSe.g.Sona-6 6.5 55,000 Back-illuminatedsCMOSe.g.Sona-11 11 85,000 EMCCD Back-illuminatedEMCCDe.g.iXon888 13 80,000 Back-illuminatedEMCCDe.g.iXon897 16 180,000 CCD Back-illuminatedCCDe.g.iKon-M 13 100,000 Back-illuminatedCCDe.g.iKon-L 13.5 150,000 Table4:Acomparisonofthewelldepthofaselectionofcameras.Notethatmaximumdynamicrangemayberestrictedtocertainmodesavailableforeachofthedifferentcameramodelssoisnotpresentedhere. Theimpactinimagingtermswillbethatusingsmallpixelsmaymeanalowdynamicrangethatcanresultinoversaturationandloseofimageinformation.Therefore,forapplicationsinwhichahighdynamicrangeisimportantawelloptimisedsensorwithlargerpixelsizesmaybemoresuitablethanonewithsmallerpixels. Imagingscenariosonemayrequirehighdynamicrangeinclude: Luminescence:experimentsmayinvolveveryfainttohighsignallevelsprovidebiologicallyrelevantinformationongeneexpression. Neurons–imagingneuronscansometimesposeachallengeastherecanbealargeintra-scenedynamicrangeduetostrongsignalsinthecentralregionoftheneuronwhichdistalregionsexhibitlowlevelfluorescence. CalciumImaging–GiventhevastnumberoffunctionsofCalciumtherearemanydifferentexperimentconditions.Forsomeofthese,Calciumsparksandcertainstudiesofcardiacandothercellstheyalsorequirewidedynamicranges. Large3Dvolumes–whenimaginglargedatasets,differentregionsofe.g.Zebrafishmayhavelargedifferencesintheirsignallevel.Thiscanmakesettingexposureandotherimagesettingsamatterofsometrialanderrortoobtainagoodimage.Asensorwithawidedynamicrangehelpshandlethedynamicnatureofsuchdatasets. Afurtherbenefitofwidedynamicrangeisfromapracticalsense–asensorwithawidedynamicrangeismoreofteninrangeandavoidssaturationrequiringlessadjustmentofsettingssuchasexposure. Conclusions Whilethereisinterestinimagingcameraswithsmallpixelstopreserveimagedetailatlowmagnificationsthereisstillverymuchtheneedforcameraswithlargerpixelsizesforlowlightapplications. Cameraswithsmallpixelsizessuitlowmagnificationstudies.Manymodelsavailablethatworkunderthesehigherlightregimes. The6.5µmpixelsizethatiscommontomanyscientificgrademicroscopycamerasprovidesagoodbalancebetweenphotoncollectionandresolutionfortypicalfluorescencebasedapplications. Whenlookingtogetthebestpossiblesensitivityfromadetector,cameraswithlargepixelsizesarethemostapplicablee.g.forsinglemoleculeimagingandluminescence BinningcanbeusedtoincreasesignaltonoiseinbothCCDandCMOSsensors,howeverforCMOSsensorsthereislessbenefitfrombinningasthereisforCCD. Thereisnoonecamerathatdoesallimagingscenariosbetterthaneveryothercamera.Thankfullytherearenowarangeofcamerasthatcanbeselectedtosuitdifferentapplicationsthatneedforexamplethehighestspatialortemporalresolution,orthebestpossiblesensitivity.Thismeansthatitisimportanttoconsidertheapplicationneedsfirstandforemostandnotassumethatthelatestcameramodelwiththelargestsensorandmostmegapixelswillbethebestoption. Date:June2021 Author:DrAlanMullan Category:TechnicalArticle Downloadaspdf Share RelatedProducts iKon-M934 ViewProduct iXonUltra888 ViewProduct SuggestedReading BalorsCMOSTestsattheAGO70TelescopeatComeniusUnivers... BALORIRIG-BGPSFeature–FAQ IntroducingthenewAndorBenchtopConfocalMicroscope-BC4... 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