NuclearIncorporationofIronduringtheEukaryoticCellCycleIanRobinsona,b,YangYangc,FucaiZhanga,b,ChristopheLyncha,b,MohammedYusufa,bandPeterCloetenscaResearchComplexatHarwell,RutherfordAppletonLaboratory,Didcot,Oxon,OX110FA,UKbLondonCentreforNanotechnology,UniversityCollegeLondon,WC1E6BT,UKcEuropeanSynchrotronRadiationFacility,38043Grenoble,FranceScanningX-rayfluorescencemicroscopyhasbeenusedtoprobethedistributionofS,PandFewithincellnuclei.Nuclei,whichmayhaveoriginatedatdifferentphasesofthecellcycle,arefoundtoshowverydifferentlevelsofFepresentwithastronglyinhomogeneousdistribution.PandSsignals,presumablyfromDNA,arehighandrelativelyuniformacrossallthenuclei;theseagreewithX-rayphasecontrastprojectionmicroscopyimagesofthesamesamples.WediscusspossiblereasonsfortheFeincorporation.1.IntroductionTheeukaryoticcellnucleuscontainsallthegeneticmaterialresponsibleforthepropagationoflifefromonecellgenerationtothenext.Allthecell’sDNAresidesthere,confinedwithinaporousnuclearenvelope,alongwithoverahundreddifferentproteinmolecules(Uchiyamaetal2005)associatedwithchromatin,theDNA-proteincomplexmakingupthechromosomes.Overthecourseofthecellcycle,themassofDNAincreasesfromtwocopiestofourduringSphase,thenreturnstotwocopiesuponcelldivisionduringmetaphase(M-phase).TheDNAmassremainsconstantduringthetwoinverveninggrowthphasesG1andG2.Forhumancells,investigatedhere,theDNAmassshouldcorrespondtothelengthofthehumangenome,whichis3.2x109basepairs.QuantitativeassessmentoftheamountsofDNAandproteincanbemadebyX-rayfluorescenceusingthePsignalforDNAandtheSsignalforprotein,notingthatmorethantwo-thirdsofthenuclearproteinisintheformofhistones,whichbindstoichimetricallytoDNAthroughthenucleosomes,andwhichcontainaknownamountofcysteineandmethionine(Uchiyamaetal2005).X-rayimagingofbiologicalmaterialshastwoimportantadvantagesoverelectronmicroscopy:theX-raypenetration,sufficienttoavoidsectioningofthesamples,anditsexcellentchemicalsensitivityforelementalanalysisusingfluorescence.Inthiswork,thenewNano-ImagingbeamlineID16A-NI,partoftheUPBL04'NINAfacility’builtintheframeworkoftheESRFUpgradeProgramme(A.Pacureanu,etal.2016)wasusedtoimagehumancellnuclei.Thesizeofahumancellnucluesisintherangeof10µm,whichfallswithinthefieldofviewofthepropagation-basedphasecontrastimagingcapabilityofID16A.Inaddition,high-resolutionsubstructureisexpected,atleastifthenucleiareclosetothemetaphasepointofthecellcyclewhentheparentcellispreparingfordivision.FortheX-rayfluorescenceimagingcapabilitiesofID16A,known

quantitiesofDNAand(toaslightlylesserextent)proteinsareexpectedtobepresentinacellnucleus,whichcanbeusedinquantitativechemicalanalysisandtoverifythecalibrationofthesensitivity.Nucleiclosetometaphaseweretargetedinthisstudybecauseofinterestinthehigher-orderstructureoftheseparatedchromosomeslocatedwithinthem,butitwasalsoappreciatedthatthisreallyneedsathree-dimensional(3D)imagingcapabilitytosegregatethem.Oursamplepreparationmethodsmakeuseofcellcyclecheckpointinhibitorstosynchronizethecellsduringculture,butthisstillallowssomenucleitoemergefromthepreparationsatotherpointsofthecellcycle.Centrifugationisusedtoremovecytoplasmandmostoftheothercellcomponents(Yusufetal,2014),soarelativelypurepreparationofwholenucleiandindividualchromosomesisobtained,manywiththenuclearmembraneintact.Thisstrictlyexcludesnucleiinlatemetaphase,oncethenuclearmembranedissolves,butdoesincludeprophasejustbeforehand,whenthe46chromosomesarefullycondensedwithinanucleus.IfthecellswereinG1phasewhenthesampleswereprepared,theywouldcontaintwodouble-strandedcopiesofallthegenomicDNA;iftheywereinG2phaseorthebeginningofmetaphase(M-phase),thereshouldbefourcopies;inSphase,therewouldbesomewherebetweentwoandfourcopies.Thefullhumangenomecontains3.2x109basepairsperdouble-strandofDNA,whichisdividedintothe23chromosomes.Associatedwitheachbasepairaretwophosphates,oneoneachstrand.ThesearethelargestexpectedcontributiontothePX-rayfluorescencesignal,withsmalladditionalamountscomingfrombuffers,thelipidsinthecellmembranesandanyresidualRNAorATP.Soacellnucleusshouldhaveawell-definedsignalfromthese2.6x1010Patomsinitsfluorescentimagesifitisinthesecondhalfofthecellcycle(G2orMphase),or1.3x1010Patomsinitsfluorescentimagesifitisinthefirsthalfofthecellcycle(G1phase).Similarly,theSX-rayfluorescencesignalwouldbemostlyattributedtoCysteine(Cys)andMethionine(Met)residuesinthenuclearproteins.Fortunately,muchisknownaboutthemake-upofthe(mostlystructural)chromosomalproteinsfoundinmetaphasefromtheworkofUchiyamaetal(2005):71%ofthetotalmassishistones,whicharethecoreproteinsaroundwhichtheDNAisspooledtomakenucleosomes.ThehistonescontainmanybasicArginineandLysinegroups,whichhelpneutralizethenegativechargecarriedbytheDNA.Onenucleosometypicallyoccupies170basepairsofDNAand,sincemostoftheDNAcanbeassumedtohavecondensedintonucleosomes,wecanusethistoestimatetheexpectedtotalamountofproteinpernucleus.Moreover,thehistonesequencesareallknown,sowecanexpecttheretobe14Satoms(2xCysand12xMet)per170basepairsofDNAassociatedwiththehistones(Mariño-Ramírezetal,2011).Wethereforeexpectacellnucleustohave2.1x109SatomsinitsX-rayfluorescenceimagesinG2orMphaseand1.0x109SatomsinG1phase.ThetotalmassofDNAandproteinexpectedinmetaphasecanalsobeestimatedfromthesizeofthehumangenome.Thiscanbecomparedwithquantitative

massesmeasuredbyX-rayphasecontrastimaging.Onebasepairweighs650Da,soonedouble-strandedcopyof3.2x109basepairsweighs3.5pg.Anucleosomeoctamer,thecoreproteinofasinglenucleosome,weighs110kDa.Sowithonenucleosomeattachedtoevery170basepairs,weexpect3.5pgofhistones;allowingfor29%ofnon-histoneproteinbringsthisestimateto4.9pgperdouble-strandedgenomecopy.Inthefirsthalfofthecellcycle(G1),weexpecttofind16.8pgofDNAandproteindirectlyassociatedwiththechromosomes.Inthesecondhalfofthecellcycle(G2/M),weexpect33.6pg.ThepresenceofFeinthecellnucleushasbeendiscussedrepeatedlyinthescientificliterature.Yagietal(1992)havesuggestedtheremaybeanevolutionaryconnectionbetweenironandDNAbecauseofthepowerfulredoxpotentialofFe.Feisanessentialelementofproteins,oftenintheformofiron-sulfur(FeS)clustersusedinelectrontransportenzymes(Johnsonetal,2005)orinhemecomplexesincytochromes(Dawson,1988).Ironcanbetoxictocellsviathegenerationoffreeradicals(Yagietal,1992).SincethepresenceofironcanleadtoDNAdamagepathways,theremaybeevolutionaryadvantagetokeepingtheDNAinitsownnuclearcompartment,awayfrommanyofthemetabolicprocesses.DespitetheviewthatFe-containingenzymeswouldnotbewidelyusedwithinthenuclearcompartmentofthecell,therehavebeenrecentreportsofFeS-containingenzymesdirectlyinvolvedwithDNAreplication.DNAprimasewasfoundtocontainanFeSdomain(Klingeretal2007,Weineretal2007)alongwithDNAhelicase(WuandBrosh,2012)andDNArepairglycosylases(WuandBrosh,2012).AreviewbyLilletal(2006)namedfiveassociationsofFeSproteinswiththecellnucleus:DNAglycosylase(Ntg2),Histoneacetyltransferase(Elp3),P-loopATPase(Nbp35),Iron-onlyhydrogenase(Nar1)andABCprotein(Rli1).AllofthesefunctionsarebelievedtobeassociatedwithDNAreplicationandrepairsoshouldbeexpressedonlyduringSphaseofthecellcycleandshouldbeabsentduringotherphases.Ferritin,theEukaryoticironstorageprotein,isnotexpectedtobecolocalizedwithDNA,yetthiswasreportedinafewdiverseexamplesbyThompsonetal(2002).NuclearferritinmightbeassociatedwiththeprotectionofDNAorconverselywithoxidativeDNAdamage.Ifnuclearferritinispresent,itmightbeexpectedtobeassociatedwiththenuclearmembrane,ratherthanmixedinwiththeDNA-containingchromatin.Oneorganellelittlediscussedinrelationtoirontransportandaccumulationisthenucleolus,asubcompartmentofthenucleuswhichappearsatcertainpointsofthecellcycle.Thereareveryfewmentionsintheliteratureofnucleolariron.Itwasrecentlyshownthatplantnucleolicontainiron(Roschzttardtzetal.,2011),purportedlyboundtoribosomalRNA(rRNA).ItwassuggestedthatironmightstabilizesecondaryRNAstructuresinthenucleolusorotherwisecatalysematurationofrRNAsubunits.Thefollowingyear,itwasshownthathumanneuronalcellsalsocontainnucleolariron(Sukhorukovaetal.,2012),althoughnoexplanationwasprovided.ItshouldbenotedthatthepresenceofnucleolarironinHeLacellswasreportedsomedecadesago(Robbinsetal.1972).Inthecaseof

HeLacells,itwasclearthatironboundtoproteinsinthenucleolusanditwassuggestedthattheorganellecouldbeanFerepositoryforiron-dependentDNAsynthesisproteins.Whetherornottheironcontentofthenucleoluschangesasafunctionofcellcyclephaseremainstobedetermined.Toaddressthesequestions,synchrotron-basedscanningX-rayfluorescence(SXRF)andphasecontrastprojectionmicroscopicimagingofhumancellnucleiwithsub-cellularresolutionwasundertakenintheworkreportedhere.2.Methods2.1SamplepreparationNucleiwerepreparedaccordingtoapreviouslypublishedfiltration-basedprotocolforchromosomes(Yusufetal,2014)withmodificationstopreservetheintactnuclei.HumanLymphocytecells(GM18507)werecultivatedat37°Cina5%CO2incubator.TheRPMI-1640medium(SigmaAldrich,UK)contained20%FetalBovineSerum(SigmaAldrich,UK)and1%l-glutamine.ThecellsweretreatedwithColcemid(0.2μgml−1,GibcoBRL)toarresttheminmetaphaseandwerefixedinof3:1,methanol/aceticacidafter0.075MKCltreatment.Followingextraction,thenucleiwerepreparedforx-rayimagingasdescribedbyShemiltetal(2015).Sampleswerewerefixedinabuffercontaining0.5%Glutaraldehyde,10mMHepes-KOHand5mMMgCl2.Thesampleswerepipettedin2µLdropsonto200nmthicksiliconnitridemembranewindowsandstainedwith150µMSybrgolddyeforopticalfluorescenceimaging.Afterwashinginwater,thesampleswerelefttodryinair.TheywereimagedusingaZeissAxioZ2microscope(usingMetaferIsissoftware)toobtainvisiblelightandopticalfluorescenceimagesforreferenceandcorrelationwiththeX-rayresults.ForX-rayimaging,severalsiliconnitridemembraneswerepreparedwiththesamesamplematerial.Theresultingsampleswerefoundtocontainalargenumberofintactnuclei,butalsochromosomespreadsandindividualchromosomesfromburstnuclei.Someofthemembrane-boundsampleswerestainedwithplatinumblue(WannerandFormanek,1995),at5mMfor30minutesandwashedinwater.NosignificantdifferenceswerefoundintheX-rayphasecontrastimages,howeverthePtX-rayfluorescenceM-line-signalwasfoundtostronglyinterferewiththefittingoftheX-rayfluorescencespectraduetooverlapwiththePK-lines.ResultsarethereforereportedfromsamplespreparedwithoutPtstaining.AftertheX-rayexperiment,thesampleswerereimagedwithanOlympusLEXT4000confocalmicroscopetoobtainfurtherreferenceimagesoftherelevantsamples.2.2X-raymeasurementsThemeasurementswereperformedundervacuum(around1e-7mbar)atroomtemperatureonthenewnanoend-stationofID16A.Thesiliconnitridemembranewindowswereclampedintotheinsertionstubsdesignedforthe

samplestageofID16A.Samplesweretransferredontoapiezo-drivenshort-rangehexapodstage.Thehexapodmovement,underthecontrolofcapacitivesensors,wasusedtomonitorthecontactforcesduringsamplechanging.TheID16AbeamlinehastwomultilayercoatedKirkpatrick-Baez(KB)focusingmirrorpairslocatedat185mfromanundulatorsourceoperatingat17keVor33.6keV(Moraweetal,2015).Theenergyof17keVwaschosenforbestexcitationoftheX-rayfluorescencesignalofallrelevantelements.TheKBsystem,withextremedemagnificationdesignedfora15nmx15nmfocus,producedameasuredfocusof25nm(H)x37nm(V)withaveryhighfluxof3.4x1011ph/sfromthebroadbandpass(1%)ofthemultilayer.Tomakecorrelativeimagingofbothmorphologyandelementalcontentinthesamesample,phasecontrastprojectionmicroscopyandX-rayfluorescencemicroscopywerecombinedinasequence.Formorphologicalmeasurements,phasecontrastimagesofthesampleswerefirstlyobtainedbymovingthemdownstreamofthefocusandrecordingFresnelprojectionimagesatfourdistances.Thesedistancesbetweenthefocusandthesamplewerefixedtoobtainamagnificationyieldinga10nmor5nmequivalentpixelsizeatthelevelofthesample.AFReLoNCCDbasedindirectdetectorwith2048x2048pixelswasusedtorecordthemagnifiedprojections,whosevisiblelightopticsgaveaneffectivedetectorpixelsizeof0.84µm.17projectionsfordifferentlateralpositionsoftheobjectwererecorded,eachtaking0.3sexposuretimeandthenaveragedtogethighqualityFresnelprojectionimages.Afull-fieldquantitativephasemapwasthenretrievedfromthefourFresneldiffractionpatternsbasedonacontrasttransferfunctionapproach(Cloetensetal,1999).Thephasemapisproportionaltoaprojectionoftherealpartoftherefractiveindexortheelectrondensityinthespecimen.Asthesampleconsistsmostlyoflightelementsthephasemapistoagoodapproximationproportionaltotheprojectionofthemassdensity.Thereforealltheprojectionphasemapswereconvertedtoarealdensity(µg/mm2).CombinedwithSXRFimages,theycanbeusedfornormalizationtoyieldtrueelementalaveragemassfractionsofspecificelements(Kosior,etal,2012).ForSXRFmeasurements,thesamesamplewasmovedbacktothefocuspositionandscannedcontinuouslyacrossthebeamwithanequivalentstepsizeof30nmandadwelltimeof50ms.TheX-rayfluorescenceemissionwascollectedontheflybyapairof6-elementsilicondriftdetectors(Sensortech,UK)positionedperpendiculartothebeampathateachsideofthesample.ThefreelyavailablesoftwarePyMca(Soleetal.,2007)wasusedfortheanalysisoftheX-rayfluorescencespectra.Ateveryscanpoint,thesummedspectrumcollectedfromthe12detectorelementswasfittedtodecomposeitintotheemissionlinesoftheindividualelements(K-emissionlinesforP,SandFe).Theabsolutecalibrationtotheelementalarealdensity(ng/mm2)wasdeterminedbyfittingthefluorescencesignalfromathinfilmstandard(AXODresdenGmbH).

3.ResultsandDiscussionFigure1showsanoverviewopticalfluorescenceimagetakenundertheexcitationconditionsforSybrgolddye,whichbindsspecificallytoDNA.Whilethenucleiareclearlywellisolatedonthemembrane,itisclearthatnotallofthemareequallybright.Thissuggeststhateitherthedyeisunabletopenetratethesamplesuniformlyor,morelikely,thatsomenucleihavebecomedepletedintheirDNAcontent.Thismighthaveoccurredduringthewashingstepsofthesamplepreparation,orpossiblyduringhandlingofthesamples.Wenotethattheimagewastakenshortlyaftersamplepreparation,beforetransportingthesamplestoESRF,sothisdoesnottakeintoaccounttheeffectsofthevacuumsampletransferintotheID16Ainstrument.Figure2showscomparisonimagesofanisolatednucleusbybothavailableX-rayimagingmethods:phasecontrastprojectionmicroscopyandscanningX-rayfluorescenceimagingoftheP,SandFeK-lines.Thetotalsignalsforthethreeelements,integratedovertheimagesandcalibratedinunitsofnumbersofatoms,arelistedinTable1,alongwiththeintegratedmass.Thefieldofviewofthisimagealsocontainsoneortwoindividualchromosomesinasmallclusterattheupperside.ThisnucleuscontainstheleastquantityofFeobserved,thelowestlevelofPandthehighestS.ThedistributionsoftheP-andS-signalsoverlaywellontopofeachotherandalsoagreewiththedistributionofthearealdensityseeninthephasemap.Thedome-shapeddistributionofallthreeimagesisroughlywhatwouldbeexpectedforasphericalorhemisphericalnucleuswithauniformdensityofchromatinmatterwithinitsvolume.ThetotalPsignalcomingfrom5.4x1010Patomsisafactor-of-twohigherthanthehigherestimateof2.6x1010Patoms,givenabove,expectedforanucleusinthesecondhalfofthecellcycle.Thisexcesssuggestsaresidualcontributionfromothersources,lipids,ATP,RNAorphosphatebuffer,unlessthereisacalibrationerror.ItisnoteworthythatPhasnotbeenlostduringthesamplepreparationandinsertionintovacuum.Wedidnotdetectanyeffectofradiationdamagebecausethesignallevelsintheimageswerefoundtobereproducibleuponrepeatedscanning.Similarly,thetotalX-rayfluorescenceSsignalof6.17x109atomsisfoundtobehigherthantheestimategivenaboveof2.1x109SatomsinG2orMphase.Sincethisappearstobehomogeneouslydistributedwithinthechromatinfilledregionofthenucleus,thissuggeststhattheextrasignalmaybecomingfromthe29%non-histoneproteins,whichmayhavehigherrelativelevelsofCysandMetaminoacids(Uchiyamaetal,2005).Itisalsopossibletherearecontributionsfromnon-chromatinproteinsormicrotubulesassociatedwiththenucleusatcertainpointsofthecellcycle.Correspondingimagesfromthreemorenuclei,aslabelledinFig1andshowninFig3,gavetheintegratedsignalslistedinTable1.ThetrendissimilarwithanoverrepresentationofbothPandS.ForthePsignalsfromthe5nucleimeasured,therearefactorsof2variationsfromonenucleustoanother,whichmightindicatethelevelofinherentmeasurementerrorsorvariationsofsamplepreparation,butitmayalsoindicatethatthenucleiarecapturedatdifferentpointsofthecellcycle.ThevariationofSsignalsissubstantiallygreater,which

suggestslessofaneffectofsamplepreparationandmorelikelyindicatingdifferentlevelsofproteininthe5nucleiassociatedwiththeirstageinthecellcycle.TheobservednuclearmassesalsoagreewellwiththeestimatesaboveforgenomicDNAandchromosomalprotein,withallvaluesfallingwithinthefactor-of-tworangeexpectedforearly/latepointsofthecellcycle.ItisnotablethatthehighermassnucleiarealsotheonesshowinghighSsignals,furthersupportingthesuggestionthatextraproteinmaybepresentinthosenuclei.MuchgreatervariationwasfoundinboththemassesanddistributionsoftheFesignal,forwhicha9.4xvariationwasfound.Fig2showsthenucleuswiththesmallestlevelofFe,whilethatofFig3(a)hasthehighestlevel.UnlikeSandP,theFesignalsarestronglyclusteredandoftenseentobelocatedattheperipheryofthenucleus.ItisthereforeconcludedthatmostoftheFesignaliscomingfromthenuclearmembranestructures,ratherthanthecentralregions,asdiscussedfurtherbelow.WealsonotethattheseparatedchromosomestructureseenatthetopofFig2hascolocalisedPandSsignalscomingfromitsdistinctarmregionsandaseparateFesignalinthecentre,whichisdepletedinPandS.ThisappearstobeanagglomerationoftwochromosomesontheleftandrightsidesandmaycontainapieceofFe-richnuclearmembraneinthecentre.FinallyinFig4,weincludetheresultofopticalconfocalmicroscopeimagingofthemembranesafterremovalfromthebeamline,showingalargerareasurroundingthenucleiofFig3(b)and3(c).ConfocalimagesofeachofthemeasurednucleiinFig3areincludedintherighthandcolumn,scaledtotherestofthepanels.ItisclearthatthestructuresseenintheconfocalimagesappeartoextendfurtherthantheX-rayfluorescenceorX-rayphasecontrastimages.However,the3DscansshowthesestructurestostandoutabovethesurfaceonlyinthesamecentralareaoftheX-rayimages,whilethebordersarelesstall.ThetwointermediatefeaturesinthemiddleofFig4arealsolowerinthickness;thesedonotshowopticalfluorescenceinFig1,sodonotcontainDNA;theymayrepresentbustnucleiwhichhavelosttheirDNA.4.ConclusionsForthehumanlymphocytecellnucleipresentedinthisstudy,thedistributionsofPandS,measuredbyX-rayfluorescencemicroscopyandassociatedwiththeDNA-proteincomplexofchromatin,arefoundtoberelativelyuniformandstructureless,exceptforafewlocalizedspotsdiscussedbelow.Thiscouldbebecausethecellsareininterphase(G1,S,orG2ofthecellcycle),whenthechromatinisdecondensed,oritcouldbebecauseofinsufficientresolutiontoisolatetheindividualchromosomesintheprojectionimages.AcertainamountofmodulatedstructurecanbedistinguishedinthePsignalsofFig3(a)and3(c),whichresemblestheexpectedpatternofcondensedmetaphasechromosomes,eventhoughtheyarenotfullyresolved.Ifso,thesenucleiareinprophase,sincetheyappeartostillpossesstheirnuclearmembranes.

ThelevelsofbothPandSarerelativelyreproduciblefromnucleustonucleus,withinafactorof2and4respectively.ThelevelofPissystematicallyhigherthanexpectedfromthenumberofPatomscontainedintheDNAofthehumangenome.WeconcludethereisanadditionalcontributionofP,possiblylipid,ATP,RNAorphosphatebuffer.ItisunfortunatethatsuchoverrepresentationshavetakenplaceasareliablePlevelmeasurementfromDNAalonecouldhavebeenausefuldeterminationofthephaseofthecellcycle.BoththelevelsofSandtheS/Pratiosarefoundtobehigherthanexpectedfromthehistoneproteinsalone,whichcomprise71%ofthetotalchromosomalprotein.Thissuggeststhatthenon-histoneproteinsmaybericherinCysandMetresiduesorthatadditionalproteinsarepresent.TheFeatomcontent,whiletwoordersofmagnitudelowerthanPorS,ismuchmorevariedamongthesamplesexamined,by9-foldamongtheintegratedsignalsinTable1.FeisnotexpectedtobeassociatedwithDNAingeneralforevolutionaryreasons(Yagietal,1992),yetsomeexceptions,particularlyduringDNAreplicationinSphase,arenotedabove.Feisseentoformsmallbrightspots,about100nmindiameter,inthesamplesshowninthelow-concentrationcasesinFigs2and3(b).Inonecase,Fig3(b),FespotsarecolocalisedwithS,perhapssuggestingthepresenceofFeSenzymes;intheothercases,Fig2,FeandSareseparatelylocalizedinspots.ThestrongercorrelationofFewithSthanwithPcanalsobeseenintheoverlayplotsofFig5.Feisseentoformshell-likecrescent-shapedplaquesaroundtheedgesofthehigh-FeconcentrationnucleiinFigs3(a)and3(c).Thisco-localisationofFeandScanbeseenespeciallyintheoverlayplotofFig5.TheseareastrongsuggestionofFebeinglocatedinthenuclearmembrane,ratherthanthechromatin-filledcentres.InmostcasestheFesignalcanbeseentosurroundthatofthePandS.ThismaythereforebeconsistentafterallwiththeevolutionaryhypothesisofYagietal(1992).Asfaraswecantell,concerningtheradiationdamagethephasecontrastimagingmeasurementsintroducedaslightshrinkage(lessthan5%)andmassloss(25%)ofthenucleus,afteronemeasurementwith0.3sand7measurementswithlongerexposures(1s).Thebeamissubstantiallyoutoffocushere,enlargedtomorethanthe20x20µmfieldofviewintheclosest-distancecase.Howevertheraster-scanningSXRFmeasurementdidcausevisiblechangestothesample,anareashrinkageofabout8%canbefoundfromthecomparisonofthephasemapsandtheSXRFmapsinFig.2andFig.3.ThedosedeliveredhereinSXRFexperimentswas3.1x109Gray,whichishigherthanthedose107Grayknowntocausestructuralchanges(Kirz,etal,1995).However,weobservednomasslossfromrepeatedSXRFscans(datanotshown).Thepost-experimentconfocalimages,recordedinFig4,showthinningofthemembraneovertheentirescannedarea,ascanbedetectedintheconfocalheightmap(greyscaleimage).Multipleoverlappingscannedareascanbeobservedfortheuppernucleus,forwhichtheimagesappearinFig3(b).However,nomasslossbetweenthesesscanswasdetectedinthefluorescencesignal.

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Fig.2 5.44x1010 6.17x109 3.89x107 8.8 19.69pgFig.3(a) 4.01x1010 11.8x109 3.65x108 3.4 33.56pgFig.3(b) 4.52x1010 11.3x109 4.06x107 4 27.70pgFig.3(c) 3.13x1010 8.07x109 2.39x108 3.9 16.30pgNucleus7 2.69x1010 3.03x109 2.49x108 8.9 17.36pgAverage 3.69x1010 8.08x109 1.87x108 22.92pgG1phase 1.3x1010 1.0x109 13 16.8pgG2/Mphase 2.6x1010 2.1x109 13 33.6pgTable1.CalibratedX-rayfluorescentsignals,integratedoverthefivemostreliablerasterscansofhumancellnuclei.DerivedmasseshavebeenconvertedintonumbersofatomsfoundwithinthenuclearregionsofthesamplesmeasuredatID16A.Thelasttworowsgivetheatomcountsexpectedfordifferentphasesofthecellcycle,asdiscussedinthetext.Thetotalmassisdeterminedfromintegrationofthephasecontrastimage.

Figure1.LowmagnificationopticalfluorescenceimagetakenundertheexcitationconditionsforSybrgolddyeusingaZeissAxioZ2microscope.Boxesandlabelsindicatethenucleithatareanalysedfurtherinthiswork.

Figure2.X-rayimagesofthenucleusoutlinedinFig1,withagroupofindividualchromosomesontheupperside.Topleft:Phasecontrastimagepresentedastotalarealdensity(unit:µg/mm2).Otherpanels:elementalarealdensitydistributionsfromscanningX-rayfluorescence(unit:ng/mm2).

Figure3.X-rayimagesofthreemorenucleioutlinedinFig1.Leftcolumn:Phasecontrastimagespresentedastotalarealdensity(unit:µg/mm2).Centrecolumns:elementalarealdensitydistributionsfromscanningX-rayfluorescence(unit:ng/mm2).Right:opticalconfocalheightmap,measuredaftertheX-rayexperiment.Thescalebarappliestoallpanels.

Figure4.Grey-scaleconfocalimagemeasuredwitha50xlensonanOlympusLEXT4000microscopeaftertheX-rayexperiment.Nuclei3(b)and3(c)canbeseen,alongwithaclearmodificationtothemembraneintheregionwherethefluorescencemappinghadtakenplace.

Figure5.NucleusinFig.3(a)shownastheoverlayoftheelementalmapsofFeandrespectivelyS(left)andP(right).TheyellowcolourontheleftindicatescolocalisationofFeandS,whileintheoverlayofFeandP,weseemoreredandgreencolours