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BASICSPECTROSCOPY
SantiNonell1andCristianoViappiani2
1InstitutQuimicdeSarria

UniversitatRamonLlull
ViaAugusta390,08017Barcelona,Spain
santi.nonell@iqs.url.edu
2DipartimentodiFisica

UniversitadegliStudidiParma
VialeG.P.Usberti7A,43100Parma,Italia
cristiano.viappiani@fis.unipr.it

1.WhatIsSpectroscopy?
Theterm"spectroscopy"definesalargenumberoftechniquesthatuse
radiationtoobtaininformationonthestructureandpropertiesofmatter.
Thebasicprinciplesharedbyallspectroscopictechniquesistoshinea
beamofelectromagneticradiationontoasample,andobservehowit
respondstosuchastimulus.Theresponseisusuallyrecordedasa
functionofradiationwavelength.Aplotoftheresponseasafunctionof
wavelengthisreferredtoasaspectrum.
Thischaptergivesanoverviewofthespectroscopictechniquesmost
commonlyusedinphotobiologyresearch.Wehaverestrictedourselves
toincludeonlythosetechniquesthatuseultravioletorvisiblelightasthe
primarystimulus.Also,withthenewcomerstudentinmind,wehave
chosentoconcentrateondescribingtheprinciplesandmainapplications
ofthetechniques,keepingthediscussionsoftechnicaldetailsandthe
numberofequationstoaminimum.
2.WhatDoPhotobiologistsUseSpectroscopyFor?
Photobiologistsuseanumberofspectroscopictechniquestounderstand
howphotobiologicalprocessesoccur.Thisinvolvesinthefirstplace
identifyingtheprimaryphotoactivemolecularentitywhose
photoexcitationbytheabsorptionoflightenergytriggersthebiological
effect.Afundamentalpropertyofsuchentitiesistheirabsorption
spectrum,whichdescribestheirabilitytoabsorblightofdifferent
wavelengths.Determiningtheabsorptionspectrumofaphotoactive
agentisthefirststepinunderstandingthephotobiologicalprocessin
whichitparticipates.
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Equallyimportantistheidentificationandcharacterizationofallother
molecularentitiesinvolvedintheprocess:excitedstates,reactive
intermediates,andphotoproducts.Spectroscopictechniquesarealsoof
greathelpforthis,andevenelusivespecieswhoselifetimemerelyspans
afewtensoffemtoseconds(1femtosecondis1015secondsorone
billionthofonemillionthofasecond)canbestudiedtoahighlevelof
detailwithspectroscopictools.
Finally,asoundunderstandingofphotobiologicalprocessesrequiresa
completeknowledgeofthemolecularmechanismthroughwhichthey
operate.Thetermmechanismreferstothesequenceofeventsthat
undergoallparticipatingspecies,theratesatwhichtheyoccur,andthe
factorsinfluencingsuchrates.Timeresolvedspectroscopictechniques
allowphotobiologiststounravelthemechanimsofphotobiological
processes.
Thischapterwilldescribethemostcommonspectroscopictechniques
availabletothephotobiologist,andwillillustratethekindofinformation
thatcanbegainedwiththesetechniques.
3.AGeneral(AndOverlySimplified)PhotobiologyScheme
Everyphotobiologicalprocessstartswiththeabsorptionoflightenergy
bytheprimarylightabsorbingspeciesMthat,asaresult,ispromotedto
anelectronicexcitedstateM*ofhigherenergy.Moleculesintheirexcited
statesaremetastablespecies,andeventuallyreturntothegroundstate
givingbacktheabsorbedenergyeitheraslightorasheat.Inaddition,
theenergycontentinultravioletandvisibleradiationishighenoughto
alsopromotecompetingmoleculartransformationsofM*thatleadtothe
formationofreactiveintermediatesI,andeventuallythephotoproductP
responsiblefortheendphotobiologicaleffect.Thecompetitionbetween
excitedstatedecayandchemicaltransformationisanessentialfeature
ofallphotochemicalandphotobiologicalprocesses,andtheirrelative
ratesdeterminethefinaloutcomeoftheprocessinitiatiatedbythe
absorptionoflightenergy,hencetheimportanceinmeasuringsuch
rates.
Photobiologistsuseenergydiagramstovisuallyorganizethesespecies,
placingthemataheightrelatedtotheirenergy(Figure1).Thisproves
veryusefulforvisualizingthesequenceofstepsformingthemechanism
andtheassociatedenergyflows.Spectrocopictechniqueshelp
photobiologistsdeterminetheenergycontentsofeachparticipating
speciesandtherateconstantsoftheprocessesrelatingthem.

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Figure1.Thebasicschemeofphotobiologicalevents:the
primaryphotoactivemoleculeMabsorbslightandispromoted
toitsexcitedstate,M*.Itthenundergoesachemical
transformationtooneormoreintermediatespeciesI,and
finallyyieldsaproductP.Theverticalpositionofthebarsinthe
diagramrepresentstheenergycontentofeachspecies.
4.WhatSpectroscopiesAreAvailableToThePhotobiologist?
Documentingthegeneralphotobiologicalschemediscussedinthe
previoussectionrequirestheidentificationandcharacterizationofall
participatingspeciesaswellasthedeterminationoftherateconstants
forthedifferentreactions.Differentspectroscopictechniquesare
availabletofulfillthisgoal,thefollowingbeingthosemostcommonly
encounteredinphotobiologylaboratories.
4.1GroundstateAbsorption
Thistechnique,explainedinmoredetailinSection5,isappliedtostable
species,"stable"meaningthattheirlifetimeisatleastofafew
milliseconds(1millisecondisonethousandthofasecond).Thisrefers
tipicallytotheprimarylightabsorbingagentandthefinalproducts.The
sampleisirradiatedwithacontinuouswave(CW)beamoflight,andthe
fractionoflightabsorbedisdeterminedthroughtransmissionor
reflectancemeasurements.Becauselightabsorptionsendsamolecule
temporarilytoanupperexcitedstate,theinformationprovidedbythis
techniqueistheabsorptionspectrumofthephotoactivespecies(Figure
2),theenergydifferencebetweentheirgroundandexcitedstates,and
theprobabilityforlightabsorption.Absorptiontransitionsaremarked
withstraightarrowsontheenergydiagram.

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Figure2.AbsorptionTransitionsInGroundstateAbsorption
Spectroscopy.
4.2TransientAbsorption
Thistechniqueisverysimilartothepreviousoneexceptthatthespecies
probedaremetastable(i.e.,transient)excitedstatesandreaction
intermediateswhoselifetimemayrangefromfemtoseconds,forthe
primaryexcitedstates,tokilosecondsforslowreactionintermediates.
Thespeciesarepromotedtemporarilytoupperexcitedstates(seeFigure
3).Measuringtheabsorptionspectrumofatransientspeciesrequiresthe
useofpulsedlasersforgeneratingameasurableconcentrationofexcited
states,andasecondbeamoflight,CWorpulsed,forprobingtheir
absorption.Inadditiontoabsorptionspectra,transientabsorption
techniquesalsoyieldthetherateconstantsoftheprocesseswherethese
speciesparticipate.

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Figure3.Absorptiontransitionsintransientabsorption
spectroscopy.
4.3EmissionTechniques
Emissiontechniquesmeasuretheelectromagneticradiationemittedupon
deactivationofexcitedstates.Whensuchexcitedstatesarecreatedby
theabsorptionoflight,thetechniquesarereferredtoasfluorescence
andphosphorescence,dependingonthenatureoftheexcitedstate.
Somechemicalandenzymaticreactionsalsoproduceexcitedstates,
whoseemissionisthenreferredtoaschemiluminescence.Emission
techniquesprovideemissionspectra,excitedstatelifetimes,andrate
constantsfortheprocesseswherethesespeciesparticipate.Emission
transitionsarealsosignalledbystraightarrowsonenergydiagrams(see
Figure4).

Figure4.Emissiontransitionsinluminescencespectroscopy.
4.4PhotothermalTechniques
Inadditionto,andsometimesinsteadof,emittinglight,excitedstates
andreactiveintermediatesgiveofftheirexcessenergythroughnon
radiativedeexcitationpathways,causinglocalchangesintemperature,
refractiveindexandvolume.Photothermaltechniquesmonitorsuch
changesandthusprovideinformationontheprocessesthatoriginate
them.Thetypicaluseofphotothermaltechniquesisfordeterminingthe
energyandvolumechangesaccompanyingthereactionsoftheexcited
statesandreactiveintermediates(seeFigure5).Thermalornon
radiativetransitionsareconventionallymarkedwithwavyarrowsonthe
energydiagrams.

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Figure5.Radiationlesstransitionsinphotothermal
spectroscopies.
4.5Timeresolvedvs.steadystatetechniques.
Optical(absorptionandemission)andthermalspectroscopiesrelyonthe
creationofexcitedstatesandreactiveintermediatesthroughthe
absorptionoflight,whichleadstotwofundamentallydifferent
approaches:insteadystatespectroscopiesthesamplesarecontinuosly
irradiatedwithabeamoflightexcitedstatesarecontinuouslycreated
andeliminatedandeventuallyasteadystateisreachedwheretheir
concentrationremainsconstant.Thisfacilitatesthemeasurementof
weaksignallevelsattheexpenseofloosingkineticinformation.Steady
statespectroscopiesarebestappliedtothemeasurementofabsorption
andemissionspectra.Ontheotherhand,timeresolvedspectroscopies
relyontheirradiationofthesamplewithalightsourcewhoseintensity
fluctuatesasafunctionoftime.Thesimplestexampleofthisisapulsed
laser,whichemitsflashesoflight.Eachflashcreatesaburstofexcited
states,whoseevolutioncanbemonitoredasafunctionoftime.Time
resolvedspectroscopiesprovidekineticinformationattheexpenseofa
lowersensitivitycomparedtosteadystatetechniques.
5.GroundStateAbsorption
Theabsorptionspectrumofstablemolecules,suchastheprimary
photoactivespeciesorthefinalproducts,ismostconvenientlyrecorded
usingasteadystatespectrophotometer.Moderninstrumentsconsistofa
combinationoflightsourcescoveringtheUVandvisibleranges,a
monochromatortoisolateanarrowwavelengthrange,asample
compartment,andadetectionsystem(Figure6).Transparentsamples
suchasfilmsorsolutionsarebestmeasuredintransmissionmode,the
instrumentmeasuringtheintensityoflighttransmittedbythesample.
Opaqueorhighlyscatteringsamplessuchassolidsorsuspensionsare
bestmeasuredinreflectancemode.

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Figure6.Typicalopticallayoutsforabsorption
spectrophotometers.Left:Indiodearrayspectrophotometers,
thespectrumisobtainedatonce.Right:insingledetector
spectrophotometers,theamountoflighttransmitted(or
reflected)isdetectedonewavelengthatatime.
Thetransmittanceofatransparentsampleisdefinedasthefractionof
lighttransmittedbythesamplewhenitisirradiatedwithabeamoflight.
Itisdeterminedbymeasuringtheintensity(moreproperlycalledradiant
power)oftheincidentandtransmittedlightbeams.Aplotofthe
transmittancevsthebeamwavelengthiscalledatransmissionspectrum.
Photobiologistsusuallyprefertoworkwithabsorptionspectra(Figure7),
wheretherelatedquantityabsorbanceisplottedinstead.Theabsorbance
ofasampleisdefinedasthenegativelogarithmofitstransmittance:
Absorbance=log(Transmittance).Forapuresubstance,the
absorbanceofasampleatagivenwavelengthisdirectlyproportionalto
itsconcentration.

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Figure7.Absorptionspectrumof(a)chlorophylland(b)Fe(II)
protoprophyrinIX,theprostheticgroupofheme.Thesharp
absorptionofchlorophyllintheredpartofthespectrummakes
grassgreen,anditslackinhememakesbloodred.
Thereflectanceofanopaquesampleislikewisedefinedasthefractionof
lightreflectedbythesamplewhenitisirradiatedwithabeamoflight.It
isdeterminedbymeasuringtheintensity(radiantpower)oftheincident
andreflectedlightbeams.Aplotofthereflectancevsthebeam
wavelengthiscalledareflectancespectrum.
Photobiologistsuseabsorption(orreflectance)spectrato:*Identifythe
wavelengthsthatasamplecanabsorbandtherelativeprobabilitiesof
theabsorptiontransitionsateachwavelength.
*Identifythemolecularspeciesresponsibleforthephotobiologicaleffect
understudy.
*Determinetheenergyofthemolecule'sexcitedstates.
*Studytheinteractionsbetweenspecies,e.g.,adrugwithahost
protein.
6.TransientAbsorption
6.1Thepumpandprobeapproach
Transientabsorptiontechniquesrelyontheuseofaprobebeamthat
interrogatesthesamplebeforeandafterexcitationwithapulsedlaser
(pumpbeam),andmonitorsabsorbancechangeseitherataselected
wavelength(kinetics)orsimultaneouslyatseveralwavelengths
(transientspectra).Thesemethodsarecollectivelyreferredtoaspump
andprobemethodsalthough,dependingonthetimescale,thetransient
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absorbancetechniquesmayhaveverydifferentexperimentallayouts.
Thesetechniquesallow,ingeneral,tostudythedisappearanceofthe
excitedstateM*,theformationofthereactiveintermediatesI,andthe
formationofphotoproductsP.
Thepumplasermustexcitethesampleatawavelengthwhereground
stateabsorptionoccurs(Section5).Thelaserpulsewidthmustbemuch
shorterthanthetimeconstantofthereactionunderinvestigation.
TheprobebeamisgenerallyabroadbandlightsourceintheUVvisNIR
spectralrange,wheretransientelectronictransitionsoccur.Theprobe
lightsourceisnormallyaCWorpulsedlampfornanoseconds(1
nanosecondisonebillionthofasecond)orlongertimescales,whilea
pulsedlaserisusedforhighertimeresolution.
Theexperimentalparameterofinterestisachangeinsampleabsorbance
( A),obtainedfromthechangeintransmittedlightintensityoftheprobe
beambefore,I(t<to),andafter,I(t),laserexcitation,accordingtothe
followingequation:
A(t)=log[I(t)/I(t<to)].
Photobiologistsusetransientabsorptionto:
*Identifyreactionintermediatesfromtheirspectralfeatures
*Assessthereactionmechanismbydetectingspectrallydistinguishable
reactionintermediates
*Determinetherateconstantsforeachkineticstep
*Determineactivationparametersfromtemperaturedependenceofrate
constants
*Determinereactionquantumyieldsinfavourablecases,where
extinctioncoefficientsofreactionintermediatesareknown.
6.2Frommillisecondstohours:steadystatespectrophotometers
Whenveryslowreactions(timeconstantsintherangeofminutesor
longer)aretobemonitored,thechangesintheabsorbanceofthe
samplefollowingphotoexcitationbythepulsedlasercanbefollowed
usingconventionalsteadystatespectrophotometers(Section5),
providedthattheabsorptionspectracanbetakenontimescalesmuch
fasterthanthecharacteristictimeofthereactionunderinvestigation.
Spectrophotometersalsoallowonetofollowtheabsorbanceataselected
wavelengthasafunctionoftime,thusprovidingakinetictimecoursefor
thephotoinducedreaction.
Forfasterreactions(downtoafewmilliseconds)multichanneldetectors,
suchaschargecoupleddevices(CCDS),andphotodiodearrays(PDA)
arenormallyusedtofollowthetimeevolutionoftheabsorption
spectrum.Intheseapplications,aCWbroadbandlightsourceisusedto
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probetheabsorptionofthesample.Thepolychromaticbeamispassed
throughaspectrographtomeasureabsorptionspectraasafunctionof
time.(Figure6,left)Specializedsetupsalsoallowtofollowthe
absorbanceataselectedwavelengthasafunctionoftime.
6.3Frommillisecondstonanoseconds:nanosecondlaserflash
photolysis
Absorbancechanges,frommillisecondstonanoseconds,following
excitationwithananosecondpulsedlasercanbemonitoredusingaCW
lightsourcesuchasaXearclamp(Figure8).Afastshutterexposesthe
sampletotheprobebeamshortlybeforethelaserpulsehitsthesample,
andisclosedaftertheendofdatacollectiontopreventits
photobleaching.(Figure9)Thepolychromaticbeamispassedthrougha
spectrograph,eithertoselectthewavelengthatwhichthereaction
kineticsaremonitoredortomeasuretransientspectraasafunctionof
time(Figure10).Examplesoftimeresolvedspectraandrebinding
kineticsaredisplayedinFigures11and12.Thespectralandkinetic
informationallowtoidentifyreactionintermediatesandtoretrieverate
constants.

Figure8.Essentialopticallayoutofananosecondlaserflash
photolysissetup.ACWlampbeammonitorschangesof
absorbance(orreflectance)inasampleafterflash
photoexcitationwithapulsedlaser.

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Figure9.Timeprofileofprobelightintensitytransmittedby
thesample.Attimezero,theshutterisclosedandthedetector
seesnolight.Whentheshutterisopenedthelighttransmitted
bythesampleisseenbythedetector.Thechangesinintensity
inducedbythepulsedlaserarehighlightedasabluesignal.At
theendoftheexperimenttheshutterisclosedagainto
preventextensivephotodegradationofthesample.

Figure10.Typicalopticallayoutofthedetectorsusedin
nansoecondlaserflashphotolysis.Thespectrographcanselect
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aspecificwavelengthandallowmonitoringtheabsorbance
changesatthatwavelength.Alternatively,aspectralrangecan
bemonitoredbyamultichanneldetectorsuchasa(CCD)and
allowthedeterminationoftransientspectraatselectedtime
delays.

Figure11.Timeresolvedchangesinabsorptionspectrum
followingphotodissociationofcarboxyhemoglobin.A
nanosecondlaserpulsephotolyzestheCOFebondleadingto
changesinthehemeabsorptionspectrumintheSoretregion.
Theligandisthenreboundbytheheme,withthecomplex
kineticsshownintheplot,extendingoverseveralordersof
magnitudeintime.Theproteinisembeddedinasilicagelto
preventquaternaryswitchingfromtheRtotheTquaternary
structureafterphotodissociation.

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Figure12.Timecourseofthechangeinabsorbanceat436
nm,followingnanosecondphotodissociationofcarboxy
hemoglobin.Thechangeinabsorbancereflectsthekineticsof
carbonmonoxiderebindingtothehemeFe.(seeViappiani,C.
etal.Proc.Natl.Acad.Sci.USA2004,101,1441414419.for
details).

6.4Thesubnanoseconddomain:thetwopulsepumpandprobe
technique
Whenabsorbancechangesaretobemonitoredinthesubnanosecond
domain,theprobebeamisobtainedbyasecondbroadbandlaserpulse,
hittingthephotoexcitedsampleatapropertimedelay(Figure13).

Figure13.Schemeforsubnanosecondpumpandprobe
methods.
Modernpumplasersemitpulsesofapprox.100femtoseconds,whichare
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tuneableacrossbroadwavelengthranges,andhaveahighrepetition
rate(80MHz).Theprobebeamisnormallyobtainedbysplittinga
portionofthepumpbeam,whichisthenfocussedontosuitablematerials
generatingspectrallybroadpulsesintheregionofinterest.Kinetic
informationisobtainedbyadjustingthetimedelaybetweenpumpand
probepulses.Byprobingthephotoexcitedsampleasafunctionoftime
delayoftheprobepulses,timeresolvedspectracanbeobtained,from
whichthekineticsatselectedwavelengthscanbereconstructed.Results
aresimilartothoserepresentedinFigures11and12,exceptforthetime
scale.
7.EmissionSpectroscopies
Inemissionspectroscopy,asampleisirradiatedwithabeamoflight(the
excitationbeam),andtheluminescenceemergingfromthecuvetteis
recordedwithasuitabledetector.Suchluminescenceiscausedbythe
radiativedecayoftheexcitedstatescreatedbytheexcitationbeam,
whichtherebyreturntotheirgroundstate.Theemissiondetectoris
placedtipicallyatrightangletotheexcitationbeamtoavoidany
transmittedlight.(Figure14).

Figure14.Opticallayoutofasteadystatespectrofluorometer.
AlightbeamproducedbyaCWlampispassedthrougha
monochromatortoselectaparticularexcitationwavelength
thatisthenfocusedontoasample.Theensuingemissionis
observedatrightangles,analyzedspectrallywiththeemission
monochromator,andrecordedbyanappropriatedetector.
Whentheexcitedstatehasthesamespinmultiplicityasthegroundstate
theradiativedecaytransitionislabelledas"spinallowed"andthe
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emissionisreferredtoasfluorescence.Whenthespinchangesthe
transitionissaidtobeforbiddenandtheemissioniscalled
phosphorescence.Fromakineticpointofview,fluorescenceemissionisa
fastprocess,typicallyinthenanosecondrange,whilephosphorescence
maylastanythingfrommicrosecondstohours.Inaddition,
phosphorescencealwaysoccursatlongerwavelengthsthanfluorescence
(Figure15).

Figure15.Fluorescence(top)andphosphorescence(bottom)
spectraoftryptophaninaprotein(black)andinsolution(red).
Noticethatphosphorescenceoccursatlongerwavelengthsthan
fluorescence.DataarecourtesyofP.Cioni,CNR,Pisa(Italy).
7.1Steadystateemissionspectroscopy
Insteadystatespectrofluorometers,thesampleisirradiatedwithaCW
beamoflight,verymuchasinabsorptionspectroscopy.Thiscreatesa
steadyconcentrationofexcitedstates,andthereforeasteadyemission
oflightfromthesample.Excitationandemissionmonochromatorsallow
theusertoselectthewavelengthsoftheexcitationandemissionbeams.
Emissionspectraareobtainedbysettingtheexcitationwavelengthtoa
constantvalueandscanningtheemissionmonochromator.Therecorded
emissionintensityisthenplottedasafunctionoftheemission
wavelength(Figure16).Alternatively,theemissionwavelengthislocked
andtheexcitationmonochromatoristhenswept.Thisproducesan
excitationspectrum.Forapuresubstanceexistinginasinglemolecular
forminthesampletheexcitationspectrummatchesitsabsorption
spectrum,whichisveryusefultoidentifytheemittingspeciesina
mixture.Differencesbetweenabsorptionandexcitationspectraindicate
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thepresenceofmorethanonespeciesinthesample,eitherdueto
differentsubstancesordifferentformsofthesamesubstance,e.g.an
acidanditsconjugatedbaseoramonomerandadimer,etc.

Figure16.Absorption,fluorescenceemission,and
fluorescenceexcitationspectraofagreenfluorescenceprotein
mutant.
Thefluorescence(orphosphorescence)quantumyieldexpressesthe
probabilityofemittinglightuponabsorptionofradiationandiscalculated
bydividingthenumberoflightquantaemittedbythenumberofquanta
absorbed.
Photobiologistsusesteadystateemissionspectroscopyto:
*Assesstheabilityofasubstancetoemitlight,andthewavelengths
wherethisemissionoccurs.Thecorrespondingpropertiesarethe
emissionquantumyieldandtheemissionspectrum.
*Identifythemolecularspeciesresponsibleforthephotobiologicaleffect
understudy.Thisisinferredfromtheexcitationspectrum.
*Determinetheenergyofthemolecule'sexcitedstates.Thewavelength
ofthefluorescenceonsetgivesagoodestimationoftheenergyofthe
firstexcitedsingletstate,whilethatofthephosphorescenceonsetgives
theenergyofthetripletstate.
*Studytheinteractionsbetweenspecies,e.g.,adrugwithahost
protein,whichoftenresultinchangesofthespectrumand/orquantum
yieldoftheemission.
7.2Timeresolvedemissionspectroscopy
Timeresolvedemissionspectroscopyprovidesinformationonthekinetic
behaviorofluminescentexcitedstatesandintermediates.Theoptical
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layoutisessentiallyidenticaltothatgiveninFigure14,butalightsource
whoseintensitychangesasafunctionoftimeisusedinstead.This
createsapopulationofluminescentspeciesthatalsochangeovertime
andthereforerendersitpossibletomonitortheemissiondecaykinetics
(Figure17).Typicallightsourcesincludepulsedormodulatedlamps,
lightemittingdiodes,orlasers.

Figure17.Decayoffluorescence(top)andphosphorescence
(bottom)oftryptophaninaprotein(black)andinsolution
(redactuallythesampleisNacetyltryptophanamide,NATA).
Noticethatphosphorescencelastsmuchlongerthan
fluorescence.DataarecourtesyofP.Cioni,CNR,Pisa(Italy)
andT.Gensch,ResearchCenterJlich(Germany).
Photobiologistsusetimeresolvedemissionspectroscopyto:
*Obtainrateconstantsforprocessesinvolvingluminescentspecies.
Theseareinmostcasesobtainedinastraightforwardwayfromthe
kineticanalysisoftheintensityvstimeluminescencedata.
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*Unravelthemechanismofaphotobiologicalprocessbycharacterizing
thereactiveintermediatesandtheirratesofformationanddecay.Thisis
usuallyachievedfromtheanalysisoftimeresolvedemissionspectra
(TRES),wheretheluminescenceisrecordedbothasafunctionoftime
andwavelength.
*Studytheinteractionsbetweenspecies,e.g.,adrugwithahost
protein.Thisisparticularlyusefulwhensuchinteractionshaveno
measurableeffectontheemissionspectraofhostorguestbutratheron
theirluminescencelifetimes.
8.PhotothermalTechniques
Photothermaltecniquesareagroupofhighsensitivitymethodsthat
monitortheeffectsinducedinsolutionafternonradiativerelaxationof
excitedstates.Thebasisofthecollectivetermphotothermalforthese
techniqueslaysinthedetectionofthermalrelaxationofexcessenergy
associatedwithphotoexcitationofthesample.Inaddition,othernon
radiativenonthermalrelaxations,suchasvolumetricchangesdueto
photoinducedstructuralchanges(isomerization,chargetransfer,solute
solventrearrangement,...)maygiverisetodetectablesignals.Themost
remarkableadvantageofthesemethodsisthat,throughaproper
analysis,thermodynamicparameters(energyandvolumechanges)for
eachstepofthephotoinducedreactioncanbeobtained.Theinherent
timeresolutionofthemethodsallowsonetoalsoretrievekinetic
parameters(rateconstants,quantumyields)forthereactionsteps.
Timeresolvedphotoacousticsisthetechniqueofchoiceforreactions
stepswithlifetimesbetweenabout10nanosecondsand10
microseconds.Forphotoinducedreactionswithlongertimeconstants,
photorefractivemethodsaremoreadequate,sincetheycanaccess
eventswithlifetimesextendingtothemilliseconds.
Photothermalmethodsgenerallyapplytoopticallytransparent,low
absorbingsamples,althoughtimeresolvedphotoacousticscanbeapplied
tostronglyabsorbingsamples,withdedicatedexperimentalsetups.
Photobiologistsusephotothermalmethodsto:
*Estimateenthalpyandvolumechangesforeachkineticstep
*Estimatetheenergycontentofreactionintermediates
*Determinethequantumyieldofphotoinitiatedreactions
*Determinetherateconstantsforeachkineticstep
8.1Timeresolvedphotoacoustics
Timeresolvedphotoacousticsmonitorsthepressurechangesinducedin
asampleafternonradiativerelaxationsfollowingexcitationwitha
(normally)nanosecondpulsedlaser.Thepressurepulsegenerallyhas
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twosources,bothleadingtoavolumechangeofthesolution,andrelated
withnonradiativerelaxationsoftheexcitedstate.Thefirstsourceisthe
thermalrelaxationassociatedwithenergychangesaccompanying
relaxationoftheexcitedstateandreactionintermediates.Thesecond
sourceisduetovolumechangesassociatedwithstructural
rearrangementsaccompanyingeachstepofthephotoinducedreaction.
Thetimeevolutionofthepressurepulseismonitoredinmicroseconds
(onemicrosecondisonemillionthofasecond)byafastpiezoelectric
microphone(Figure18).Calibrationofthesetuptoretrieve
thermodynamicandkineticparametersimpliescomparisonofthe
photoacousticsignalwiththatobtainedforaphotocalorimetricreference
compound(i.e.,acompoundthatundergoesthermalrelaxationwithunit
efficiencywithinafewnanoseconds).(Figure19)Theresonant
configurationofthedetectorrequirestheuseofnumericaldeconvolution
analysistoretrievekineticinformationfromexperimentalsignals.

Figure18.Essentialschemeofatimeresolvedphotoacoustics
setup.

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Figure19.Photoacousticsignalsfordegassedacetonitrile
solutionsofthephotocalorimetricreferencecompound
2,hydroxybenzophenone(R(t))andbenzophenone(S(t))at
roomtemperature.Boththeamplitudeandtheshapeofthe
signalforbenzophenonearedifferentfromthosefor
2,hydroxybenzophenone.Thesignalforbenzophenone,of
thermalorigin,isbestdescribedbyadoubleexponentialdecay
withafastcomponent(lifetimebelow(10ns),duetotriplet
formation,andaslowerrelaxation(lifetimeof(6.7
microseconds),duetotripletdecay.
8.2Photorefractivetechniques
Photothermallensing,beamdeflection,andgratingtechniquesdetect
changesinrefractiveindexuponchangesindensity(resultingfroma
changeinvolumeofeitherthermalofstructuralorigin),inabsorbance,
andintemperature.Whenasampleisexcitedwithalightbeam,which
usuallyhasaGaussianshape,beingabsorbedbythesample,the
concentrationoftheexcitedstatemoleculesreflectsthepumpbeam
geometry.Thenonradiativerelaxationsoftheexcitedstatesheatthe
sampleresultinginatemperatureprofile,whichreflectstheGaussian
intensityprofileoftheexcitinglaserbeam.Adecreaseindensityresults
fromheating,eventuallyleadingtoadecreaseintherefractiveindexof
thesample.Thesemethodsallowonetoretrieveenthalpyandvolume
changesassociatedwiththerelaxationofthephotexcitedmolecules,and
therateconstantsoftheseprocesses.
Threebasicexperimentalapproachesareusedtodetecttherefractive
indexchange:thetransientlens,thebeamdeflection,andthetransient
grating.
Transientlens.ACWprobebeamtravelsthroughtheilluminatedregion
(bythepumplaser)andisexpandedbytheGaussianprofileofthe
refractiveindex.Thiseffectcanbedetectedfromthechangeinlight
intensitythroughapinhole(Figure20).
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Figure20.Schematicoftheexperimentalsetupintransient
lensexperiments.Thepumpbluebeamgeneratesachangein
refractiveindex.Theredprobebeamisexpandedbythe
refractiveindexprofile.Adetectorplacedafterapinhole
sensesthedefocusingasachangeinlightintensity.
BeamDeflection.TheCWprobebeamisnotperfectlyconcentricwith
thepumpbeam.Thespatialprofileoftherefractiveindexchangeresults
inadeflectionoftheprobebeam.Apositionsensitivedetectormonitors
thedisplacementoftheprobebeam(Figure21).

Figure21.Schematicoftheexperimentalsetupinbeam
deflectionexperiments.Thepumpbluebeamgeneratesa
changeinrefractiveindex.Theredprobebeamisdeflectedby
therefractiveindexprofile.Apositionsensitivedetectorsenses
thedeflection.
TransientGrating.Twopumplaserbeamswithparallelpolarizationare
broughtintothesampleatanangleandfromtheirinterferencepatterna
modulationoftheexcitationintensityresults.Therefractiveindex
changesoriginatingfromthephotoinducedprocessesreflectthis
modulation,andcanbemonitoredbyathirdlaserbeam,whichis
diffractedtoanextentdependingonthegratingproperties.An
advantageofthismethodisthatitcanaccesssubnanosecondevents
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(Figure22).

Figure22.Schematicoftheexperimentalsetupintransient
gratingexperiments.
Therateofformationofthedensitylens(107s1)setsthetime
resolutionintransientlensandbeamdeflection.Ontheotherhand,the
techniquesareverysensitiveindetectingslowerkinetics,extendingto
milliseconds.Inordertoretrievequantitativethermodynamicinformation
fromthelensandbeamdeflectionsignals,aphotocalorimetricreference
mustbeusedtocalibratetheinstrumentalresponse,inessentiallythe
samewayasalreadydescribedforthephotoacousticsignals.The
advantageofthesemethodsisthattheyrequirenodeconvolution
analysistoretrievethekineticinformation.
9.ConcludingRemarks
Thephotobiologistofthe21stcenturymightratherbecalledamolecular
photobiologistinthatthemolecularapproachprovidesthemostaccurate
albeitcomplexdescriptionofphotobiologicalphenomena.Spectroscopy
offersawonderfulwindowintosuchamolecularworld,andprovidesus
withauniqueyetpowerfulsetoftoolstoexploretheintricaciesof
photobiologicalphenomena.Techniquesthatwererathersophisticated
justadecadeagoarenowbeingroutinelyusedinmanylaboratories
worldwide,revealingawealthofdatawhichischangingthewaywe
understandnature.
10.FurtherReading
NanosecondLaserFlashPhotolysis
BonneauR.,WirzJ.,ZuberbuehlerA.D.(1997)Methodsfortheanalysis
oftransientabsorbancedata,Pure&Appl.Chem.1997,69,979992.
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ChenE.,GoldbergR.A.,KligerD.S.Nanosecondtimeresolved
spectroscopyofbiomolecularprocesses,Ann.Rev.Biophys.Biomol.
Struct.26,327355.
TetreauC.,LavaletteD.(2005)Dominantfeaturesofproteinreaction
dynamics:Conformationalrelaxationandligandmigration,Biochim.
Biophys.Acta1724,411424.
AbbruzzettiS.,BrunoS.,FaggianoS.,GrandiE.,Mozzarelli,A.,
Viappiani,C.(2006)TimeresolvedmethodsinBiophysics.2.Monitoring
haemproteinsatworkwithnanosecondlaserflashphotolysis,
Photochem.Photobiol.Sci.,5,11091120.
PicosecondTransientAbsorbance
Cerullo,G.,ManzoniC.,LuerL.,Polli,D.(2007)Timeresolvedmethods
inbiophysics.4.Broadbandpumpprobespectroscopysystemwithsub
20fstemporalresolutionforthestudyofenergytransferprocessesin
photosynthesis,Photochem.Photobiol.Sci.,6,135144.
Groot,M.L.,vanWilderen,L.J.G.W.,DiDonato,M.(2007)Timeresolved
methodsinbiophysics.5.Femtosecondtimeresolvedanddispersed
infraredspectroscopyonproteins,Photochem.Photobiol.Sci.,6,501
507.
EmissionSpectroscopy
Lakowicz,J.R.(2006)PrinciplesofFluorescenceSpectroscopy,3rded.,
KluwerAcademic/PlenumPublishers,NewYork.
Becker,W.(2005),AdvancedTimeCorrelatedSinglePhotonCounting
Techniques,Springer,Germany.
TimeResolvedPhotoacustics
Braslavsky,S.E.,Heibel,G.E.(1992)Timeresolvedphotothermaland
photoacousticsmethodsappliedtophotoinducedprocessesinsolution,
Chem.Rev.92,13811410.
Gensch,T.,Viappiani,C.(2003)Timeresolvedphotothermalmethods:
accessingtimeresolvedthermodynamicsofphotoinducedprocessesin
chemistryandbiology,Photochem.Photobiol.Sci.2,699721.
PhotorefractiveTechniques
Schulenberg,P.J.Braslavsky,S.E.(1997)Timeresolvedphotothermal
studieswithbiologicalsupramolecularsystems,inProgressin
PhotothermalandPhotoacousticScienceandTechnology,Mandelis,A.,
Hess,P.,Eds,SPIEOpticalEngineeringPress,Washington,pp.5781.
Terazima,M.(2001)Proteindynamicsdetectedbythetimeresolved
transientgratingtechnique,Pure&Appl.Chem.73,513517.
10/16/08
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