Measurement of Absorption and Scattering

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Volume14 ,N u mbe r2, March-April 2009JournalofResearchofth eNationalInstituteofStandardsan dTechnology[J. Res.Natl.Inst.Stand.Technol.14 ,69-81(2009)]

MeasurementofAbsorptionandScatteringWithanIntegratingSphereDetector:ApplicationtoMicroalgae

V o lu me14 Number2 March-April20 0 9A. K.Gaigalas,Hua-JunHe, andLiliWangBiochemicalScienceDivision,NationalInstituteofStandardsan dTechnology,Gaithersburg,M D20899

[email protected]@[email protected]

Aspectrometerwithanintegratingsphere(IS)detectorwa susedto measurethe absorbancedu eto scatteringan dabsorption.Analysisofthe measurementprocessshowed thattwo measurementsofthe absorbance,on ewiththe cuvetteplacedinthe normalspectrometerposition,an dthe secondwiththe cuvetteplacednextto the entrance aperture oftheISdetector,provide enoughinformation toseparatethe contributionsfromscatteringan dmolecularabsorption.Measurementswerecarriedou twithmixturesofmicrosphere and chromophoresolutions.Tw ocaseswereexamined: icrospheressuspendedinanaqueousfluoresceinsolution,an dmicrospheres suspended inanaqueousholmium oxidesolution.Inbothcases,the proposedmeasurementmod elgaveresultswhichwereingoodagreementwiththe expectedresponse.Measurementson microalgae suspensions

yielded amolecularabsorptioncontribu-tionand ascatteringcontribution.Th escatteringcontributionha dsignificantspectralstructurewhichwa s inverselyrelatedto the molecularabsorptioncontribution. Th eabsorptionan dscatteringcontributionsm ayprovide independentinformation onthe statusofchlorophyllmolec ulesan dthe structureofchloroplastsinmicroalgae.

Key words:bsorbance;integratingsphere detector;microalgae;scattering.Accepted:March6,2009Availableonline:ttp://www.nist.gov/jres

1. Introduction Th emeasurementofabsorptionndcatteringnturbidnvironmentsnmportantroblem.uch

measurementsrese doharacterizempuritiesnwater1]nd toharacterizequaticparticles2] .nthesepplications, pectrometerwithnntegratingspheredetectorIS )sdealinceheSaneduceth effectofscatteringndnhanceheffectdu eomolecularabsorption.Descriptionofth euseofISwithacuvetteholderinside theISwa sgivenbyNelson[3].Adescriptionofth eISwithaninternal sampleisgivenbyabsphere4].Anpproachha tplacesamplesoutside th eISwa salsodescribed5] .AlthoughtheISwithnnternalamplee e m sorovideheeastsensitivityocattering,hisrrangementesultsn

annon-idealS6]ndasroblemsithtirringan dtemperaturecontrol.An ISwithth esamplefillingth eompletevolumeofth eShavebeendescribedinth eliterature[7,8].Thislatertechnique isno tpracti-ca loriologicalamplesithequirementsorsmallampleol um e .Microalgaeultivationsnimportantre aherebsorptionmeasurementsillgain in importance.Th ebsorptionspectraofchloro-phyllinplantsisarichsourceofinformationaboutth estateofth emicroorganism [9].Inthiswork,we devel-opam o r egeneralan dsystematicmodelof th eISmeas-urementrocess10]ndhenterpretationfheresultingata.hisorkppliesheeasurementm o d e ltoth emeasurementofabsorbancein mixturesofmicrospheresan dchromophoresan dto asuspensionofmicroalgae.

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2. InterpretationofIntegratingSphere IS )MeasurementsTh eproposedmethodormeasuringhecatteringan dmolecularabsorption utilizesaspectrophotometerwithanintegratingsphere(IS)detector.Fo rclarity,th emeasurementwillbe described withaspecificnstru-mentthePerkinElmerLambda850'.Figure 1 shows

th epathsof th esamplean dreferencebeamsusedinth emeasurementofabsorbance.Th ewopathsreverysimilar;bothndn th ewalloftheSdetector.Thepathf th eamplebeamontainshreemechanicalholdersfo racuvette labeled,2, an d3 inFig..Tw ocuvette holders, nd 2, are locatedoutsideofthe ISdetectorwhileth ecuvetteholder3 islocatedinsideth eISdetector.Th e lensan dth emirrorsshapeth esamplebeamsothatitpassesunobstructedthroughth ecuvetteholders.herefore,woeasurementsaneer -formedwithth ecuvetteplacedoutsideth eIS,an dth ethirdmeasurementwithth ecuvetteinsideth eIS .Inth efollowing,wediscussam o d e lof th eresponseof th eISdetectororheas ewhereheamplesplacedncuvette holders nd 2. Th ecasewhereth esampleisplaced incuvetteholder3 isno ttreated.The responseinholder3scomplicatedby th eincreasedsensitivitytofluorescencean dth einability tostirthesuspensioninholder.Th euseofholder 3o rmeasurementoffluorescenceuantumieldaseenescribedpreviously[11].2 .1 Responseofth eISDetectorWithSamplesin CuvetteHolder

Anncidentlux,0,(W-Watts),passeshroughhecuvetteplacednholder,ntersheShroughhesampleapertureofareaA,(m^),an dhitsth ewallofth eIS. referencelux,0,,(W),ntersheShroughreferenceapertureofarea ^Cm^)an dhits th eISwallwithoutassinghroughheuvette.heeferencebeamwillenterth eIS, hitth eISwallan dundergo th efirstreflection.A baffleinsideth eISpreventsth edetec-to rfrom seeing thefirstreflectionoftheincidentrefer-encebeam.Th eeferenceightbeam undergoesmanymo re reflections resulting inan averageflux,O , * ,incidentontheISsurface.Th eaveragefluxwillbewrittenas

O M,,,0, (1 )Certainommercialquipment,nstruments,raterialsreidentified in thispapertoosterunderstanding.Suchdentification

doesno tmplyecommendationrndorsementbyheNationalInstituteofStandardsndTechnology,no roe stmplyha thematerialsorequipmentdentifiedare necessarily the bestavailableforthe purpose.

Th esymbolM,^^represents th emagnificationofth eincidentreferencefluxdu e to th emanyreflectionsonth e ISwall.Neglectingth epresence oft hebaffle,heidealSehavioreadsoheollowingxpressionfo rheagnification,^ ^=p(l-/)/(l-p(l-/)).Th econstant/isdefinedasth eratioofareasgivenby/={A i+ A,+ Aj)lAgwhereA^and Agareth eareasofth edetectoraperturendotalphereurfaceespec-tively.Th everageeflectanceofth ematerialiningth eurfaceof th eSsgivenbyp[12].Thephoto-multiplier(PM)detectorismountedflushwithth esur-faceofth eIS.Thereforeth ePM detectorresponsewillbeproportionaloheveragelu xncidentpe runitsurfaceareaofth e ISmultipliedby th edetectorarea.Explicitly,hedetectorignal,),.,willbegivenbyEq . (2 )

D ^=i?(A)0,M, =Ea)M,^^0^2)A,where(X )sheadiantensitivityfhehoto-multipliercathode.Th eratioofth edetectorareato theareaofth eentireSsaconstantwhichca nbe com -binedwithth eradiantsensitivityto giveoveralldetec-tionefficiencyE{X).Itwillbeassumed thatt hedepend-enceoft hedetectionefficiencyon wavelengthcom e smainly from th eradiantsensitivity ofth ephotomulti-plierathode.Accordingoheanufacturer,hereflectanceofth eSurfacevariesbyes sha n%overth epectralregionconsideredin thiswork.Thisvariationhas weakdependencenwavelengthndm ayntroduce ystematicncertaintynheinalresult.AnequationsimilartoEq .(2 )ca nbewrittenrelatingth edetectorresponse,D,to th eincidentflux,O,enter-in gthroughthesampleport

A, 3

Th eubscripton th emagnification factor indicatesthatth emagnificationm aybe slightly differentfo rth efluxenteringthrought hesample porttothatfor thefluxenteringhroughheeferenceort.heautozerofunctionofth espectrometerisusedtocompensate fo ran ydifferencesinth eISdetectorresponsebetweenth ereferencean dsample beamsinth ecasewherethereisnoamplen th euvette holder.W ewillssume thatth edetectionefficiency isdenticalo rbothcases,othatth e autozero function insures theequality givenbyEq .(4 )below

M O=MO 470


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o

JO.(jc)dx =-


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Volume14 ,Number2, March-April 2009JournalofResearchofth eNationalInstituteofStandardsan dTechnology

inEq .8)suggests that it isno tpossible toobtain th escatteringndmolecularbsorptione rm simplybysubtractingthe bufferabsorptionfromthe measurementof totalabsorption.The measuredabsorbanceshould b everydifferento ramplesplacednuvetteholder2since thedetectoracceptance angle ism u c h largerfo rholder2.W ediscussthiscasenext.2 .2 Responseofth eISDetectorWithSampleinCuvetteHolder2

Th eesponsefhenstrumentoheamplencuvetteholder2ca nbemodeledinth esamewa yasth eresponse to th ecuvette inholder.The onlychangesarein th evalueof th einstrumentacceptanceapertureinEq .(7),an dth emagnificationfactorchangedu eto th epresenceofacuvettet th eamplentranceport.Toaccountfo rdifferentacceptance aperture, thequantitya ^ p in Eq .(8 )ischangedtoa'^wherethe primeindicatesthat th eacceptance aperture isppropriate fo rcuvetteinholder2.W h e n uvettesplacednrontofth eentranceaperture,th emagnificationfactorm aybe dif-ferentbecausehentrancepertureno wha s finitereflectance.Thisac tsmadexplicitbywritinghemagnificationfactorswithaprime inEq .8).TheautozerorelationinEq .(4 )isno tappropriatefo rt hecaseofacuvette inholder2. Itwillbe assumedthatth em ag-nificationactorso rheransmittedndcatteredfluxesareequal.Differencesbetweenth etw omagnifi-cationactorswillea doystematicuncertaintiesnth einalesults.Al lf th etheronsiderationsreidenticaltothosediscussedfo rholder 1othatam o d -ifiedformofEq .(8 )ca nbeusedtom o d e ltheresponsefo racuvette inholder2

ample M'O.+M'O,D., M: ..sample ^^^2jo-0.434 , e -(1-.j^-0.434 a ,+a,^))dMsample =10- ' (9 )

whereyefinifionM=(M',^/A/'^)(M,^/M,JwhereAf ^shemagnificationactororhebeamenteringthesampleaperturewithacuvette infrontofit,nd M^fishemagnificationactoro rhebeamentering through th ereferenceaperturewithacuvetteplacedinfrontofth esample aperture.Th equantitya'^isargerha n,poha theesponsepredictedbyEq .9)sxpectedobedifferentro mheesponsepredictedby Eq .8) .Th emagnitude oft he factor5Mca nbe estimatedbymeasuringwaterfilledcuvette in

holders nd2afterperformingan autozerofunctionwithallholdersempty.To averygoodapproximationth eabsorbance an dscatteringofwateriscloseto zero.Thereforeomparingq.8)ndq.9)orhecasewhere,ndavanish,webtainheelation5M=lO '*^10 ^'whereAian dA2are th eabsorbenciesmeasurednholder ndholder espectively.Th edataisshowninFig.3 aan dth eresultingfactor5MisshowninFig.3b. The factordMdiffersfrom 1by lessthan%ve rheangefwavelengths00 nmo700 nm .Th efactoris slightlygreaterthan sw ouldbe expectedfo rareflectingsurfaceplaced infrontofth esampleport.Th efactor5M willbeneglectedinth esubsequentdiscussion.

1 rf

holder2

wavelength,nm

b)

son500050 wavelength,nm

Fig. .igurea,heolidracehowshemeasuredbsorbancefrom acuvettefilledwithwaterand placed inholder.Th edottedtracenFig.3ahowsheeasuredbsorbancewhenheam ecuvette isplacedinholder2. Th ediscussionfollowingEq .(9 )inthetextindicatesho wthe tw omeasurementscan beusedtoestimate thechangeinISmagnification factordu etothe presenceofacuvetteattheamplentranceperture.Th eolidracenFig.3bhowsheestimatedchange inmagnificationfactor5Mobtainedfromthe datainFig.3a .Th echangeislessthan% .Th edottedfrace inFig.3b isafittothe datausingasecondorderpolynomial.

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2 .3 Summationofth eMeasurementModelThewoquations,q. (8),q. (9),roviden

explicitelationbetweenheeasuredbsorbenciesan dth efluxesthatexistinth eISdetector.The discus-sionofth em o d e l responsewa sgeneralndontainsparameterscharacterizingth escatteringan dmolecularabsorption.Th emeasurementmodelprovidesamethodfo rxtractinghepparentbsorption,,,ueoscatteringan dth emolecularabsorption,a.Inth ecaseofholder1 ,hephereccepts smallportionofth escattered fluxexiting th euvettewhilen th eas eofholder2 th esphereacceptsalmostall oft hefluxexit-in gth ecuvette.Fluorescencewas neglected.Equation(8) canbe used todescribe bufferabsorbancewhich isassumedto haveno scattering(a ,=0)an dnomolecularabsorption (a =0) .Equation8)uggestsha t fo r th ecaseofabuffer,heransmissionoefficientanbeevaluatedusingEq .(10)an dth eassumptionofnormalincidence

10 100.434(a,+a -a ) +2 log(()

10^ 10 (10)However,heormofEq .8)ndEq .9)uggests

thatingeneralth ebufferabsorbance ca nno tbe simplysubtractedfromth esampleabsorbancetoyieldth etrueabsorbance.Th eobjectiveof th efollowingdiscussionisouggest ethodorsingeasurementsfabsorbanceinholders 1 an d2toextractseparatevaluesfo rth eapparentscatteringabsorption,a,,an dmolecu-la rabsorptiona.3. MeasurementofAbsorbancein ScatteringSuspensions

Inorderoes thetilityofEq .8)ndEq .9) ,measurementswereperformednmicrospheresus -pended inanaqueoussolution ofholmium oxide,an dmicrospheresuspendednnqueousolutionffluorescein.Measurementswerelsoperformedonsuspensionfmicroalgae.om ef th euspensionswereufficientlydiluteoha tpproximateorm sofEqs.(8 )an d(9 )couldbeutilized.Inth efollowing,th eapproximateformsare presentedan dth edataanalyzed.

Asdiscussedpreviously,o goodpproximationth eagnificationactorsothangedppreciablywhen uvettesplacednrontofth eSntranceaperture.Inthatcase,Eq .(8 )an dEq .(9 )haveidenticalform withdifferentvaluesofa,p.W euse th eapproxi-mation 1 0 = 1 -j:ln(10),whichisvahdfo rJ:1 ,an dobtain th e following approximaterelationsfo rEq .8)an dEq .(9 )

10 10-0.434(0,+a-a'+21og(0 (11)Equation(10)ca nbeusedtorelatet toth emeasuredabsorbancefheuffernheuvettelacednholder1 .Subtractinghe measuredbufferabsorbance

fromth eabsorbance inholder 1ndholder2givesthefinal relationbetweenmeasuredabsorbenciesnd th esuspensionpropertiesV' / 0A34(a,+a^-a^p):0.434(a+aJ-0.434a (12)

2- f 0.434(a,+a -a;)=0.434a+0.434(a^-


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isno tsensitivetoa,.Th eaboveprocedureisapplicabletoargeraluesfnd,ndxtendsheanalysisusedinEq .(12).3.1 MixtureofaMolecularAbsorber andaScatterer

Figure4 ashowsthemeasured absorbance ofcarboxylmodifiedolystyrenepheresBangsaboratories,PC0 5 N)ofdiameter2.04|amuspended in fluoresceinsolution.he icrosphereser eilutedy0 '*ndsuspendedinasolutioncontaininga5 0folddilution ofSR M1932fluoresceinsolution) inacetatebuffer.Th epHofthecetatebuffer isloseo hu sminimizingfluoresceinfluorescence.Th esolidtraceinFig.4 ashowstheeasuredbsorbancenolder ndheotted

0 .1

tracehowshe measuredabsorbancenholder2.Th evalueofabsorbanceaftercorrectionfo rbuffer) in thedottedtraceat700nm wa s takenastheestimateofthecontributionfrom scattering inholder 2.naccordancewithEq .(12),thescatteringcontribution, {a, a'.^,),wassubtractedfrom th edottedtrace toyieldan estimateofa .Th eestimated valueofaisshownbythesolid traceinFig.4b .Th edottedtraceinFig.4 bshowsthemeasuredabsorptioninholder1orafluoresceinsolutioninacetatebuffer(withoutmicrospheres)where the fluoresceincon-centrationisapproximately equaltothe fluoresceinconcen-trationinthe suspensioncontainingthemicrospheres.Th ecorrespondence between the estimated aobtainedfrom thesuspensionndhebtainedro mhequivalentfluoresceinolutionsood.Th eolidracenFig.c

5 5 55 6 65 7

wavelength,nm Fig.4.Th esolidtrace inFig.4 ashowsthe measuredabsorbance inholder ro m acuvette containingamixtureofmicrospheres and fluores-ceininanacetatebuffer.Th edottedtrace inFig.4ashowsthe measuredabsorbancew henthe cuvette istransferredtoholder2.Th esolidtraceinFig.4 bshowsthe derivedmolecularabsorptioncoefficientusingEq .(8) and Eq .(9) and the datainFig.4 a.Th edottedtrace inFig.4 bshowsthemeasuredmolecularabsorptioncoefficientofanequivalentsolution offluoresceinwithoutthe microspheres.Th esolidtraceinFig.4 cshowstheapparentscatteringabsorptioncoefficientobtainedusingEq .(8),Eq .(9 )an dthe datainFig.4 a.Th edotted traceinFig.4 cshowsthe measuredapparentscatteringabsorptioncoefficientofanequivalent suspensionofmicrosphereswithoutfluorescein.

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showstheresult of subtractingafromth esolidtraceinFig.4 a (seeEq .12)).The dotted trace inFig.4 c isameasurementfbsorptionueocatteringfnequivalentuspensionofmicrospheresnwater.Thevaluesfheworacesnig .4 cifferlightly,how ever th eunctionalormofth eworacesshesameshowing thatonlyscatteringiscontributing.Theuncertainties inth emeasurementofabsorbance du etoinstrumentnoisewereofth eorderof0.001bsorbanceunits.hereasndditionalncertaintyfbout0.001nsuspensionmeasurementsdu e to th e fluctua-tionofmicrosphereoncentration.Changesnmeas-uredvaluesdu etoinstrumentdriftwerenegligibleoveraperiodof10m into15 min,th etimerequiredtoaccu-mulateheetfdatahownnig .4 a.ow everinstrumentdrift couldintroducedifferencesofth eorderof0.002betweendataaccumulated at timesseparatedbyhours.Th eystematicuncertaintiesdu eonstru-m e n tdriftare difficulttocharacterize.The differencesbetweenhemeasurementsisplayednFig.4 bndFig.4 creofth eorderof0.01,ndhu sregreaterthan th edifferencesxpected from instrumentalnoisean drifts.urthermoreheifferencesetweenhetracesin Fig.4 ban dFig.4 cshowacleartrendsuggest-in gasmallsystematicbias inth eanalysisoft hedata.Nevertheless,heverallonsistencyfheesultsobtainedor icrospheresuspendednluoresceinsolutionndicatehat th ereatmentofthedatausingEq .(8 )an dEq .(9 )doespermitth eseparationofcontri-butionsro mcatteringndmolecularbsorption,albeitsomerefinementsm aybenecessary.

Figureaho wheesultso r uspensionfcarboxylodifiedolystyrenepheresBangsLaboratories,PC05N)ofdiameter2.04 |am inaaque-ousolutionontainingolmiumxide4%as sfraction)nd perchloriccid1 0%volumeraction).The aqueoussolution isequivalent toSR M2 0 3 4 .Th esolidracendhedottedracenFig.5 aho whemeasuredabsorbenciesinholders nd2respectively.The absorbance at 700 nm in th esolidtrace inFig.5 awasusedtoestimatethecontributionfromscatteringa.

inEq .(14).Following th eproceduredescribedboveyieldsnstimatefhemolecularbsorptionshownbyheolidracenFig.5b.ThedottedraceinFig.5b,whichoverlapsheolidrace,howshemeasuredbsorptionfnquivalentolmiumsolutionithoutmicrosphere.heorrespondencebetweenhew oracesnig .5 bsxcellent.Followingherocedureutlinednq. (13 )ndEq .(14) ,hestimatedasubtractedromAi Ai * ' ^ t ogivetheapparentabsorptiondu etoscatter-in gwhich ishownby th eolid tracenFig.5 c.Th edotted tracenFig.5 chowshepparentbsorptionmeasuredfo ra nequivalentsuspensionofmicrospheresinwater.Th eoverallcorrespondence isgood suggest-in gha thenalysissingheethodutlinedorlargervaluesofa,ndsvalid.However,sdis-cussedinconjunctionwithFig.4 ,th esystematicdiffer-encesbetweenheworacesnFig.5 creutsideinstrumentaluncertaintiesan dindicateasmallsystem-aticbiasinth eanalysis.3.2 PropertiesofMicroalgaeSuspension

ChlorellafuscaanfuscaU TE Xnumber3 4 3 )wa sobtained from theCultureCollection ofAlgae, UniversityofTexasat Austin.Thisorganismwa sselectedaccordingtotscologicalelevancendaseofculturenhelaboratory14].CulturesweremaintainednProteoseMedium,hichshemediumro mBristol'secipemodifiedy..old2.94mmol/LNaNOj,0.17m m ol /LCaClj,.3m m ol /LMgS04,.4 3m m ol /LK2HPO4,1.29m m ol /LKH2PO4an d0.43 m m ol /LNaCl,pH 6.8)ndupplementedithg/LfroteosePeptone.Al lulturesweretartedro mtockulturesinitiatedfrom singlealgagrownon semi-solidplatesoftheappropriate mediacontaining3 % agar.Newlytrans-ferredcultureswereincubated at20C to 25 C underacool-white fluorescentlampfo r2han d theninthedarkor2h,llowingellsogrowuntil desiredpopulationdensitywa sachieved, usuallyat,000,000cellspe rm L.Th epossiblecontaminationan dcellden-sityweremonitoredan dcountedwithamicroscope.

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d

o

1.2

1.0


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diameter,jim

Fig.6.a)Th edistributionofparticlesizeswhenadilutedsuspen-sionofmicroalgae ispassedthroughaCoulterMultisizer 3particlecounter.Th epeakat7^mcorresponds to singlemicroalgae passingthroughthe orifice.Th epeakat8^mcorresponds toaggregates ofmicroalgaeontainingro m0o0individualmicroalga.Therem ay be evenargeraggregatesn themicroalgaeuspension;how-everheilutionndtirringnheMultisizer nstrumentm aydestroyheseargerggregates,b)ifferentialcatteringrosssectioncalculatedfo raparticleof diameter7^im,relative indexofrefractionequal to.03,nd 60 0nm illumination.Th escattering ismostlyinthe forwarddirection.becauseheuspensionontains argeumberfaggregated icroalgaeellshichrexpectedoscatterdifferently.urthermoreheelativendexofrefractioncouldbedifferentfo rthisspecific speciesofmicroalgae.

3.3 MeasurementofAbsorbanceofMicroalgaeSuspension Duringth emeasurementsofabsorbanceofasuspen-

sionofmicroalgaeincuvetteplacedinholders 1 an d2,th esuspensionwas stirredtokeepth esuspensionfromsettling.Measurementsarriedutfterheapseofabouthalfhour indicate thattheabsorbancevaluesareverylosendicatinghatettlingasotccurred.Fluorescencefromchlorophyllmoleculesisno tamajorsourceofuncertaintysincethequantum yieldfromalgaeisfoundtobeoftheorderof0.02[17].Furthermorethefluorescenceemissionisisotropicresultinginnegligiblysmallluorescencelu xntoheSdetector.Th eolidtracenFig.7ahowshebsorbancefmicroalgaesuspensionmeasured inholder while thedottedtraceshowshebsorbanceeasurednolder 2.learlythere isalargedifference inthemeasuredabsorbenciesinthetw oholders.Th eprocedure outlinedinEqs.1 3)an d (14)wa susedtoanalyze thedatainFig.7a .nallcases,hemeasuredbsorbanceromherowthmediumbuffer)asubtracted.heolidracenFig.7bhowsheesultorndheolidracenFig.7cshowstheresultinga,.Thereissubstantialstruc-tureintheresult fora,and thestructureisamirrorimageof th eabsorptionspectrumshowninFig.7b .Althoughitisno tpossibleoomparehevaluesofa,ndoindependentmeasurementsofscatteringan dabsorption,itcanbe expectedthatthe traces shown inFig.7b an dFig.7carerepresentativeof the true valueswithsomequalificationsasdiscussedincormectionwithFig.4an dFig.5.Measurementswereperformed indilutedsuspensionofmicroalgaeoha tllmeasuredbsorbencieswereless than0. 2nd th emeasurementscouldbe analyzedusingEq . (12).Th eresultsweresimilartothoseshowninFig.7. Thereforeneitherth edegreeofapproximationintheanalysis no rthemicroalgaeconcentrationseemstoinfluenceheinalesults.Measurementswerearriedou ton microalgaeuspensionswitheducedllumina-tion.Th elluminationevelwa seducedbynsertingneutraldensity filters in the incidentbeam.Th eresultsfo r% illuminationwere thesameas thosefo r00%illumination.

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0 .5

0 .4

0 .3

0 .2

0.1

E OC C

0 .2

0 .3

holdera)

holder2

5 5 55 6wavelength,nm 65 0 700

Fig. .heolidracenFig.7ahowshemeasuredbsorbancenholder or uvetteontainingsuspensionofmicroalgaenagrowthmed ium.Th edottedtracenFig.7a showshe measuredabsorbancew henthe cuvette istransferredto holder2. Th esolidtrace inFig.7b showsthe molecularabsorptioncoeffi-cientbtainedusingEq .8),Eq .9)nd theata inFig.7a .Th eolidracenFig.7chowshepparentscatteringabsorptioncoefficientobtainedusingEq .(8),Eq .(9) an dthe datainFig.7a .

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4. Discussion Th eresultsfo rmixturesofmicrospheresan dabsorb-

in gsolutions,hown inFig.4an dFig.5,uggestthatth enalysisbasedonEq .1 2)rEqs.1 3 )nd1 4 )givesareasonablygoodseparationofth econtributionsfrommolecularbsorptionndcattering.hewosystemsstudiedconsistedofamixtureofnonabsorbingmicrospheresan dabsorbingchromophoremoleculesinsolution.Thereforeitissafeto assumethatth escatter-in gprocess,whicharisesro m th emicrospheres,ndmolecularbsorptionprocess,whichrisesro mhechromophores,er eompletelyndependent.nhecaseofmicroalgae,hebsorbinghromophoresrepackednsideheell.nhisase,heontributionsfromcatteringndmolecularbsorptionreo tindependent.Infact, itisexpectedthatmicroalgaescat-teringndmolecularbsorptionrehighlyorrelated.Th eescriptionofcatteringgivenbyq. (6 )ndEq .(7 ) isapplicableto microsphereswhichcontainanabsorbing material (e.g.,algaecellswithchlorophyll).Usinghiscatteringormalismtsossibleodescribeheotalnergyos sro m beamof lightpassing throughacelluspension asarisingfrom tw oprocesses:helasticcatteringbyheel lnterfacean dmolecularabsorptionbyth echlorophyllmoleculesinside th ecell18].The cruxof th ematteris that th emeasurementm o d e linEq .5) isequallyapplicabletomicroalgaendomixturesofindependentcatterersan dbsorbers.hereforehemicroalgaeesponseshowninFig.7b, an dFig.7cm u s tbe ofequalvalidityasheesponseoundormixturesfmicrospheresan dchromophores.

Th epresenceofstrongspectralfeaturesinth emeas-uredcatteredightro mmicroalgaeuspensionsha sbeennotedpreviously[19].Earlyworkinthisareawa sreviewedbyButler20].Th etrongpectraleaturesinhecatteredightse eFig.7c)havebeenalledselectivecattering ndtwa snotedheelectivescatteringeffectdependedon th ecceptancepertureofhenstrumentse dnheeasurement.ecentattempts tocompensateor nonselective an dselectivescatteringan dtoobtainth ecorrectabsorptionspectrumaredescribed by Merzlyak21].The selectivescatter-in gresponseoriginatesfrom th e largeenhancementofvariousmoleculartransitionmatrixelementswheneverth eenergyoft heincidentphotonsmatches th eenergyof molecularlectronicransition.hereforeheexpressionselectivecattering synonymouswithresonancelightscattering. Resonancelightscatteringha seenidelybservednamancattering.How everthe resonanceeffectsare equallyapplicableto

luminescencean dRayleighscattering[22].Resonancescatteringshouldbeminimalinth ecaseofmixturesofmicrospheresan dchromophoressincescatteringfromth eindividualchromophores isnegligiblecomparedtoscatteringromhemicrospheres.nheas efmicroalgae,scatteringan dabsorption are highlycorre-latedhu seadingohebservationfesonancescattering.Naqvimade th eassumption thatcatteringan dmolecularabsorptionrewopartsofaunctioncharacterizingheesponsefellsoight19].ApplyingheKramers-Kronigelationohebsorp-tionspectrum,Naqviobtained th escatteringspectrumverysimilarto th epairshowninFig.7b ,an dFig.7c .Th echlorophyll molecules inalgaeare packed tightlyan dayormrraysfnteractinghromophores.Suchnteractingrraysayxhibitdditionalenhancedcattering23].heonnectionetweenresonancecatteringan dchromophore packingnsideth eel lwa susedby Bialek toprobehetructureofchloroplasts24].W een dwithafe wqualifyingcom -mentsregarding t heinterpretationof th edatain Fig.7c .Ifth eesonancecatteringontributionhasheam eangulardistributionasthenonresonanceM iescatteringthenthe resonancescatteringshouldenhanceth evaluesofa,ndo tecreasehe mshownnig .7c .How everiftheresonancescatteringishighlypeakedinth e forwarddirection,he n th enstrumentwillno tbeableto distinguishth eresonancescatteringfromtrans-mittedightndnterpretheontributionro meso-nancecatterings reductionnbsorbance.n thiscaseheeasuredaluesfa,willecreasesnFig.7c ,nd th e resonancecatteringcontribution ca nbe estimatedfrom th edifferenceofth evaluesofa,atresonancean dnonresonancewavelengths.Apossibili-tyxistsha t hirdphenomenonu chstimulatedemissionsccurringndeducinghebservedabsorbance.However,stimulatedemissionisexpectedonlytargel luminationpower.tsikelyha tnadditionalmeasurementisneededtointerpretth edatainFig.7c intermsofamolecularprocess.

5. Conclusion Ananalysisof th emeasurementprocessinaspectro-

meterwithanintegratingsphere (IS)detectorleadto aprocedurefo rseparatingth econtributionsto themeas-uredbsorbancedu eocatteringndhemolecularabsorption.Thenalysishingesnhenterpretationofabsorbancemeasuredor uvetteplacednwoholdersnhepectrometer.older 1sheormalposition,an dholder2placesth ecuvetteatth eentrance

80


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apertureoftheISdetector.Eq .(8 )an dEq .(9) give therelationshipbetweenthemeasuredabsorbencies{Ai,A- ^ an dtheanalyteproperties{a,,a).Approximate formsof thetw oequationsareusedtoanalyze severalsystemsconsistingfcatterersndbsorbers.heesultssuggestthatitispossibletoseparateth eabsorptionan dscatteringcontributions.Furthermore,he resultswithmicroalgaeuggestha thewoquantitiesa,,reindependentcharacteristicsofth emicroalgaeuspen-sion.heuantityivesnformationboutheelectronictatesf th ebsorbinghlorophyllmole-cules,whilehequantitya,m ayprovidenformationaboutheackingfhehromophoresnsidehemicroalgaecells.Furtherworkisneeded toclarify th esystematicuncertaintiesnherentnhemeasurementmodel .Th emostsignificantoftheseuncertaintiesareth eestimatesofth epartialcrosssectionsa,pan da .^ inEq .(8 )ndq. (9).othstimatesependnheinstrumentonfigurationsellshengulardistributionofth escatteredradiation.6. References

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[3 ].B.Nelsonan dB.B.Prezelin,Calibrationofa nintegratingspherefo rdetermining the absorptioncoefficientof scatteringsuspensions.Applied Optics3 2, 67 10 - 67 17(1993).

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[19] P .Latimer and E. Rabinowitch,Selective ScatteringofLightb yPigmentsnVivo,ArchivesofBiochemistryndBiophysics84 ,42 8- 4411959).[20] W ..Butler,AbsorptionSpectroscopynivo:TheoryndApplication, AnnualReview ofPlantPhysiology5, 451- 460(1964).

[21 ] M.N. Merzlyak,0.B. Chivkunova,I.P.Maslova,K .R.Naqvi,A. E. Solovchenko, an d I. Klyachko-Gurvich,ightbsorptionndcatteringyel luspensionsfom eCyanobacteriandMicroalgae, Russianournal oflantPhysiology 55 ,42 0 - 42 5 (2008).

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Aboutth eauthors:il iWan gs researchhemist,A. K .Gaigalas isascientistemeritus,an dHua-JunHeisabiologist,all in th eBiochemicalScienceDivisionofheISThemicalciencendechnologyLaboratory.heNationalnstitutefStandardsndTechnologysngencyfhe.S .epartmentofCommerce.

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Measurement of Absorption and Scattering