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The fascinating diatom frustule—can it play a role for attenuation of UV radiation?

Ellegaard, Marianne; Lenau, Torben Anker; Lundholm, Nina; Maibohm, Christian; Friis, Søren MichaelMørk; Rottwitt, Karsten; Su, Yanyan

Published in:Journal of Applied Phycology

Link to article, DOI:10.1007/s10811-016-0893-5

Publication date:2016

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Ellegaard, M., Lenau, T. A., Lundholm, N., Maibohm, C., Friis, S. M. M., Rottwitt, K., & Su, Y. (2016). Thefascinating diatom frustule—can it play a role for attenuation of UV radiation? Journal of Applied Phycology,28(6), 3295–3306. https://doi.org/10.1007/s10811-016-0893-5

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Thefascinatingdiatomfrustule–canitplayaroleforattenuationofUVradiation?

MarianneEllegaard1,TorbenLenau2,NinaLundholm3,ChristianMaibohm4,SørenMichaelMørkFriis4,KarstenRottwitt4,YanyanSu1

1DepartmentofPlantandEnvironmentalSciences,UniversityofCopenhagen,Thorvaldsensvej40,1871Frederiksberg,Denmark.CorrespondingauthorMarianneEllegaardme@plen.ku.dk;phone:+4535320024

2DepartmentofMechanicalEngineering,TechnicalUniversityofDenmark,Produktionstorvet,Building426,2800KongensLyngby,Denmark

3TheNaturalHistoryMuseumofDenmark,UniversityofCopenhagen,Sølvgade83S,DK-1307CopenhagenK,Denmark

4DepartmentofPhotonicsEngineering,TechnicalUniversityofDenmark,ØrstedsPlads343,2800KongensLyngby,Denmark

Keywords:UVprotection,photonics,photobiology,nano-patterning,diatom

Reference: Ellegaard, M., Lenau, T. A., Lundholm, N., Maibohm, C., Friis, S. M. M., Rottwitt, K., & Su, Y. (2016). The fascinating diatom frustule—can it play a role for attenuation of UV radiation? Journal of Applied Phycology. doi:10.1007/s10811-016-0893-5

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Abstract

Diatomsareubiquitousorganismsinaquaticenvironmentsandareestimatedtoberesponsiblefor20-25%ofthetotalglobalprimaryproduction.Auniquefeatureofdiatomsisthesilicawall,calledthefrustule.Thefrustuleischaracterizedbyspeciesspecificintricatenano-patterninginthesamesizerangeaswavelengthsofvisibleandultraviolet(UV)light.Thishaspromptedresearchintothepossibleroleofthefrustuleinmediatinglightforthediatoms´photosynthesisaswellasintopossiblephotonicapplicationsofthediatomfrustule.Oneofthepossiblebiologicalroles,aswellasareaofpotentialapplication,isUVprotection.InthisreviewweexplorethepossibleadaptivevalueofthesilicafrustulewithfocusonresearchontheeffectofUVlightondiatoms.WealsoexplorethepossibleeffectofthefrustulesonUVlight,fromatheoretical,biologicalandanappliedperspective,includingrecentexperimentaldataonUVtransmissionofdiatomfrustules.

Introduction

Thediatomsareuniqueamongalgaeinthatthecelliscompletelyenclosedinacontinuoussilica-basedcover,thefrustule.Thefrustuleisasarulebuiltmoreorlesslikeapetridish,withaninnerandoutervalve,connectedbygirdlebands(Fig.1).Diatomsarealsospecialamongeukaryoticmicroalgaeasduetothefrustuletheyhaveafixedcellshape,andtheylackflagellainthevegetativestages.Diatomsareubiquitousorganismsinaquaticenvironmentsandcanbedominantprimaryproducersinallaquatichabitats,includingice.Theyarethusestimatedtoberesponsiblefor20-25%ofthetotalglobalprimaryproduction(Nelsonetal.1995).Diatomspeciesdiversityisfarfromdiscoveredatpresent.Some10,000specieshavebeenformallydescribed,butitisestimatedthattherealnumberofspeciesis10-20timeshigher(Guiry2012).Traditionally,speciesdescriptionshavebeenbasedondetailsintheintricatenanoscalepatternofholesinthefrustuleandeachspeciesthushasitsowndistinctpatternwhichisreproducedatcelldivision.Whilebenthicdiatomshaveevolvedaspecializedmodeofmovementviaaslitthroughthefrustulecalledtheraphe,plankticdiatomsarenon-motileandarethereforedependentonbuoyancyandwatermovementtostaysuspendedinthephoticzone.Asthefrustuleisgenerallydenserthanbothseawaterandfreshwater,itwouldseemtobeanimpedimenttophotosyntheticorganismsdependentonstayingupinthephoticzone.Possibleadaptiverolesofthediatomfrustulehavethereforebeenexplored,fromthepointofviewthatthefrustulemustgiveadaptiveadvantagesthatoff-setthisseemingdisadvantage.Wewillfocusontheevidenceforaroleinprotectingthediatomcellfromultravioletradiation(UV)damage.Thatthefrustuleinteractswithlightisclearalreadywhenstudyingfrustulesunderthelightmicroscope(Fig.2b).This,andthefactthatsomefrustules,suchasthatshowninFig.2b,lookverysimilartoaverythinsliceofaphotoniccrystalfiberormicro-structuredopticalfiber,haspromptedintensiveresearchintopossiblephotonicapplicationsofdiatomfrustule.InthisreviewwewillfocusonpotentialforUVprotectiveapplications.

Review

Thesuggestedadaptivefunctionsofthediatomfrustule

Thediatomfrustulehasbeensuggestedtobeaby-productofotheradaptationsratherthangivinganadaptiveadvantageperse.Thussomeauthorsstatethatitismetabolicallycheaptoproducerelativetoothertypesofcellcovering(only2%oftheenergybudgetofthecell;Raven1983)althoughotherauthors

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questionthis(FinkelandKotrc2010;Lavoieetal2016).Medlin(2002)putforwardthehypothesisthat,becausesilicametabolismplaysanimportantroleinresistingaging,thesilicafrustuleismainlyaby-productofthis.Otheradaptiveadvantageshavebeensuggested,includingadeliberateincreaseofthedensityofcells,alteringsinkingrateviaspinedevelopment,resistingturgorchangesandresistancetoparasites(reviewedbyRavenandWaite2004).However,thesuggestionswhichhavereceivedmostsupportare:1.defenseagainstpredation,2.proton/pHbufferingand3.modulationoflightwithinthecell.Totestthefirsthypothesis(defenseagainstpredation)Hammetal.(2003)performedastudywheretheytestedthestrengthofdifferentlyshapedfrustules(threespecies),usingglassmicroneedlestopressthecellandmeasuringtheforcenecessarytobreaksinglecells.Theyconcludethatallthreeshapesofcellstestedareremarkablystrongduetotheirarchitectureandthematerialpropertiesofthesilica.Thesecondhypothesis,pHbuffering,hasreceivedsupportfrombothlaboratorytestingoftheefficacyofdiatombiosilicaasapHbuffer(MilliganandMorel2002)andfromtheorieslinkingmajoreventswithintheevolutionaryhistoryofdiatomstochangesinatmosphericCO2levels,andtherebypHinaquaticsystems(e.g.Armbrust2009).Athirdsuiteofhypothesesdealwithdifferentaspectsofcontrollingthelightregimewithinthediatomcell(Fuhrmanetal.2004),byfunctioningasaphotoniccrystal(Fuhrmanetal.2004),byscatteringvisiblelight(RavenandWaite2004)orbyreflecting,absorbingorscatteringlightintheUVrange(below380nm;Davidsonetal.1994;RavenandWaite2004).InthisreviewwefocusonthepossibleroleoffrustulesinmodulatingUVlight,aswellasdiscussingwhichcharacteristicsofthediatomfrustulearecentraltothephotoniceffects.

Photonicapplicationsordiatombiology?

Whendiscussingpotentialphotonicutilizationsofdiatomfrustules,thelightenvironmentthatdiatomsareadaptedtoundernaturalconditionsshouldbetakenintoaccount.Whenlightenterswater,itbecomespartiallypolarizedandisabsorbedmorestronglythaninairinawavelengthdependentway.Red,orangeandyellowarequicklyabsorbedbythewater,onlypenetratingmetersbelowthesurfacewhilebluelightisnotstronglyabsorbedbywaterandcanpenetratemuchdeeper,dependingontheconcentrationofdissolvedmatterandparticles(Depauwetal.2012;seealsoFig.2).AcoupleofmetersbelowthesurfacealmosthalfoftheintegratedlightintensitywillbeintheUVspectralregion,i.e.below400nm,comparedtoonlyabout3%atgroundlevel(JohnsenandSosik2004).DiatomsthusliveinalightenvironmentdominatedbyUVandbluelight,supportingtheideathatthefrustulemayplayaroleinUVlightprotection.Asmoststudiesaimedatcharacterizingthephotonicpropertiesofdiatomfrustulesandelucidatingtheirindustrialpotentialaredoneonrinsedfrustules,theorganicmaterialinthecellcontentistakenintoaccountinneithermeasurementsnorsimulations.Further,mostofthesestudiesareperformedorsimulatedinair,contrarytotheaquaticnaturalhabitatofthediatoms(althoughthereareexceptions,e.g.DeTommasietal.2010;DiCaprioetal.2014;Hsuetal.2012;LeDuffetal.2016).Thereforethecalculatedeffectsoftheseexperimentsandsimulationsapplytothefrustule,andforuseinapplicationsofrinsedfrustules,butcannotgivefullbiologicalanswersastohowthelivingdiatomsbenefitbytheinteractionsbetweenthefrustuleandlight.Thesedifferencesbetweenmeasurementsonrinsed(andoftenseparated)frustulesandthediatominitsnaturalhabitataresummarizedinFig.2.

Anotheraspecttobeconsideredistheorientationofthefrustule.Undernaturalconditions,inaquaticplankticenvironments,lightwillhitthediatomcellfromtheoutsideofthefrustule(theconvexside),butsomeofthelightmayalsobeaffectedbythe(concave)insideoftheoppositevalveasitpassesthroughthe

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cell.Underexperimentalconditionstotesttheeffectofthefrustulenano-patternonincidentlight,thefrustuleswilltypicallyhavebeencleanedoforganicmaterial(cellcontentandanyorganiccovering)andthefrustulewillbeseparatedintoitsparts–thetwovalvesandthegirdlebands(seeFig.1).Typically,measurements(andsimulations)havebeendoneonasinglevalve.Itisnotalwaysclearfromwhichdirectionthelightishittingthevalve,andthequestionishowtheorientationofthevalveaffectsthemeasurements.Dothephotonicpropertiesdifferdependingonwhethertheincidentlighthitsthevalvefromwhatwastheoutsideortheinsideofthecell?Formeasurementsonalayeroffrustules,anotherquestioniswhetheristhereapatterntohowasinglevalvewillnormallyfallwheninadropofwateroranotherliquidmatrix.Basedonthe(sparse)literaturetodateonthis,andonourownunpublisheddata,theanswertoboththesequestionsappearstobeyes.Preliminaryresultsindicatethatinadropofliquidthevalvesfallapproximatelyonethirdmoreoftenwiththeinsideup(Gösslingetal.pers.comm.).Thiscan,however,becontrolledasshownbyWangetal.(2012),wherethevalvesaremadetoallfaceinthesamedirectionbysuspensionandsubsequentlyextractingtheliquid.Wehaveobserveddifferencesinourmeasurementswithregardtointerferencepoints,dependentonthewaythevalvesarefacing(unpublisheddata).ThisisingoodagreementwithfindingsbyRomannetal.(2015),wherenointerferencepoints(seee.g.Maibohmetal.2015a)areobservedwhenthevalveisfacingwiththeoutsidetowardsthemicroscopeintransmissionexperiments.

EffectsofUV-light

UVlightisingeneraldetrimentaltolivingorganisms(althoughnearUVlightmaybephotosyntheticallyactive;XuandGao2010)aswellasmanyman-madematerials;theshorterthewavelength,themoredamaging(withtheeffectfurtherenhancedbyresonantbehavioratspecificwavelengthsfrompossibleabsorptionpeaks).EffectsofincreasedUVlevelsonlivingdiatomshavebeenstudiedinachangedglobalclimatescenariosincehuman-inducedreductionsintheozonelayerofthestratospherewerereporteddecadesago(e.g.Wuetal.2014).Polarregions(receivingincreasingUVlight)aswellasmountainhabitats(naturallyhighUVradiation)havebeenthepreferredhabitatsforUVresearchonalgae.AsmostUVradiationisnotphotosyntheticallyactive,cellstrategieswouldthusbeexpectedtobeeithertoavoidortolerateUVradiation(HolzingerandLütz2006).AUV-inducedeffectisdependentonthelevelofUV,thedurationoftheexposure,thespectralcompositionofthelightintheUVrange,thesensitivityoftheorganism(s)investigated,physiologicalstatus,ambientnutrientconditionsandtemperature(Bumaetal.1996;CullenandLesser1991;LitchmanandNeale2005;Hessenetal.2012).UVradiationharmscellsthroughabsorptionbynucleicacidsandproteinsandhenceinhibitingDNAtranslation,replicationandtranscription(Bumaetal.1995;HolzingerandLütz2006).ButcomparisonsamongstudiesofUVeffectsonalgaearehamperedbydifficultiesincomparingradiationconditionswhichcannotberecalculatedandthuscompared(HolzingerandLütz2006).Thus,infuturestudiesofthepossibleeffectsofthefrustuleonUVlight,itwouldbeadvantageoustostandardizethemethodofregisteringUVintensityandwavelength.

AdecreaseinphotosynthesishasbeenthemostprominentUVeffectinalgae(Waringetal.2006;HolzingerandLütz2006),anddecreasesinbiomassand/orgrowthrateofdiatomsexposedtoUVradiationarecommonlyreported(e.g.Reizopoulouetal.2000;Nahonetal.2010),buttheeffectandtherecoverycapabilityafterUVexposuresapparentlyvaryamongspecies(Bumaetal.1996),asspeciescompositioninexposedcommunitiesarereportedtochangewithtime(Reizopoulouetal.2000).ThisagreeswithvaryingeffectsofUVamongcultureddiatomspeciesbothwithregardtoresponseandsensitivitytoUV(Karentzet

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al.1991;Davidsonetal.1994;Waringetal.2006;Hessenetal.2012;Scholzetal.2014),evenamongspecieswithinagenus(Hargravesetal.1993).ThishasraisedspeculationsastowhydifferencesinUV-effectsvarymoreindiatomsthaninotheralgalgroupswheretheresponsescanoftenbecorrelatedtocellsizeduetosizerelatedeffectsonself-shadingandUV-absorbingcompounds(Waringetal.2006andreferencesherein).Onepossibleexplanationcouldbethatthisdifferenceisduetospeciesspecificdifferencesinthepatternofholesorothercharacteristicsofthefrustule.

ChronicUV-Bexposureondiatomshasoftenbeenrelatedtoincreasesincellvolume(duetoadecouplingbetweencellgrowthandcelldivision)anddecreasesingrowthrates(Karentzetal.1991;Bumaetal.1995;1996;HolzingerandLütz2006andreferencestherein;Nahonetal.2010;Scholzetal.2014).Anincreaseinnumberofchloroplastsandthusincellularpigmentcontenthasbeenobservedinsomediatoms(Bumaetal.1996),whereasothershavefoundareductioninpigmentation(Lohmannetal.1998).Othersagainshowedanincreaseinpotentialphotoprotectivepigmentslikediadinoxanthinanddiatoxanthin(Döhler1995;Leuetal.2006).Inastudyonlong-termacclimation,severaldiatomspeciesacclimatedbychangingphotosynthesisrelatedpigments,whereasonespecies,Amphoracoffeaeformis,lackedthesepigmentchanges(Rechetal.2005).IthasbeensuggestedthatUVabsorbingcompounds,mycosporine-likeaminoacids(MAAs),chemicallyboundtothediatomfrustuleprovidediatomswithUVprotectingproperties(Ingallsetal.2010),andtheyexplainalackofincreaseinMMAsindiatomsinresponsetoUV,incontrasttoflagellates(Davidsonetal.1994),toberelatedtothetightassociationwiththefrustule(Ingallsetal.2010).ThishypothesisissupportedbyhighMAAcontentintwocentricdiatomspeciesrelatedtolackofUVeffectonphotosynthesis,asopposedtolowerMAAconcentrationsintwopennatediatomsaffectedbyUV(Helblingetal.1996).

Nitrate-limitationincreasesthesensitivityofdiatomstoUVlightduetoadecreaseinphotochemicalquantumyield(CullenandLesser1991;Scholzetal.2014).Moreover,ithasbeenreportedthatthesensitivityofdiatomstoUVradiationcouldbeincreasedbyelevatedCO2duetoareductioninrelativeelectrontransportrate(Wuetal.2012).AnadditionalstressfordiatomshasbeenobservedundercertainsalinityconditionscombinedwithUVradiation,andthiscombinedeffectisspecies-dependent(Döhler1984;Rijstenbil2005).

InspiteofthereportedeffectsofUVradiation,somestudiesfindnoeffectofUVondiatoms,orlesseffectthanonflagellates(Villafaneetal.1995;Vernet2000;LitchmanandNeale2005;Guihéneufetal.2010;Hessenetal.2012:Wuetal.2012).Sofarnoexplanationhasbeenfoundforthisdifference.WehypothesizethatitisthediatomfrustuleanditscharacteristicswhichreflectorabsorbtheUVandthereforeprotectthediatomcellsagainstUV.Thisissupportede.g.byobservationsofdiatomsnotrespondingtoUVwithachangeinfattyacidcompositionorhavingverylowamountsofMMAsincontrasttomanyothermicroalgaelikedinoflagellates(Jeffreyetal.1999,Guihéneufetal.2010andreferencesherein).AlsoJeffreyetal(1994)underlinedthatthesurvivalofdiatomsexposedtoUVdidnotcorrelatewithabsorptionbye.g.pigments.Potentialindustrialapplicationsofdiatomfrustules

Similartootheralgalgroups,potentialindustrialapplicationsofdiatomsincludebiofuels(e.g.Levithanetal.2014),bioplastics(Hempeletal.2011)andotherusesoftheorganiccellcontents(Bozartetal.2009).Uniqueamongthealgae,however,istheadditionalpotentialforapplicationsofinorganicmaterial:the

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silicafrustule.Geologicaldepositsrichinprehistoricdiatomremains,calleddiatomaceousearth,arealreadyindustriallyexploitedfore.g.filtering(ParkinsonandGordon1999).

Inrecentdecades,diatomfrustuleshavebeenthefocalpointforintenseopticalstudiesduetotheirinherentstronginteractionwithlight(includingUVradiation)andpotentialindustrialusesofspecificcharacteristics.Theideasforpotentialapplicationsforthesilicafrustuleincludephotoniccrystals(Fuhrmanetal.2004,Yamanakeetal.2008;deStefanoetal.2009),chemicalsensors(deStefanoetal.2009),microlenses(deStefanoetal.2007a),enhancedlightharvestinginsolarcells(Ottesen2011),lightfocusingcapabilitiesandapplicationsofthis,suchasopticalswitches(DeTommasietal.2010;Maibohmetal.2015a;Ferraraetal.2014).DuetothedestructiveeffectsofUVlightonlivingorganismsandmanytypesofmaterials,andduetotheunwantedeffectsassociatedwithmanytypesofUVprotectionthereisgreatinterestinfindingnovelmeansofUVprotectionwithoutdeleteriousaspects.UVprotectionisimportante.g.toprotectpolymersfromdeteriorationwhenexposedtosunlightandfortheprotectionofhumanskininsunlotions.ThepotentialadvantageofusingdiatomsasUV-filterswouldbethatitmaybepossibletoselectivelyreflectorabsorbcertainwavelengths.Suchafilterthereforehasthepotentialnottoimpactthecolorofthematerial.Itwouldforinstancebepossibletomakedurabletransparentpolymersandlacquerswithlastingpropertiesinsunlight.ThemosteffectiveUV-filterusedforpolymersiscarbonblackwhichabsorbsnotonlyUVbutalsootherwavelengthsoflightandthereforeappearblack(Coleman2011).ThereareotheradditivestopolymersthatpreventdegradationfromUV-lightincludingHinderedAmineLightStabilizers(HALS)thatworkbytrappingfreeradicalsformedduringphoto-oxidationofthepolymer(Gijsman2011)thusextendingthelifetimeofthepolymer.SunlotionstypicallyprotectstheskinfrombothUVA(400-320nm)andUVB(320-290nm)light.

Parameterscentraltophotonicapplicationsofdiatomfrustules

Forapplicationpurposesitisnotnecessarytoknowthefullinteractionbetweenthelightandthefrustule.Itisenoughtoknowtheeffectswhichareimportanttotheapplicationneeded.

Developmentofsophisticatedmethodsandequipment,whichhaverevealedmaterialcompositionandthreedimensional(3D)structure,havestrengthenedthepotentialforapplicationsofthefrustule.Whenlightinteractswiththefrustule,someeffectscaneasilybeobservedinalightmicroscope(Fig.2b,c).Theintricate,semitransparent,nano-structuredfrustulecauseslighttobetransmittedormultiplereflectedfromthedifferentinterfacesgivingrisetophaseshiftsaswellasfrequencydependentconstructiveanddestructiveinterference(ParkerandTownlee2007;Gordonetal.2009;Maibohmetal.2015a,b).Inadditiontotheseeasilyobservedopticalphenomenamoresophisticatedopticaleffectshavebeentheorized,i.e.wave-guidingandband-gaps(Fuhrmannetal.2004).Ifdiatomfrustulesaretobeusedinindustrialapplicationsweneedtodeterminewhichaspectsarecentralforthedesiredapplication.Althoughthereishugemorphologicalvariationamongtheindividualspeciesofdiatoms,thisreviewdiscusscommontraitsaswellasidentifydifferences(seee.g.Maibohmetal.2015b)whichcanbeexploitedforapplicationpurposes.

Structuralpropertiesofthediatomfrustule

Diatomscanroughlybedividedintotwodifferentgroupsbasedontheirvalvesymmetry,thecentrics(radiallysymmetrical)andthepennates(bilaterallysymmetrical).Forbothforms,thevalveisavery

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complex3Dstructure,asseeninFig.1andingraphenereproductions(Panetal.2014).ThesizefeaturesofnanopatternofthevalvevaryfromspeciestospeciesandareintheorderofwavelengthsofUV-Ctonearinfraredlight.Thisincludesphotosyntheticallyactiveradiation(PAR)(400-700nm),indicatingthatPARlightwillbestronglyinfluencedwhenitinteractswiththefrustule.ThisisseenwhenlightinthePARwavelengthrangeistransmittedthroughthefrustule;theporestructureinteractswiththelightandcreatesawavelengthdependentinterferencepattern(DeTommasietal.2010;Maibohmetal.2015a).Thestructurealsointeractswiththelightinawaythatitredirectslighttowardsthecellinteriorevenwhenlightisstrikingthefrustuleatanotheranglethannormalincidence(Maibohmetal.2015c).

Materialpropertiesofthediatomfrustule

Thefrustulematerialispredominantlysilicaprecipitatedbydiatomsfromsilicicacidwithspecies-specificsizeandpatternofholes.ThespecificsilicastructureofthefrustuleisusualcharacterizedasamorphoushydratedsilicabuttherehavealsobeenindicationsfromX-raydiffractionstudiesthatpartofthefrustulecouldbecomposedofα-quartzwithagrainsizeoffewtenthsofmicrometers(Goswamietal.2012).Inadditiontothis,traceimpuritiessuchasaluminum,magnesium,ironandtitaniumcanbepartofthefrustulematerial(Butcheretal.2005).Thesetraceimpuritiesaretakenupfromthewaterenvironmentandincorporatedintothefrustulebythediatomcell.

Simulationoflightinteractionwiththefrustulestructure

ThetransmissionoflightthroughadiatomvalvecanbesimulatedusingMaxwell’sequations,whichgovernthepropagatingelectromagneticfield.Ageometricalopticsapproachisinsufficientbecause,asdescribedabove,thestructuresofthevalveareonthesamescaleasthewavelengthofvisibleandUVlight;hence,diffractionplaysanimportantrole,andsomeinterferencepatternsarereadilyobservedalreadyunderanormallightmicroscope(Fig.2b,c).Accuratenumericalmodellingofthethree-dimensionallystructuredvalverequiresmanycomputationalresourcessoitisdesirabletosimplifythemodelofthevalveandfocusonfewofitsfeatures.Forsuchsimulations,thevalveisgenerallyassumedtobeaplaneglasssurfacewithapatternofairholes,whichresemblesthelargerholesononesideofthevalveasseeninFig.2b.Somestudieshavealsomodelledtheotherplaneofthevalve,eitherincludingthemoreintricateholestructure(DeTommasietal.2010),orasahomogeneoussurfaceabsentofholes(Yamanakaetal.2008).Intheliterature,twodifferentschemesofsimulationapproacheshavebeenpursued:oneconsidersthetransmissionoflightthroughtheplaneofthevalvewherelightpassesthroughtheholes,andthesilicawallsareconsideredopaqueorpartlytransparent;theedgesoftheholesareconsequentlyresponsiblefordiffractionofthepropagatingbeam.ThetheorybehindthisandanumericalalgorithmtocarryoutthisapproachwasdescribedbyDelenetal.(1998).Theaimofthisapproachistoexplainthelens-lesslightfocusingobservedbyDeStefanoetal.(2007a),whereitwasfoundthatthearrayoflargerholesinthevalveplaysacriticalroleinunderstandingthecomplexdiffractionpatternbehindthevalve;thesmallerholesontheotherplaneofthevalve(inthecribrum)wereignoredinthatmodel.Later,thesameapproach,includingthesmallerholeshowever,wasusedtopredictthat,forvisiblelight,multiplefocuspointsarefoundascloseas100-200µmfromtheplaneofthevalve,butthatnosuchfocusingisfoundforshorterwavelengthsintheUVregime(DeTommasietal.2010).Whilethelatterpredictionremainsunconfirmedbyexperimentalinvestigations,arecentstudyshowedexcellentagreementbetweenexperimentaldataandtheoryandwithoutthesmallerholesinthevisibleregime(Maibohmetal.2015a),thusvalidatingthisnumericalapproach.

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TheothercommonapproachfoundintheliteratureisthepropagationoflightintheplaneofthevalveasfirstpresentedbyFuhrmannetal.(2004);itisshownthatthevalvemayactasawaveguideinwhichtheopticalpropertiesaredeterminedbytheperiodicstructureofthelargeairholesinthedirectionofpropagation.Suchastructuremayleadtophotonicbandgaps(PBG;seelater)dependingontheparametersofthespecificstructureandtherefractiveindexcontrastbetweenthevalvematerialandthematerialsintheholes(airorwater).ThestudybyFuhrmannetal.(2004)andalaterstudybyKieuetal.(2014)shownoPBGforaccuratemodelsofdiatomfrustules.Ontheotherhand,ithasbeenshownthatiftherefractiveindexofthefrustuleisincreasedto2.4(whichmayberealizedbymaterialsubstitutionasmentionedbelow)afullPBGisfoundatawavelengthof2.2µm(DeStefanoetal.2009).Thissuggeststhatthefrustulemayfinduseinindustryasanewopticalchemicalsensor(DeStefanoetal.2009).

BesidesthediscoveryofPBGs,thestudyofphotonicbandsgivesmoreinformationabouttheinteractionoflightwiththediatomfrustule:inanotherstudy,strongabsorptionwasfoundintheblueregionofvisiblelightandpartoftheUVAregionbytransmittinglightthroughafrustule(Yamanakaetal.2008).Simulationsofthephotonicbandsofanaccuratemodelofthefrustuleshowedthatastronginteractionbetweenthefrustuleandlightofwavelengthsintheshorterpartofthevisiblespectrum.

Photoluminescence

Poroussilica,oneofthemainconstituentfrustulematerials,isknowntoemitphotoluminescence(PL)inthevisiblespectralregionwhenirradiatedwithUV-light(Cullisetal.1997).ThediatomfrustulescanalsoexhibitPLwithoneormorespectralpeakswhichcanbemoreorlesspronounced(Butcheretal.2003,2005;deStefanoetal.2007b;Mazumderetal.2010;Vijietal.2014).Thusresearchershaveforexcitationusedwavelengthsof325nm,withemissionat539.4nm(deStefanoetal.2009)and300,370and380–allwithemissionat440nm(Mazumderetal.2010;Vijietal.2014).ExperimentsshowgreatdifferencesinPLactivityamongdiatomspeciesandevenamongdiatomsfromsametheculture,fromnoPLactivitytointensitiesmeasurablewiththenakedeye(Butcher2005;Maibohmetal.2015c).Ourownexperimentswithcultivateddiatoms,fromthesameculture,showlowPLactivityorsometimesnoPLatall;underliningthattheuptakeofmineralsfromthesurroundingsandotherinfluencingparameterstothePLactivityareofimportanceontheindividualcelllevel(Maibohmetal.2015c).Forapplicationpurposesthelocalenvironmentofthecleanedfrustuleshasahugeinfluence.Anoxygen-containingatmosphereworksasafastquencherofPLactivitywhileacontrolledatmosphere,asinaultrahighvacuumsystem,thefrustulePLactivityhasbeenshowntoactasagassensorbybindingvarioustypesofgassesandtherebyeitherquenchorenhancethePLactivity(deStefanoetal.2007b).ThepositionandshapeofthePLspectrumisdependentontheUVexcitationwavelength,andthepresenceofmultiplepeaks,i.e.multiplede-excitationpathways,isattributedtonon-uniformcrystallitesize,trappingstatesandimpuritiesinthefrustules(Goswamietal.2012).ThePLspectrumisdominatedbypeaksfromtheamorphoussilica,butitisalsoaffectedbythesmallamountsoftraceimpuritiesinthefrustule.Uptakeoftraceelementswillchangetheabsorptionandtransmissioncharacteristicsofthefrustuleagain,thedegreedependingontheelementandtheconcentrationofit.Thenaturalamountoftraceimpuritiesvaries,dependingonthehabitat,andthetraceimpuritiescaneitherbedepositedinthefrustulebythecelloradsorbedtothefrustuledirectlyfromtheenvironment(deJongeetal.2010).ThePLcanalsobeaffectedbyadsorptionofmaterialwhichquenches,enhancesoraltersthePLspectra(deStefanoetal.2007b;Galeetal.2009;Vijietal.2014).ChangesinPLspectrahavebeenusedasanoveltypeofgassensorwherebindingabsorptionofmolecules

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onthefrustulesleadstoameasureablechange(deStefanoetal.2007b).Metaldopedfrustulescanalsobestimulatedbyanelectriccurrentandsubsequentlyemitelectroluminescence(Jeffryesetal.2008).

Photonicbandgaps,wave-guidingandBraggscattering

Thesethreephotonicphenomenaareofcentralrelevanceandinterestforstudyingdiatomfrustules,particularlywithregardtothepossibilityforusingdiatomfrustulestoselectivelyscreenoutspecificwavelengthsoflight.

Photoniccrystalsareperiodicstructuresthatphotonsofcertainwavelengthscannotpropagatethrough.Thenon-propagatingwavelengthorusualthebandwidthofnon-propagatingwavelengthsconstitutestheso-calledphotonicbandgap(PBG).FormationofaPBGinastructurecanbeviewedasthesynergeticinterplaybetweenthemacroscopicBraggscatteringresonance(whichstemsfromtheperiodicstructureofalternatingmaterialandcreatesstopgapswhentheperiodofthestructurecoincideswithanintegernumberofhalfthewavelengthofthelight)andthemicroscopicscatteringresonance,whichstemsfromthesingleunitcellofthestructure,calledtheBrillouinzoneandisrelatedtohowthestructureisordered.Inthecaseofthediatomfrustule,themacroscopicscatteringwouldbefromtheholes(consistingofwateror,whencleaned,air)alternatingwiththesilicawalls.Themicroscopicscatteringwouldbefromhowtheholesarearrangedinthefrustule,forinstanceahexagonalstructure.IfboththemacroscopicandmicroscopicresonanceoccursatthesamewavelengthformationofaPBGisgreatlyenhanced.Thewavelengthorratherthebandwidthoverwhichlightisnottransmitteddependsalsoonthedifferenceinrefractiveindexbetweenthetwomaterialsofthemicrostructure.Ingeneral,thelargerthedifferenceinrefractiveindex,theeasieritistoobtainaPBG.ThecenterwavelengthandthebandwidthofthePBGarethereforedeterminedbythemacroscopicperiodicity(i.e.thedistancebetweentheholesandthesizeoftheholes),microscopicsymmetry(howtheholesarearrangedcomparedtoeachother),materialcompositionandpolarizationofthelight.Thepropagationdirectionofthelightintheabovesectionispresumedtobeintheplaneofthealternatingstructuresandthereforelightneedstobecoupledto(orredirectedto)thestructureinthisplane.Thecomplexityofthephotoniccrystal,andtherebytherelationbetweenthebeforementionedparametersincreaseswiththeneedofmultiplepropagationdirectionswhichincludesaPBG.WavelengthsincludedinthePBGandinthepropagationdirectionofthePBGisreflectedfromthestructureandmanyapplicationsbenefitfromthestrongwavelength-dependentpropagationoflight.Manyoftheseapplicationsdonotevenrequirethattheperiodicallymodulatedmaterialexhibitallpropertiesofphotoniccrystalsbutonlythat,lightisreflecteddifferentlydependingonitswavelength,i.e.themacroscopicBraggscattering.Thiseffectcanbeseeninthecolorsofcertainbutterflieswherethescatteringiswavelengthdependentandthereforethespectralresponsealsodependsontheobserver.

Diatomwallshaverepeatednanostructures(Figs1,2b)intherelevantsizerangeofPARandUVlightandhavebeenshowntohavecertaincharacteristicsofphotoniccrystals.Theorderedstructureoftheholeshasindicatedthepossibilityofphotonicbandgapswhenlightispropagatinginthevalveplane.Thishasbeencalculatedforbothvalvesandgirdlebandsbuthasneverbeenshownexperimentally(Fuhrmannetal.2004;Yamanakaetal.2008).IftheaimistopreventtheshortwaveUV-light(280-380nm)fromenteringthecell,diatomwallswithaperiodicityofholesoftheorderof200-250nmmaybeexpectedtodothiswhileallowingvisiblelighttopassthrough.However,calculationsshowthattherefractiveindexcontrastbetweensilica(1.45)andeitherwater(1.33)orair(1)togetherwithperiodicity,symmetryand

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polarizationisnotenoughtoformafullPBGinthefrustule.Furthermore,therefractiveindexcontrastbetweenwaterandthefrustuleinnaturalenvironmentofdiatomsisalsotoolowforefficientBraggscatteringbuttheperiodicstructureisstillshowntohaveastrongabsorptioninthebluespectralregion(Fuhrmannetal.2004;Yamanakaetal.2008).Whenacleanedfrustuleisilluminatedinair,therefractiveindexcontrastislargeenoughforefficientBraggscatteringandatleastpartialphotonicbandgapsoccur.Thisisseenintransmissionmeasurementsandcalculationswherespecificwavelengthsareattenuatedmorethanothers(Fuhrmannetal.2004;Yamanakaetal.2008;Kieuetal.2014).

Inexperimentalmeasurementswheretheincidentlightstrikesthefrustulenormaltothesurface,onlyaweakcouplingoflighttothevalveplaneoccursandtheindexcontrastoftheperiodicstructuregivingrisetoasmalleffect.Furthermore,onlythedirecttransmittedandscatteredoutofplanecontributionofthelightfromthefrustuleisobservedandnotthelightguidedinthevalveplane,makingitdifficulttoprobethetruephotonicbandgapstructureofthefrustule.Iftheangleofincidenceisvariedfromnormal,awavelengthdependentredirectionofthelighttowardsthecellinteriorisobserved.Thisindicatesastronginteractionbetweenthelightandthefrustuleindicatingastrongeffectoftheperiodicstructure(Romannetal.2015,Maibohmetal.2015c).Tofurtherincreasetherefractiveindexcontrastandtherebyenhancetheinteractioneffectbetweenlightandthefrustule,silicacanbesubstitutedinthegrowthmediumwithhighlevelsofthetracesubstances(Townleyetal.2007;Jeffryesetal,2008;Langetal.2013).Duetothefactthateventhetraceamountofimpuritiesmayinfluencephotoniccharacteristicsofthefrustule,cultivationconditionsmustbecontrolledtoensurereproducibleproductsforindustrialapplications.Oritcanbedoneby3Dstructuralpreservingchemicalsubstitutionafterremovaloforganicmaterialinthecleaningprocess(Sandhageetal.2002;Baoetal.2009;vanEyndeetal.2013).Tofurtherquantifyandunderstandthelight-frustuleinteraction,itsimplicationonbiologicalfunctionsofthediatomandthepossibleindustrialapplications,moreadvancedphotonicstudieshavetobeconducted.

TestsoftheeffectofthefrustuleonUVlight

Wehavefoundthatfourofthestudiesaimedatdeterminingpotentialindustrialapplicationsofdiatomfrustuleshaveanalyzedormodelledtheeffectofthefrustulesilicaandnano-patternonUVlight,experimentallyinvestigatingatotalofthreecentricspeciesaswellasperformingsimulations.InastudyontheopticalpropertiesofthecentricdiatomArachnoidiscussp.,Ferreraetal.(2014)simulatedthefocusingeffectofafrustuleinaironUV-lightof300nmandfoundthatthefocusingspotsatthiswavelengthstartedca.900µmfromthevalve,incontrasttofocusingspotsofPARlight(640nm),whichstartedatca.200µm.TheyalsomeasuredtransmittanceintensityprofileatUVBandfoundthespotintensitylowanddistancefarfromthevalve.Inapreviousstudy(onCoscinodiscuswailesii;DeTomassietal.2010)theyshowedbysimulationthatinwaterorcytoplasm,thefocusingspotsforPARwouldbeclosertothevalve.DeTomassietal.(2010)furthertestedtheeffectofavalveofCoscinodiscuswailesiionlightat280nm,andshowedthat,incontrasttolightatPARwavelengthsandabove(532nm,633nm&1000nm),thevalveformednofocusingspotsatthetestedUVwavelength.Goswamietal.(2010)studiedUV-luminescencepropertiesofCyclotellameneghiniana.InaUV-visiblespectroscopyanalysisofasinglevalvetheyfoundpeakabsorbanceat274nm.DeStefanoetal(2007b)simulatedUVshieldingpropertiesofpennateandcentricdiatomsbydigitalizingscanningelectronmicrographsoftwodifferentfrustules(fromThallassiosirarotulaorCoscinodiscuswailesiiandCocconeisscutellum).Davidsonetal.(1994)studiedtheeffectofUV-BirradiationongrowthandsurvivalofsixdifferentAntarcticdiatoms(Nitzschialecointei,N.curta,Proboscisalata,

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Thallasiosiratumida,Odentellaweisflogii,Chaetocerossimplex).Theirfocuswasthusonthebiologyofthediatoms(seediscussionabove)ratherthanonpotentialindustrialapplications;however,oneoftheparameterswhichtheyanalyzedwasabsorptionspectraoftherinsedvalves.Theyfoundthatgenerallyforallsixspeciesabsorbancefellalongthespectrum250-800nm.Theycalculatedthat,oftotalUV-Babsorbance,ca.20%wasduetothefrustule.Thiscould,however,beduestrictlytothematerialpropertiesofthesiliceousfrustuleandnottotheporestructure.MostofthesestudiesweredoneoncleanedfrustulesinairwheretheindexcontrastisgreaterthatinwaterandthereforethereispotentialforstrongerBragg-scattering.Therefore,asdiscussedpreviously,itcanbedifficulttoextrapolatetheseresultstoUV-protectionforlivediatomsinawateryenvironment.

TestsofUVandPARspectraofsinglefrustulesandlayersoffrustulesandwholecells

Experimentsaimedattesting,forpotentialapplications,methodsforcoatingasurfacewithbothrinsedfrustulesandcellsdriedwithcellcontentwerecarriedouttoexploreifthetransparencywasdifferentforUVandvisiblewavelengthswhendiatomswerearbitrarilydriedontoaglassplate(Fig.3).

LighttransmissionwasmeasuredfortwospeciesofCoscinodiscus:C.graniiandC.concinnus.Bothdriedintactdiatomswithcellcontents(dead,butwithremnantsofcellcontents)anddiatomvalvesrinsedoforganicmaterialwereused.ThediatomsweredrippedontoaUV-transparentquartzsilicamicroscopeglassplateanddriedatroomtemperature.Theprocesswasrepeatedseveraltimestoincreasetheconcentration.Inspiteofthis,wedidnotalwaysachieveahighdegreeofcoverageontheglassplate.TransparencyspectraweremeasuredusingaShimadzuUV-2600spectrometerwithbuild-inlightsourcesandintegratingsphere.MicroscopepicturesweretakenusinganOlympusGX41lightmicroscope.Allmeasurementswererepeatedatleasttwice.

AllsampleshadthehighesttransmissionforthelongestwavelengthsandthelowestintheshortwaveUV-spectrumasshowninFig.4.ForrinseddriedfrustulesofC.concinnusthetransmittancewasreducedfromca.67%toca.60%inthevisiblepartofthespectrumfrom700-400nm.IntheUV-Bregion(from320-280nm)thereductionwasfromca.57%toca.53%,i.e.theslopeofthetransmittancereductionwassteeperintheUV-Bregion.AsimilartendencywasseenforC.granii,buttheconcentrationoffrustuleswaslowandthereforethesignalwaslesspronounced.Forthedrieddiatomswithcellcontents,thereductionintransparencyissteeperandstartsat500nm.ForC.granii,thereductionwithintheUV-AandUV-Bspectrum(400-280nm)isfrom14to3%.ForC.concinnus,thereductionintheUV-spectrumisfrom24to9%.Thetransmissionvariedforthedifferentsamplesdependingontheconcentrationofthediatoms.Fortherinseddiatomfrustulestheconcentrationvariedbetween45and85%andforthedriedlivingdiatomsitwas85-95%asshownintable1andillustratedinFig.3.

ThesepreliminaryresultsindicatethatthevalvesaremoretransparenttolightinthevisiblespectrumthanintheUVspectrum,orinotherwordsthatthevalvesfiltersomeoftheUVlight.Thistendencybecomesmorepronouncedtheshorterthewavelength.Forthecleanedvalves,thefilteringeffectintheUVspectrumisseeminglyrathersmall(below10%).However,thiseffectislikelytobecomelargerifacompletecoverageisachieved.Inthemeasuredsamplesonlyabout45-95%ofthesurfaceareawascovered.Toestimatethesizeoftheexpectedspectraltransmissionforacompletecoveragethedatawasnormalizedtothedegreeofcoverageonthequartzmicroscopeglassbymultiplyingwiththefoundcoveragepercentage.TheresultingcurvesinFig.5showthattransparencyishigherforC.concinnus

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comparedtoC.graniiforcleanedfrustulesandfordiatomsdriedwithcontent.C.concinnusalsohasarelativelylowertransparenceintheUV-area.ThetransparencylevelforC.graniiwas30-40%(lowestfortheshortestwavelengths)whichwassimilartothetransmissionobservedthroughasinglevalvedrop-castedonquartz(Fig.6).ThelightbeamwasinthiscasemaskedwithapinholetoonlyallowlightthroughasinglefrustuleasshowninFig.1a.

Formanypotentialindustrialapplicationsitwillbenecessarytoincorporatethediatomsintosomekindofamatrix,e.g.paintorcreme.Thisraisesquestionsofhowtoachievethis,howhighadegreeofcoverageisnecessaryforthedifferentapplicationsandwhetherthelayermustbeuniformand/orflat.

Forthediatomsdriedintactwithcellcontent,amuchmorepronouncedfilteringcanbeobserved.ForC.graniionly3%ofshortwaveUV-Bat280nmisallowedthroughthesampleincontrasttothe28%transmittanceforred700nmlight.Inadditiontothepresenceofcellcontents,anotherdifferencebetweenthetwotreatmentsisthatinthecaseofthediatomsdriedintactwithcellcontents,thefrustuleisintactwiththetwovalvesstilltogether.Thismeansthatthelightwillalwaysstrikethesideofthevalvefacingtotheoutsideofthecell,andasmentionedpreviously,thereappeartobedifferencesinthephotonicpropertiesdependingonwhichsideofthevalveisfacingtowardsthelight.

ThetendencyinthesefindingsisinconcordancewiththefindingsbyNoyesandcolleagues(2008).TheymeasuredlighttransparencyinthediatomC.wailesiiandfoundapproximately80%forredlight(633nm),30%forgreenlight(543nm)and20%forbluelight(472nm).Theyexplainthevaryingdegreeoftransparencywithdiffractionintheholestructureandpointoutthatitisthelargeholesontheinsideofthevalvethatareresponsibleforthediffraction.Similarly,asstatedpreviously,Goswamietal.(2010)foundpeakabsorbanceofthefrustuleofCyclotellameneghinianaat274nmandDavidsonetal.(1994)foundthatthefrustulesofsixdifferentspeciesaccountedforca.20%ofthetotalUVabsorbance.

Summary/Conclusions

Ouroverallconclusionisthat,whileonlyafewspecieshavebeentestedsofar,theresearchintotheroleofdiatomfrustulesinUVprotectionshowgreatpotential,bothwithregardtodiatombiologyandpotentialapplications.

- LessthantendiatomspecieshavebeentestedforeffectsofthefrustuleonUVlight.- Studiesshowthatthefrustuleaccountsforupto20%reflectance/absorbanceintheUVspectrum- Lightfocusingdistancedependsonthewavelength,withlongerdistancesforUVlight.Thiscould,

however,beveryspeciesdependent,becauseofthedifferencesinperiodicstructuresbetweenspecies.

- Therearetwogeneralapproachestomodelingphotoniceffectsofdiatomfrustules.Oneconsidersthetransmissionoflightthroughtheplaneofthevalvewithlightpassingthroughtheholes,andthesilicawallsmoreorlessopaque.Theotherconsidersthepropagationoflightintheplaneofthevalveandfocussesontheperiodicstructureofthelargerholes.

- PhotoluminescenceeffectsareseenwhenilluminatingfrustuleswithUVlight.Thesearestrongestundervacuumconditions.

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- Theabilitytocompletelyexcludelightofcertainwavelengths(PBGs)dependsontherefractiveindexbetweenthefrustuleandthematrixaswellastheperiodicityofthenanostructure.SofarcompletePGBshavenotbeenexperimentallyshownindiatoms.

- LivingdiatomshaveamorevariableresponsetoUVlightthanothergroupsofalgae–thiscouldbeduetodifferencesinUVpropertiesofthefrustule

- ImportantelementstoconsiderforUV-mediatingeffectsare:theperiodicityofthelargeholes(areolae),therefractivecontrastbetweenthefrustuleandthemedium,theorientationofthevalves,thedegreeofcoverageofalayeroffrustules.Thesmallholes(inthecribrum;seefigure1)andcentralareaappeartobelessimportant.

Acknowledgements

ThisworkwasfundedbyTheDanishResearchCouncil(projectALPHA12-127569).SandraWalbyhelpedwithlaboratoryworkandJacobSnebjørnBrøggerKristensendidpreliminarystudiesontransmissionthroughaspin-coatedlayeroffrustules.TomasBenzonhelpedwithgraphicsinfigure2.Threeanonymouseviewersarethankedfortheircommentsthathelpedimprovethemanuscript.

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Figure1:ScanningelectronimagesofCoscinodiscusgranii,showingavalveseenfromtheoutsideofthecellandgirdlebandspartlydetached.Insert:Scalebar500nm;smallsectionofadiatomvalveshowingthe3Dstructure.

Figure2:Overviewoverrelevantfeaturesthatdifferbetweenthediatominitsnaturalhabitatandarinseddiatomvalveinatypicalexperimentalphotonicset-up(a).b:Adiatomvalverinsedoforganicmaterialandseenunderlightmicroscope.c:Alivediatominwater(lightmicrograph).d:Aschematicofspectralchangesinlightthroughawatercolumn

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Figure3:LightmicroscopepicturesofdrieddiatomsonaUV-transparentquartzsilicamicroscopeglassplate.Scalebar0.2mm.Coverage(seetable1)isestimatedwithinthewhitesquare.a:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.

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Figure4:Transmittancespectrafora:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.ThereferencespectrumistheUV-transparentquartzsilicamicroscopeglassplatewithnothingontopofit.

Figure5:TransmittancespectrafromFigure4normalizedtodegreeofcoverageonthequartzmicroscopeglass.a:Coscinodiscusconcinnus,cleanedfrustulesb:C.granii,cleanedfrustules.c:C.concinnus,driedcellswithcellcontent.d:C.granii,driedcellswithcellcontent.

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Figure6:TransmissionthroughasinglevalveofC.Graniinormalizedtothebackground.

Table1:AveragediatomdiameterandcoveragepercentagefordiatomsonquartzglassshowninFig.3.

Approximatediameterofdiatom

valve(µm)

Standarddeviationdiameterofdiatom

%coverage

1.Cconcinnuscleaned

49 6.09 Ca85

2.Cgraniicleaned

62 2.61 Ca45

3.Cconcinusdriedwithcellcontent

43 12.18 Ca85

4.Cgraniidriedwithcellcontent

146 1.71 Ca95