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BASICPHOTOPHYSICS
AntonieJ.W.G.Visser1,2andOlafJ.Rolinski2
1LaboratoryofBiochemistry,MicrospectroscopyCentre

WageningenUniversity,P.O.Box8128
6700ETWageningen,TheNetherlands
antoniejvisser@gmail.com

2DepartmentofPhysics,UniversityofStrathclyde

ScottishUniversitiesPhysicsAlliance,PhotophysicsGroup
GlasgowG40NG,UK
o.j.rolinski@strath.ac.uk

Introduction
Basicphotophysicsintheframeworkofphotobiologyisconcernedwith
processesthatoccurwhensunlight,filteredthroughtheEarth's
atmosphere,interactswithmatter(atomsandmolecules)presenton
Earth.ThespectrumofsolarradiationstrikingtheEarth(Figure1)spans
100nmto106nanometers(1nm=109m)andcanbedividedintothe
ultraviolet(UV)range(100nmto400nm),visiblerange(400nmto700
nm)andinfrared(IR)range(700nmto106nm).UVradiationhasboth
damagingandbeneficialeffectsonlivingmatter.UVradiationisalso
responsibleforthephotochemicalreactionleadingtoproductionofthe
protectiveozonelayerintheatmosphere.Asthenamesuggeststhe
visiblepartofthespectrumisthelightthathumaneyescandetect.An
importantpartofelectromagneticradiationreachingtheEarthisIR
radiation.

Figure1.Solarradiationspectrumabovetheatmosphere,and
atthesurfaceoftheearth.[FromWikipedia:Sunlight]
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Thespectrumofsolarradiationisclosetothatofablackbodywitha
temperatureof5600K.Ablackbodyisanidealisedobjectthatabsorbs
allelectromagneticradiationfallingonit.Becausenolightisreflectedor
transmitted,theobjectappearsblackwhenitiscold.However,ablack
bodyemitsatemperaturedependentspectrumoflight,whichistermed
blackbodyradiation.Allhotobjectsradiateamixtureofheatandlight,
withintensityandcolorvaryingwithtemperature.Thinkofanironpoker
inafirethatchangescolorwhenitbecomeshotter,andaftertakingit
outtheglowstartstodisappear,asdoestheheat.Historically,studying
thelawsofblackbodyradiationhasledtothedevelopmentofquantum
mechanics.
Visiblelightisonlyonesmallpartoftheelectromagneticspectrum,
whichisclassifiedbywavelengthintoradiowave,microwave,infrared,
visible,ultraviolet,Xraysandgammarays(Figure2).

Figure2.Electromagneticspectrum,withthevisiblelight
spectrumenlarged.[FromWikipedia:Electromagnetic
Radiation]
Thebehaviourofelectromagneticradiationdependsonitswavelengthor
energy(i.e.,frequency).Higherfrequencieshaveshorterwavelengths
and,viceversa,lowerfrequencieshavelongerwavelengths.The
interactionofelectromagneticradiationwithatomsormoleculesdepends
ontheenergy.Spectroscopycanbedetectedatamuchwiderrangethan
thevisiblerangeof400nmto700nm.Dependingonthespectralregion
ofinterest,theunitsusedtodescribetheradiationmaybefrequency,
energy,wavelengthorinversewavelength(wavenumber).Table1
comparesdifferentspectralregions.

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Table1.CharacteristicsoftheElectromagneticSpectrum.
Wavelengthrangesoftheelectromagneticspectrumhavebeen
classifiedbytheirspectroscopicuses.Forexample,veryhigh
frequency(VHF)radiationisusedinnuclearmagnetic
resonance(NMR),whileultrahighfrequency(UHF)radiationis
usedinelectronparamagneticresonance(EPR).Theotherrows
ofTable1provideinformationonfrequency,wavelength,wave
numberandenergyforacenterwavelengthofthespectral
range.Thelastrowgivestheenergyprovidedbyonemoleof
photonsofgivenwavelength,thatistheenergypereinstein.
Electromagneticradiationexhibitsbothwavelikeandparticlelike
properties,aconceptknownaswaveparticleduality.Bothwaveand
particlecharacteristicshavebeendemonstratedbyalargenumberof
experiments.Electromagneticwavesarecomposedofanelectricfield
(E),andamagneticfield(B)perpendiculartoit,oscillatinginphasein
thepropagationdirection(k).Anarrowontopofalettergivesthe
directionofthevector(Figure3).

Figure3.Electromagneticwavesasoscillatingelectricand
magneticfields.(qand+qrepresentelectricalcharges)
[FromWikipedia:ElectromagneticRadiation]
Thesinusoidalwavesarecharacterizedbyfrequency,whichisinversely
proportionaltothewavelengthaccordingtotheequation:

wherecisthespeedoflight(invacuumc=300,000km/slighttravels
inonenanosecond30cm1ns=109s).Thewavemodelcanexplain
severalphenomena.Interferenceisthesuperpositionoftwowavesgiving
anewwavepatternthatcanbeconstructivewhentwowaveshavethe
samedirectionordestructivewhentheyhavetheoppositedirection.
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Anotherexampleislightdispersionintoavisiblespectrumwhenwhite
lightisshonethroughaprism,becauseofthewavelengthdependent
refractiveindexoftheprismmaterial.Electromagneticwavesalsohave
particlelikeproperties,aspacketsofenergyorquanta,calledphotons.
Theenergyperphoton,E,canbecalculatedfromthePlanckEinstein
equation:

wherehisPlanck'sconstantandisfrequency.Theparticlemodelcan
explaintheabsorptionandemissionspectraoflightbyatomsor
molecules.Theabsorptionofelectromagneticradiationishowphoton
energyistakenup.Typicallyanelectroniselevatedtoahigherenergy
levelbylightabsorption.Whenanexcitedelectronreturnstothelowest
energylevel,alightphotonisemitted.
MolecularPhotophysicalProcessesRelevantforPhotobiology
Molecularphotophysicalprocessesrelevantforphotobiologyinclude
absorptionandemissionofUV,visibleornearIRlight,byaromatic
molecules.Intheremainderofthismodulewewillfocusonthese
processes.
JablonskiDiagram:Basicprinciplesofmolecularphotophysicscanbe
clarifiedwiththehelpoftheJablonskidiagram,namedafterthePolish
physicistAleksanderJablonski(Figure4).

Figure4.Jablonskidiagramrepresentingenergylevelsand
spectra.Solidarrowsindicateradiativetransitionsasoccurring
byabsorption(violet,blue)oremission(greenfor
fluorescenceredforphosphorescence)ofaphoton.Dashed
arrowsrepresentnonradiativetransitions(violet,blue,green,
red).Internalconversionisanonradiativetransition,which
occurswhenavibrationalstateofahigherelectronicstateis
coupledtoavibrationalstateofalowerelectronicstate.Inthe
notationof,forexample,S1,0,thefirstsubscriptreferstothe
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electronicstate(firstexcited)andthesecondonetothe
vibrationalsublevel(v=0).Inthediagramthefollowing
internalconversionsareindicated:S2,4S1,0,S2,2S1,0,
S2,0S1,0andS1,0S0,0.ThedottedarrowfromS1,0T1,0isa
nonradiativetransitioncalledintersystemcrossing,becauseit
isatransitionbetweenstatesofdifferentspinmultiplicity.
Belowthediagramsketchesofabsorption,fluorescenceand
phosphorescencespectraareshown.
Thediagramillustratestheelectronicstatesofamoleculeandthe
transitionsbetweenthem.Theelectronicstatesarearrangedverticallyby
energy.Theyaregroupedhorizontallybyspinmultiplicity.Intheleftpart
ofthediagramthreesingletstateswithantiparallelspinsareshown:the
singletgroundstate(S0)andtwohighersingletexcitedstates(S1and
S2).Singletstatesarediamagnetic,astheydonotinteractwithan
externalmagneticfield.Thetripletstate(T1)istheelectronicstatewith
parallelspins.Amoleculeinthetripletstateinteractswithanexternal
magneticfield.Transitionsbetweenelectronicstatesofthesamespin
multiplicityareallowed.Transitionsbetweenstateswithdifferentspin
multiplicityareformallyforbidden,butmayoccurowingtoaprocess
calledspinorbitcoupling.Thistransitioniscalledintersystemcrossing.
Superimposedontheseelectronicstatesarethevibrationalstates,which
areofmuchsmallerenergy.Inthefollowingsectionswewilladdressthe
sequenceofprocesses,whichoccurwhenanaromaticmoleculeabsorbs
aphotonofsufficientenergy.
Itrequiressomeefforttocalculatetheenergylevelsandwavefunctions
ofanaromaticmoleculeofaveragesize.BornandOppenheimerhave
madethistaskconsiderablyeasierbybreakingupthequantum
mechanicalwavefunctionofamoleculeintoitselectronicandnuclear
(vibrationalandrotational)components:

InthefirststepofthisBornOppenheimerapproximationtheelectronic
Schrdingerequationissolvedyieldingtheelectronicwavefunction,
,dependingonelectronsonlywiththenucleifixedinan
equilibriumconfiguration.
LightAbsorption:Whenamoleculeabsorbsaphotonofappropriate
energy,avalenceelectronispromotedfromthegroundstatetosome
vibrationallevelintheexcitedsingletmanifold(Figure4).Theprocessof
lightabsorptionisextremelyrapid,intheorderofonefemtosecond(1
fs=1015s).Itmeansthatthenucleiofthemoleculearefixedduring
thetransition,becauseoftheirmuchlargermass,andthattheBorn
Oppenheimerapproximationisvalid.
Itistobenotedthatthepointofdepartureisalwaysthelowest
vibrationallevelofS0designatedS0,0.Wecanunderstandthisby
referringtotheBoltzmanndistributionfunction,whichisameasurefor
thedistributionof(inthiscase)vibrationalstatesofthemolecule:

whereN1isthenumberofmoleculesinthefirsthighervibrationalstate
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(S0,1),andN0thatforthelowestvibrationallevel(S0,0),Evibisthe
energydifferencebetweenthetwovibrationalstates,kBistheBoltzmann
constant,andTtheabsolutetemperature.Atroomtemperature,Evibis
muchlargerthankBT,implyingthatonlythelowestvibrationallevelis
populated.
Afterlightabsorptiontheexcitedmoleculeendsupatthelowest
vibrationallevelofS1(S1,0)viavibrationalrelaxationandinternal
conversion.Thisradiationlessprocesstakesplaceinaboutone
picosecond(1ps=1012s).
Asketchofanabsorptionspectrumconsistingoftwobandsisshownin
Figure4.Inthecondensedphase,broadabsorption(andalsoemission)
bandsareobserved,notthesharptransitionsseenforatomsor
moleculesinthegaseousphase.Thereasonisduetophenomenaknown
ashomogeneousbroadeningandinhomogeneousbroadening.
Homogeneousbroadeningarisesfromthemultitudeofvibrationalstates
(manymorethanshowninFigure4)androtationalstates(superimposed
onthevibrationalstates),whichallaresuperimposedontheelectronic
transitionspreventingtheobservationofsharptransitions.
Inhomogeneousbroadeningarisesfromsolventeffects,whichwillbe
detailedlaterforfluorescence.
Thestrengthofthelowestopticaltransitionisveryoftenexpressedin
termsofthe(dimensionless)oscillatorstrengthf:

whereisthemolarextinctioncoefficientconnectedwiththelowest
electronictransition,isthewavenumberandtheintegralisoverall
wavenumbersoftheabsorptionband.Forintense(stronglyallowed)
transitionsf1.Theoscillatorstrengthhasadirectrelationshipwiththe
electronictransitiondipoleeg,whichcouplesthewavefunctionsofthe
ground(g)andexcited(e)electronicstates:

with=er(eischargeofelectron),andtheintegrationtakesplaceover
spatialcoordinatesr.
Thetransitiondipoleisameasureofthedipolemomentassociatedwith
theshiftofchargethatoccurswhenelectronsareredistributedinthe
moleculeuponexcitation.Theoscillatorstrengthisproportionaltothe
magnitudeofthetransitiondipole:

Fluorescence:In1852,theBritishscientistSirGeorgeG.Stokescoined
theterm"fluorescence"afterobservingblueluminescenceinthemineral
fluorite.Stokesalsodiscoveredtheredshiftinbandmaximumofthe
fluorescencespectrumrelativetothebandmaximumofabsorption
(Stokesshift).
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ThelowestvibrationallevelofS1isthestartingpointforfluorescence
emissiontothegroundstateS0,nonradiativedecaytoS0(internal
conversion),andtransitiontothelowesttripletstate(intersystem
crossing)(Figure4).Fluorescencetakesplaceonthenanosecond
timescale(1ns=109s)and,dependingonthemolecularspecies,its
durationamountsto1100nanoseconds.ItisclearfromtheJablonski
diagramthatfluorescencealwaysoriginatesfromthesamelevel,
irrespectiveofwhichelectronicenergylevelisexcited.Theemittingstate
isthezerothvibrationallevelofthefirstexcitedstateS1,0.Itisforthis
reasonthatthefluorescencespectrumisshiftedtolowerenergythanthe
correspondingabsorptionspectrum(Stokesshift).TheStokesshiftcan
beenhancedbysolventinteractions,examplesofwhichwillbegiven
later.WecanalsoconcludefromthesketchedspectrainFigure4that
vibrationalfinestructureinafluorescencespectrumreportsabout
vibrationsinthegroundstate,andvibronicbandsinanabsorption
spectrumprovidesinformationonvibrationsinhigherelectronicexcited
states.
Anotherfactorthathastobeconsideredinfluorescencespectroscopyis
theFranckCondonfactor.IfwelookattheJablonskischemeinFigure4,
itcanbeseenthatthefluorescencetransitionS1,0S0,0isnotthemost
intenseone.TheFranckCondonprinciplestatesthatthemostintense
vibronictransitionisfromthevibrationalstateinthegroundstatetothat
vibrationalstateintheexcitedstateverticallyaboveit(Figure5,blue
arrow).

Figure5.EnergydiagramforexplanationoftheFranck
Condonprinciple.Thepotentialwellsshowfavoredtransitions
betweenvibrationalsublevels=0andv=2bothfor
absorption(bluearrow)andemission(greenarrow).
[FromWikipedia:FranckCondonPrinciple]
Theschemes(forabsorptionandemission)inFigure5aresimplified
twodimensionalpotentialenergydiagrams,butforthesakeof
argument,theyareasufficientrepresentation,sincewearedealingwith
(an)harmonicoscillators.Sincetheexcitedstateisdifferentfromthe
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groundstate,adisplacedminimumnuclearnormalcoordinatecanbe
expected.Itshouldbenotedfurtherthatthetimetoreachtheexcited
stateissoshort(femtoseconds)thatthenucleipositionsarevirtually
unchangedduringtheelectronictransition.Inthevibronicwavefunctions,
thenuclearcoordinatescanthenbeuncoupledfromtheelectronic
coordinates(BornOppenheimerprinciple).Thetransitiondipolecanthen
befactoredintoanelectronicandanuclearpart:

ThesecondintegralisthesocalledFranckCondonvibrationaloverlap,
whichalsodeterminesthestrengthoftheelectronictransition(or
oscillatorstrength).InthefluorescentpartoftheschemeinFigure4,the
secondandthirdvibrationaltransitions(S1,0S0,1andS1,0S0,2)have
largerFranckCondonfactorsthantheonebetweenfundamental
vibrationalwavefunctions(S1,0S0,0).
Phosphorescence:InFigure4,thetripletstateisalsodrawn.Oncethe
moleculehasreachedthisstate,itwillresideforaverylongtimethere
(frommicrosecondstoseconds)beforeitwilldecaytothegroundstate.
Thisisduetothespinforbiddentransitionsinvolvedinthe(excited)
singlettripletandtripletsinglet(groundstate)transitions.Inrigid
solutionorindeoxygenatedsolutions,longlivedphosphorescence
(millisecondstoseconds)fromthisstatecanbeobserved.Thename
"phosphorescence"probablyoriginatesfromtheFrenchphysicistEdmond
Becquerel,whodevisedin1857aninstrumentthathecalleda
phosphoroscope.Withthisinstrumenthecouldmeasurehowlongit
takesaphosphorescentsampletostopglowingafterexcitation.
Becauseofitslonglifetime,thetripletstateofanaromaticmoleculeis
thestartingpointforphotochemicalreactions.Onereactioninparticular
isveryprominent,namelytheproductionofveryreactivesingletoxygen.
Theoxygenmolecule,O2,possessesatripletgroundstate.Insolution,
frequentcollisionsbetweenanaromaticmoleculeinthetripletstateand
oxygenresultinenergytransfer,andgenerationofsingletoxygen,which
canoxidise(anddestroy)thearomaticmolecule.
PracticalAspectsofFluorescence
[seeModuleonBasicSpectroscopy]
AbsorptionSpectrum:Themeasurementofanabsorptionspectrum
(Figure6)isbasedontheLambertBeerlaw,andshowstheabilityofthe
investigatedsampletoabsorblightatdifferentwavelengths.Aslight
absorptionoccursalmostalwaysfromthelowestvibrationallevelofthe
electronicgroundstate,theabsorptionspectrumcharacterizesthe
energeticstructuresoftheelectronicexcitedstatesofanaromatic
molecule.

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Figure6.Measurementprincipleoflightabsorptionspectra.
Thevaluesmeasureddirectlyinthespectrophotometerarethe
intensitiesoflighttransmittedthroughsampleandreference
cuvettes.ThelightsourceSgeneratesabroadspectrumoflight
andthedispersiondevice(gratingorprism)Mselectsa
monochromaticlightofspecificwavelength.Thelightisthen
dividedintotwoidenticalbeamsdirectedtotwocuvettes,a
samplecuvette,SC,containingthesolutionofthearomatic
compoundandareferencecuvette,RC,withsolventonly.
Changingthewavelengthovertherequiredrangeenables
measuringtheintensitiesofthelighttransmittedthroughboth
cuvettes,I()andI0().
TheabsorbanceA atwavelengthisthendefinedas:

andisequalaccordingtotheLambertBeerlawto:

where()istheextinctioncoefficientatwavelengthfortheparticular
molecule,citsconcentration,anddthepathlengthofthecuvette,for
instance1cm.
Examplesofabsorption(absorbance,leftside)andfluorescence(right
side)spectraofthreewidelyinvestigatedfluorophoresarepresentedin
Figure7.

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Figure7.Normalisedabsorption(left),andfluorescence
(right)spectraofthreefluorophores.Shownarethespectraof
2,5diphenyloxazole(PPO,amoleculeusedinscintillation
counting)inanorganicsolvent,Nacetyltryptophanamide
(NATA),andtheproteinhumanserumalbumin(HSA)in
aqueousbuffer.
FluorescenceSpectrum:AfluorescencespectrumIflu()representsthe
intensityofthefluorescencelightemittedbythesampleasafunctionof
emissionwavelength(Figure8).Asfluorescencetransitionsstartinmost
casesfromthelowestvibrationallevelofthefirstelectronicexcited
state,Iflu()characterisestheenergeticstructureoftheelectronicground
state.

Figure8.Measurementoffluorescencespectra.
Fluorescenceisdetectedatrightangletotheexcitationbeam.
Amonochromaticlightbeamofselectedwavelengthexc
excitesthemoleculesinthesample.Fluorescencelightis
emittedfromtheexcitedstateofthesemoleculesinall
directions.Fluorescenceisusuallycollectedatrightanglesto
theexcitationdirectioninordertominimizethepresenceof
scatteredexcitationlightinthefluorescencechannel.The
monochromatorM2allowsmeasurementsoffluorescence
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intensityasafunctionoftheemissionwavelength.The
fluorescencespectraofPPO,NATAandHSAareshowninFigure
7.
Sometimesitisusefultomeasurehowfluorescenceintensitydependson
theexcitationwavelength.Inthiscasewewilldetectfluorescence
intensityatafixedwavelength(forinstanceatthewavelengthof
maximalfluorescence)asafunctionoftheexcitationwavelength(M1),
thusobtaininganexcitationspectrum.Anexcitationspectrumis
essentiallyanabsorptionspectrum,becausethefluorescenceintensity
Iflu()isproportionalto:

where()istheextinctioncoefficientatexcitationwavelength,I0is
themonochromaticlightintensity,andisthequantumyieldof
fluorescence(seebelow).Thisequationisvalidonlyfordilute
fluorophoresolutions,inwhichtheabsorbanceofthearomaticcompound
neverexceeds0.05.Excitationspectraturnouttobeusefulinobtaining
absorptionspectraofverydilutesamples.
QuantumYieldofFluorescence:Thequantumyield,,isdefinedas
theratioofthenumberoffluorescencephotonsemittedbythesamplenE
tothenumberofphotonsabsorbednA.Itcanbeshownalsothatisthe
ratiooftherateoftheradiativetransition(kr)totheratesofall
transitions(kr+knr),inwhichtheexcitedstateisinvolved.Therefore,any
molecularmechanismleadingtoanonradiativedepopulationofthe
excitedstatereducesthequantumyield:

Thequantumyieldsofsomepopularfluorophores,naturallyoccurring
monoanddinucleotides,wildtypegreenfluorescentprotein(GFP),and
othervisiblefluorescentproteinvariants,arecollectedinTable2.

Table2.Quantumyieldsofselectedfluorophores.
FluorescenceLifetime:Theotherimportantcharacteristicfeatureof
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fluorescenceisitstimeresponse,namelythedecayoffluorescence
intensityfollowinginfinitesimallyshortortypeexcitation.Thescheme
presentedinFigure9isasimplifiedJablonskidiagram,whichcanbe
usedtoexplainthebasickineticsoffluorescence.

Figure9.Kineticschemetoexplainfluorescencelifetime.(0)
isexcitationwithapulse,krisrateconstantofradiative
transition,andknrisrateconstantofnonradiativetransition.
Thepopulationoftheexcitedmolecules[M*]generatedatthemoment
ofexcitation,t=0,startstodecreaseexponentiallythroughtheradiative
(kr)andnonradiative(knr)transitionstothegroundstate.
Thecharacteristictimeofthisprocess,
lifetime:

,iscalledthefluorescence

Theintensityoffluorescence,F(t),emittedatanymomentofthis
processisproportionalto[M*](t),thus:

NotethatF(t)isashorthandnotationforIflu(t).Thefluorescencelifetime
0hasthephysicalmeaningofthetimeneededforthefluorescence
intensitytodecreaseto1/e(=1/2.71)ofitsinitialvalueF0(Figure10).

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Figure10.FluorescencedecayF(t)=F0exp(t/5)withtinns.
F(t)isthefluorescenceintensitythatdecaysexponentiallywith
afluorescencelifetime, 0,equalto5ns.Thepulse
representsanexcitationlightpulseofinfinitesimallysmall
duration,forinstance,adurationof1femtosecond(1015
second).
Inpracticethegenerationofapulseforexcitationisnotpossible,
becausetheinstrumentalresponsetimeneededtodetectfluorescence
decaysisoftencomparabletothefluorescencelifetimesthatwewantto
measure.Theexperimentallydetectedfluorescencedecaysarenotpure
decayslikeF(t)inFigure10,butconvolutionsoftheidealdecayfunction
withthesocalledpromptfunction(orinstrumentalresponsefunction).
Measuringthepromptfunction,andthenextractingtheundistorted
fluorescencedecayfunctionwillbedescribedlaterinthismodule.
FluorescenceAnisotropy:Photonscanbeabsorbedonlywhentheir
energyfitstotheenergygapbetweenthegroundandexcitedenergy
levelsofaparticularmolecule.Anotherconditionforlightabsorptionis
thattheelectriccomponentorvectoroftheelectromagneticwavemust
beparallelorclosetoparallel,tothetransitionmomentofthemolecule.
Insolution,theorientationsofthetransitionmomentsarecompletely
random.Therefore,ifweexcitesuchasystemwithlinearlypolarized
light,theexcitationwillbeefficientonlyforthosemoleculeswhose
transitionmomentsare,atthemomentofexcitation,orientedsimilarly
tothedirectionofpolarization.Theinitialdistributionoforientationsof
theexcitedmoleculeswillthenbehighlyanisotropic.Thisorderingeffect
iscalled'photoselection'.
Afterexcitation,themoleculesstarttofluorescewiththeircharacteristic
fluorescencelifetime 0,and,simultaneously,Brownianrotationalmotion
causestheinitialorientationalorderoftheexcitedmoleculestovanish.
Polarizationoffluorescenceisdeterminedbytheorientationofthe
fluorophores'transitionmomentattheinstantoffluorescenceemission.
Thisgivesanopportunitytodeterminerotationaldiffusionof
fluorophoresbydetectingtheanisotropyoftheirfluorescence(Figure
11).

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Figure11.Geometryoffluorescenceanisotropyexperiment.
Fluorophoresinthecuvetteareexcitedwithshortpulsesof
verticallypolarizedlight(z)causingphotoselection.Apolarizer
inthefluorescencechannel(x)canberotatedfromthevertical
tothehorizontalposition.Inthefirstpartoftheexperiment,
thedecayofintensityofverticallypolarizedfluorescenceIvv(t)
ismeasured.Thenthepolarizerismovedtohorizontal
orientation,andIvh(t)isdetected.
Bothdecays,Ivv(t)andIvh(t),arecombinedtoformthefluorescence
anisotropyfunctionr(t),definedas:

Forsphericalandfreelyrotatingfluorophorestheanisotropydecays
exponentially:

withthecharacteristicdecayconstant r,calledrotationalcorrelation
time,andr0=2/5forrandomlydistributedmolecularsystems.According
tothetheoryofrotationaldiffusion, risrelatedtotheradiusrofa
rotatingspherebytheequation:

Here,kBTistheBoltzmannfactor,andistheviscosityofthesolvent.
Therelationbetweenfluorescenceanisotropydecays,andthedimensions
ofrotatingmoleculesisoneofthefundamentalsofthenewlyemerging
researchdisciplineof'nanometrology'.
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SolventEffectsonFluorescence:Theconceptofsolvationcanbe
understoodfrominteractionsbetweenafluorophore(thesolute),andthe
surroundingsolventmolecules.Thedominatingsolutesolvent
interactionsarisefromelectrostaticdipoledipoleinteractions,whichlead
toloweringthepotentialenergiesofallenergylevelsinvolvedin
absorptionandfluorescenceprocesses.Thiseffectcanbeexplainedby
Onsager'smodelofsolvation(Figure12).

Figure12.Changesinsolutesolventinteractionsleadto
solvatochromicshiftsinabsorptionandfluorescencespectraof
thesamefluorophore.[seetextfordefinitionsofsymbols]
Accordingtothismodel,thedipolemomentofthefluorophoreinthe
groundstate,g,interactswiththedipolemomentsofthesurrounding
solventmolecules,rearrangingtheminawaythatminimisesthe
potentialenergyofthewholesystem.Ifwewould"freeze"themolecules
forawhileandremovethefluorophore,thespecialarrangementofthe
solventdipolemomentswouldresultinanonbalancedelectricfieldRg,
calledthe"reactionfield".
InOnsager'smodel,thesolutesolventinteractionisidentifiedasan
interactionofthefluorophoredipolemoment,g,withthereactionfield
Rg,namely:

Theenergylevelofthegroundstateisthereforeloweredbythisvalue.
Thesymbol'rel'indicatesthatthesolventisinastateofthermodynamic
equilibrium(relaxed).Electronicexcitationofthefluorophore(Figure12)
causesarapid(~1015s)changeofitsdipolemomenttoe.Thistimeis
muchtooshortforthesolventmoleculestorearrangetheirorientations.
Thus,immediatelyafterexcitationtheinteractionenergywillbe:

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indicatingthatthereactionfieldwillbestillthesameasitwasbefore
excitation.Thesymbol'FC'indicatesanonequilibrated,FranckCondon
state.Thesolventmoleculesneedusuallypicoseconds(10121010s)to
performsolventrelaxationachievingfinallythesolutesolventinteraction
energy:

Theprocessoffluorescencebringsthefluorophoredipolemomentback
toitsgroundstatevalueg,sojustafterfluorescence:

whichfinallyevolvesduringgroundstatesolventrelaxationto

Directconsequencesofthedifferentsolutesolventinteractionenergies
atdifferentstagesofabsorptionandfluorescenceeventsarethespectral
shiftsinabsorption(Uabs)andfluorescence(Uflu)spectra:

Thefluorescencespectraofthesolventsensitivefluorophore"badan"in
anumberofsolventsareshowninFigure13.For"badan",eismuch
largerthang.Thelargerthedipolemomentofthesolventmoleculethe
strongeristhe"redshift"ofthespectrum.

Figure13.Fluorescencespectraof"badan"(i.e.,the2
mercaptoethanoladductof6bromoacetyl2
dimethylaminonaphtalene)in:(1)toluene,(2)chloroform,(3)
acetonitrile,(4)ethanol,(5)methanoland(6)water.Figure
fromHandbookofMolecularProbes.
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QuenchingofFluorescencebyExternalMolecules:Anymolecule
thatinteractswiththefluorophoreandreducesitsquantumyield,is
calledaquencher.Thereareanumberofdifferentmolecularmechanisms
ofquenching.
Incaseofstaticquenching,externalmoleculesQ,simplyformground
statecomplexeswiththefluorophoreM(Figure14).Thesecomplexes
canbeexcited,butarenotfluorescent.

Figure14.Schematicillustrationofstaticquenching.TheMQ
complexisnotfluorescentyieldingreductioninfluorescence
intensity,butnochangeinfluorescencelifetime(seetext).
Theequilibriumbetweenfreeandcomplexedfluorophoresiscontrolled
byanassociationconstantKs:

where[M],[Q]and[MQ]aretheconcentrationsoffluorophore,quencher
andfluorophorequenchercomplexes,respectively.[M]0isthetotal
concentrationofthefluorophore.Theaboveequationcanbeeasily
convertedintothewellknownSternVolmerequation,whichdescribes
thedecreaseoffluorescenceintensitywithincreasingquencher
concentration:

Here,F0andFarethefluorescenceintensitiesmeasuredwithout,andin
thepresenceofthequencher.Duetothenatureofstaticquenching,
thereisnochangeinfluorescencelifetime,sinceonlythenoncomplexed
fluorophoreisfluorescent.However,thequencherfluorophorecomplex
canstillbefluorescent,butdisplaysanultrashortfluorescencelifetime
inthepicosecondtimerangeyieldingnosignificantsteadystate
fluorescenceintensity.Thestaticquenchingarisesfromcompeting
processesthatinducenonradiativepathwaystothegroundstate.The
nonradiativerateconstant(k'nr)ismuchlargerthantheradiativerate
constant(kr),andemissionisalmostcompletelyeliminated.
InthecaseofdynamicquenchingthequenchingmoleculeQcollideswith
theexcitedfluorophoreM*(Figure15).Theexcitedstatekineticsare
affectedbyforminganadditionalwayofdepopulatingtheexcitedstate.
Consequently,thefluorescencedecayismodified.Bothsteadystateand
timeresolvedfluorescenceyieldthefamousSternVolmerconstant
KSV= 0kq,inwhich oisthefluorescencelifetimewithoutquencher,and
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kqisthediffusioncontrolledrateconstantofquenching.KSVhasthe
dimensionM1.
Severalcompoundsbehaveasdynamicquenchingagentswhenpresent
atsufficientlyhighconcentration,includingoxygen,heavyatomhalides
suchasbromideandiodide,mercuryandcaesiumions,tetrachloro
methane(CCl4),aminesandacrylamide.

Figure15.Schematicillustrationofdynamicquenchingand
relevantrateequations.Thesteadystatefluorescenceintensity
ratio(F0/F)(Fisthefluorescenceintensityinpresenceof
quencherQ)isthesameastheratiooffluorescence
lifetimes 0/ q( qisthefluorescencelifetimeinthepresence
ofquencherQ).TheSternVolmerconstantKSVisequalto
0kq.
FrsterResonanceEnergyTransfer(FRET):TheodorFrster
developedthequantitativetheoryforresonanceenergytransferinthe
late1940s.Therefore,wecallthisprocessFrsterresonanceenergy
transferorFRET.FRETisaphotophysicalprocesswheretheexcitedstate
energyfromadonormoleculeistransferrednonradiativelytoan
acceptormoleculeatclosedistanceviaweakdipoledipolecoupling
(Figure16).FRETissometimesreferredtoas"fluorescence"resonance
energytransfer,butthisisamisconception,sincenofluorescencetakes
partinthecoupledtransition.

Figure16.KineticschemeofFRET.

Frsterderivedthefollowingexpressionfortherateconstantoftransfer
kT:

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inwhichRisthedistancebetweendonorandacceptormoleculesand D
isthefluorescencelifetimeofthedonor(withoutacceptor).Sincethe
transferrateisproportionaltotheinverse6thpowerofthedistanceR,
thetransferrateisanextremelysensitiveparameterforobtaining
distancesintherangeof110nm(or10100).Thedistanceatwhich
theexcitationenergyofthedonoristransferredtotheacceptorwith
probability0.5,iscalledtheFrsterorcriticaldistanceR0(inunitsof
cm),andcanbecalculatedusingtherelevantspectroscopicpropertiesof
theparticipatingmolecules:

inwhich2istheorientationfactor,0thequantumyieldofdonor
fluorescence(withoutacceptor),NAisAvogadro'snumber,andnisthe
refractiveindexoftheinterveningmedium.Theintegral(J,inunitsof
M1cm3)isthedegreeofspectraloverlapbetweendonorfluorescence
spectrum(FD,itsspectrumnormalizedsothattheintegralisequalto
one),andacceptorabsorptionspectrum(scaledtoitsmaximummolar
extinctioncoefficient,A,inunitsofM1cm1),givenbyeither
wavenumber()orwavelength()scale(Figure17):

Figure17.Illustrationofspectraloverlapintegralbetweenthe
fluorescencespectrumofcyanfluorescentprotein(CFP),and
theabsorptionspectrumofyellowfluorescentprotein(YFP).
CFPandYFParemutantsofGreenFluorescentProtein.

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Theorientationfactor isgivenby:
2

ForadefinitionoftheangleswerefertoFigure18,inwhichsome
limitingsituationshavebeendepictedillustratingthat0<2<4.For
systemswithoutany3dimensional,spatialinformationtheorientation
factoristheindeterminateparameterinR0.Allotherparameterscanbe
measuredorevaluated.

Figure18.TheorientationfactorinFRET.DandAarethe
transitionmomentsofdonorandacceptormoleculesandRis
theseparationvector.
Whenthecriticaltransferdistance,R0(seeequationabove),isexpressed

inunitsof,thescalingconstant
isequalto0.2108,the
molarextinctioncoefficient,A,inunitsofM1cm1,thewavelength,,in
unitsofnm,andtheoverlapintegral,J,inunitsof
M1cm1nm4,andR0isgivenby:

Foranexercisehowtocalculatethecriticaltransferdistancefrom
spectroscopicdataseeModuleinExperimentsforStudents:(A.J.W.G.
Visser,E.S.VysotskiandJ.Lee)
FrsteralsointroducedthetransferefficiencyE,whichisonlyafunction
ofactual(R)andcritical(R0)distances:

ThereareseveralmethodsavailableforquantificationofFRET,ofwhich
theonebasedondonorfluorescencelifetimesisthemost
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straightforward,becausethefluorescencelifetimeisaconcentration
independentproperty,whilefluorescenceintensityisnot.Donor
fluorescencelifetimesintheabsence( D)andpresence( DA)ofacceptor
moleculesareoftenmeasuredfortheobservationofFRETanda
decreasedfluorescencelifetimeofthedonoristhenanindicationof
molecularinteractions(Figure19).Fromthisreductioninlifetimeswe
immediatelyobtaintheexperimentalFRETefficiency:

Figure19.TimeresolvedfluorescencedetectionofFRET.
ShownareadonormoleculeDandanacceptormoleculeA
separatedatdistanceRinamacromolecule.Averyshortlight
pulse((0))excitesdonorD.Analysisofthegenerateddonor
fluorescencedecay(F(t))givesinformationontherate
constantoftransferTfromwhichthedistanceRcanbe
obtained.
InFigure20wehavesummarisedthemainconceptsofFRET,whichare
illustratedwiththewidelyusedFRETcoupleCFP(cyanfluorescent
protein,donor)andYFP(yellowfluorescentprotein,acceptor).FRETis
usedextensivelyformonitoringinteractionsandconformationalchanges
betweenorwithinbiologicalmacromoleculesconjugatedwithsuitable
donoracceptorpairs.BecauseofitssensitivityFRETalsoformsthebasis
for"sensing"importantbiologicalmoleculesinmanyapplications.

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Figure20.MainconceptsofFRET.TheoccurrenceofFRET
dependsondistance(upperleftpanel)andthespectraloverlap
(upperrightpanel).Thereductionofalifetimefrom2.5to2.0
nscorrespondstoatransferefficiencyof0.2(20%)andtoa
distancebetweenCFPandYFPof6.3nm.
TimeResolvedFluorescenceInstrumentation:Measurementofthe
fluorescencelifetime,orgenerallyoftheparametersthatgovernthe
courseoffluorescenceintensitywithtimecanbeperformedwithtwo
differenttechniquesthatarebothwidespread:thepulsemethodandthe
phasemodulationmethod.
Thepulsemethodisfrequentlyusedincombinationwithtimecorrelated
singlephotoncounting(TCSPC).Thesampleisexcitedwithashortpulse
oflight,andthetimebetweenthepulseanddetectionofthefirstemitted
photonismeasured.Thecourseoffluorescenceintensitywithtime
(Figure21)isrecordedbymultiplerepetitionofthisprocedure.

Figure21.Schematicsoftimecorrelatedsinglephoton
counting.Thearrivaltimeofthefirstphotonafteranexcitation
pulseismeasuredandstoredinmemory.Thehistogramof
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pulseismeasuredandstoredinmemory.Thehistogramof
manyarrivaltimesofphotonsrepresentsthe"fluorescence
intensityversustime"curve.

Theresultingsignalisaconvolutionoftherealdecayovertime(i.e.,
followingthepulseofexcitation)offluorescenceintensitywiththe
instrumentresponsefunction(Figure22).

Figure22.Illustrationoftheconvolutionprinciple.L(t)isthe
instrumentalresponsefunctionandF(t)istheexperimental
decayfunction.Thechannelnumbergivesthetimeaxis,asit
correspondstoacertaintimeincrement.F(t)isaconvolution
productofL(t)withtherealdecayfunctionFc(t):

Analysisofthemeasuredfluorescenceintensitycurvesanddetermination
offluorescencelifetimesareoftenperformedbyanonlinearleast
squaresfittingmethod.Letusassumethatamodelfunctionofasumof
twoexponentialtermsadequatelydescribesthecourseoffluorescence
intensitywithtime.Suchsituationmayoccur,whenthefluorophore
existsintwodistinctenvironmentseachcharacterisedbyitsown
fluorescencelifetime.

inwhichn=2,and 1and 1aretheamplitudeandlifetimeof

component1,and 2and 2aretheamplitudeandlifetimeof
component2.Theresultoftheconvolutionofthemodelfunctionwith
theinstrumentresponsefunctionisfittedtothemeasuredfluorescence
intensitycurvesbyoptimisingtheparameters 1, 1and 2, 2.
TheresolutionthatcanbeachievedwithTCSPCdependsmuchonthe
lightsourceanddetector.Usingmodelockedlasersincombinationwith
microchannelplatephotomultipliers,instrumentresponsefunctionwidths
ofthirtypicosecondsarepossible.Thisallowsforthemeasurementof
decaytimesintherangeoftentotwentypicoseconds.Muchcheaper
diodelasersandlightemittingdiodes(LEDs)givelongerexcitation
pulses(widthintherange50200ps),butdecaytimesofseveral
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hundredpicosecondscanstillberesolved.
Inthephasemodulationmethod,thesampleisexcitedbyanintensity
modulatedlightsource,whichcaneitherbeaxenonarclamp,LED(see
below)oralaser(pulsedorCW).Theexcitationissinusoidalmodulated
atangularfrequency (rad/s).Thefluorescenceisdelayedinphaseand
partiallydemodulated.Theexperimentalquantitiestobedeterminedare
thephasedifference( ),andthemodulationratio( ),asshownin
Figure23.

Figure23.Determinationofthephasedifference( )and
modulationratio( )inthephasemodulationmethod.
Forasinglefluorescencelifetime,phasedifferenceandmodulationratio
arerelatedtothelifetime( )by:

Fortheprecisedeterminationoffluorescencelifetimes,thephase
difference( )andthemodulationratio( )aremeasuredasfunction
ofdifferentfrequencies .Thecurvescanbeanalysedbythemethodof
nonlinearleastsquaresusingtheoreticalexpressionsofthesineand
cosineFouriertransformsofthepulseresponse.Theresolutionofthe
phasemodulationmethoddependsonthemodulationfrequency.
Fluorescencelifetimesinthepicosecondrangecanbemeasuredusing
modulationfrequenciesbetween2and10GHz.Forlifetimesinthe110
nanosecondrange,modulationfrequenciesbetween2and200MHzare
appropriate.
LuminescenceFromSemiconductor(Nano)Crystals
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Semiconductors:Untilnowwehavetreatedlightemissionfrom
aromaticmoleculessuchasfluorescenceand,inlessdetail,
phosphorescence.Ageneraltermoflightemissionisluminescence,a
termderivedfromtheLatin"lumen",light,andintroducedbythe
GermanscientistEilhardWiedemannin1880("Lumineszenz").Because
luminescencefromcrystallinesemiconductormaterialshasacompletely
differentoriginthanthatoffluorescence,itisworthwhiletopayattention
toit,aswearesurroundednowadaysbymanyapplicationsofthem.
Tounderstandhowsemiconductorsworkwemusthavebackground
knowledgeinsolidstatephysics.Thatisbeyondthescopeofthis
module,andweprefertogiveaqualitativepictureofthephysicsbehind
semiconductors.Asemiconductorisamaterialthathasanelectrical
conductivitybetweenthatofaconductorandaninsulator,thatis,
generallyintherange103Siemens/cm(conducting)to108S/cm(non
conducting).Devicesmadefromsemiconductormaterialsareusedin
modernelectronics,includingcomputers,telephones,LCDdisplays,etc.
Semiconductorshavealsofoundapplicationsinoptoelectronicsand
photophysics.Belowwewillbrieflydescribetheworkingprinciplesof
quantumdots,lightemittingdiodes(LEDs),diodelasersandsolarcells.
Thereisanothermotivationtoknowmoreaboutsemiconductors,
especiallysolarcells.Theworkingprincipleofsolarcellsshowsa
remarkableanalogywiththatofthephotosyntheticapparatus.Plantsuse
radiationinthewavelengthrangeof400700nmtoreduceCO2to
glucose,withtheconcomitantoxidationofwatertoO2.Theradiation
energyisintherangeofthebandgapofmanysemiconductormaterials
(Table3).

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Table3.Somesemiconductormaterialsandtheirbandgaps.
[adaptedfromWikipedia:BandGap]
Thisphotosystemconsistsofproteincomplexes,whichcombinelight
harvestingproteinsandreactioncenters.Thelightharvestingcomplex
havingboundalargenumberofchlorophyllsandcarotenoids,absorbs
solarenergyandrapidlytransfersthisenergytothereactioncenter,
whichisaspecialpairoftwochlorophyllmolecules.Efficientlight
harvestingrequiresthatenergymustbetransferredviaweak(Frster
type)andstrong(excitontype)couplingmechanisms.Thisisanalogous
tothecreationofelectrons,holesandexcitonsinsemiconductor
materialsafterillumination,whicharetransportedefficientlyinthe
crystallatticetoapnjunction(seebelow).Thefunctionofapn
junctionmaybecomparedtothatofthereactioncenterofthe
photosystem,sincechargecarriersareseparatedinbothsystems.
Negativechargecarriersareelectronsinbothsystems.Positivecarriers
areholesinsemiconductors,andprotonsinreactioncentres.Both
photosystemsandsolarcellsmustoperatereversibly.Theyshare,
therefore,thesameprocessofrecombinationofchargecarriers.
Impregnatingimpuritiesintotheircrystallatticemayeasilymodifythe
conductivityofsemiconductors.Theprocessofthecontrolledaddingof
impuritiestoasemiconductorisknownas'doping'.Theamountof
impurity,ordopant,addedtoapuresemiconductorvariesitslevelof
conductivity,andalsoitsbandgap.Byincorporatingimpurityatomsinto
thecrystallatticeofpuresemiconductors,theelectricalconductivitymay
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bevariednotonlybythenumberofimpurityatomsbutalso,bythetype
ofimpurityatom.
Ingeneral,dopantsthatproducethedesiredcontrolledchangesare
classifiedaseitherelectronacceptorsordonors.Adonoratomdonates
weaklyboundvalenceelectronstothematerialcreatingexcessnegative
chargecarriers.Theseweaklyboundelectronscanmoveinthecrystal
latticerelativelyfreely,andcanfacilitateconductioninthepresenceofan
electricfield.Conversely,anactivatedacceptorproducesanelectronhole
(usuallyreferredtosimplyasahole),whichcarriesapositivecharge.
Theholesdonotactuallymove,butaneighboringelectroncanmoveto
fillthehole,leavingaholeattheplacethattheelectronoccupied
previously.Inthisway,holescanalsomovefreelyinthecrystallattice.
Bothconductionbandelectronsandvalencebandholescontributeto
electricalconductivity.Semiconductorsdopedwithdonorimpuritiesare
calledntype(nmeaningnegative),whilethosedopedwithacceptor
impuritiesareknownasptype(pmeaningpositive).
Quantummechanicaltheorypredictsthatelectronsinsemiconductors
canhaveenergiesonlywithincertainbandscorrespondingtoelectrons
tightlyboundtotheatomicnucleiofthematerial,andthefreeelectron
energy,whichistheenergyrequiredforanelectrontoescapeentirely
fromthematerial.Eachenergybandcorrespondstoalargenumberof
discretequantumstatesoftheelectrons.Mostofthestateswithlow
energy(closertothenucleus)arefilledwithelectrons,uptoaparticular
band,whichiscalledthevalenceband,andwhichisalsofilledwith
electrons.Semiconductorsandinsulatorsaredistinguishedfrommetals
becausethevalencebandinthesemiconductormaterialsisnearlyfilled
underusualoperatingconditions,thuscausingmoreelectronstobe
availableintheconductionband,whichisthebandimmediatelyabove
thevalenceband(andemptyatabsolutezerotemperature,0K).The
easewithwhichelectronsinasemiconductorcanbeexcitedfromthe
valencebandtotheconductionbanddependsonthebandgapbetween
thebands,anditisthesizeofthisenergybandgapthatservesasan
arbitrarydividingline(roughly4eV)betweensemiconductorsand
insulators(Figure24).

Figure24.Energybandsofelectronsinametal,
semiconductorandinsulator.TheFermilevelreferstothe
energyofthehighestoccupiedquantumstateinasystemof
fermionsatabsolutezerotemperature.Afermionisany
particlethathasanoddhalfinteger(like1/2,3/2,andso
forth)spin.[FromWikipedia:ElectronicBandStructiure]
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pnJunctions:Apnjunctionisajunctionformedbyjoiningptypeand
ntypesemiconductorstogetherinveryclosecontact(Figure25).The
termjunctionreferstotheboundaryinterfacewherethetworegionsof
thesemiconductormeet.Althoughtheycanbeconstructedoftwo
separatepieces,pnjunctionsaremoreoftencreatedinasinglecrystal
ofsemiconductorbygrowingalayerofcrystaldopedwithonetypeof
dopantontopofalayerofcrystaldopedwithanothertypeofdopant.

Figure25.pnJunctionsinsiliconinanopencircuit(top),
forwardbias(bottom,left),andreversebiascircuits(bottom,
right).[FromWikipedia:pnJunction]
Thepnjunctionpossessessomeinterestingproperties,whichhave
usefulapplicationsinmodernelectronics.Apdopedsemiconductoris
relativelyconductive.Thesameistrueofanndopedsemiconductor,but
thejunctionbetweenthemisanonconductor.Thisnonconductinglayer,
calledthedepletion(orspacecharge)region,arisesbecausethe
electricalchargecarriersindopedntypeandptypesemiconductors
(electronsandholes,respectively)attractandeliminateeachotherina
processcalledrecombination.Transportofchargecarrierscanoccurin
twoways.Onemodeoftransportisby'drift',whichiscausedbyan
electrostaticfieldacrossthedevice.Theothermodeoftransportisby
diffusionofcarriersfromzonesofhighcarrierconcentrationtozonesof
lowcarrierconcentration.Driftanddiffusionhaveoppositedirections.
Figure26givesaschematicpictureofapnjunction.Pnjunctionsare
essentiallyelectronicdiodesandareelementarybuildingblocksofalmost
alloptoelectronicdevicessuchasLEDs,diodelasers,solarcells,etc.

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Figure26.Apnjunctioninthermalequilibriumwithzerobias
voltageapplied.Blueandredlinesindicatetheconcentrationof
electronsandholes,respectively.Grayandwhiteregionshave
neutralcharges.Thelightredzoneispositivelycharged
(holes).Thelightbluezoneisnegativelycharged(electrons).
Thedirectionoftheelectricfieldisshownonthebottom.Also
shownaretheelectrostaticforceonelectronsandholes(drift),
andthedirectioninwhichthediffusiontendstomoveelectrons
andholes.[FromWikipedia:pnJunction]
LuminescenceofSemiconductors:Bytheabsorptionofaphoton,an
electroncanbepromotedfromthevalencebandintotheconduction
band.Electronsexcitedtotheconductionbandalsoleavebehindelectron
holesinthevalenceband.
Luminescencecanoriginatefromdirectlightabsorption,since
recombinationoftheexcitedelectronwiththeholeinthevalencebandis
accompaniedbylightemission.Luminescencecanalsobecreatedby
excitons.Anexcitonisanelectronholepair,boundbyCoulombforces,
withtheelectronintheconductionband,andtheholeinthevalence
band.Sinceanexcitonisaboundstateofanelectronandahole,the
overallchargeforthisquasiparticleiszero.Henceitcarriesnoelectric
current.Thewholeexcitoncanmovethroughthemolecularcrystal,and
impurities(socalledcarriertraps)aresometimesaddedtostabilizethe
excitonatoneposition.Withthisadditionalkineticenergytheexcitonic
energymaylieabovethebandgap.Thisextraenergycanbereleased
byradiation.Thislightemissioninsemiconductorsistakingplaceat
lowertemperature,whentheBoltzmannfactor(BT)islessthanthe
excitonbindingenergy.
InFigure27,wehaveredrawnthequantummechanicalbandschemeto
understandtheluminescenceofsemiconductors.

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Figure27.Bandschemeofsemiconductors,andthetwoways
oflightemission.
QuantumDots:Nanometersizedcrystalsofcertainsemiconductors,
suchascadmiumselenide(CdSe),haveinterestingluminescent
properties.Afterphotonabsorption,thewavelengthofluminescence
dependsonthenatureofthesemiconductormaterial,andonthesizeof
thecrystal.Thelightemissionisduetoaphenomenonknownas
quantumconfinement.Thereforethesenanocrystalsarecalled'quantum
dots'.Inquantumdots,electrons,holesandexcitonsareconfinedinall
threespatialdimensions.Thesmallerthequantumdotis,theshorteris
theemissionwavelength.ThecolorofluminescenceofCdSequantum
dotsvariesfromviolettored,whenthesizeincreasesfrom2nmto7
nm.Theemissioncolorofquantumdotscanbetunedfromthevisible
throughouttheinfraredbythecarefulchoiceofmaterialsandsizes
(Figure28).Thequantumyieldofluminescencecanbeincreasedby
coatingthequantumdotwithalayerofzincsulfide(ZnS),whichhasa
muchlargerbandgap(seeTable3),andfinallywithapolymer.This
protectivelayerwillreducenonradiativedecayprocessesresponsiblefor
thelowquantumyield.

Figure28.Luminescenceofvariousquantumdotsilluminated
withUVlight.
Overthepastdecade,quantumdotshavebeenusedintheconfocal
fluorescencemicroscopyofcells,therebyreplacingorganicfluorescent
dyes.Theimprovedphotostabilityofquantumdotsallowsacquisitionof
manyconsecutivetwodimensionalconfocalimagesthatcanbe
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reconstructedintoahighresolutionthreedimensionalconfocalimage.
Anotherapplicationtakingadvantageoftheirphotostabilityisthe
trackingofcertainbiologicalmoleculesattachedtoquantumdots
("biosensors")inlivingcellsandanimals(mice)overextendedperiodsof
time(evenmonths).Firstattemptshavebeenmadetousequantum
dotsfortumortargeting.Oneoftheremainingissueswithquantumdot
probesistheirinvivotoxicity,astheyhavetobeexcitedwithUVlight.
Quantumdotswithastablepolymercoatingseemtobeessentially
nontoxic.
LightEmittingDiode(LED):AnLEDconsistsofachipof
semiconductingmaterialdopedwithimpuritiestocreateapnjunction.
Whenthepnjunctionisforwardbiased(switchedon)(Figure25),
currentflowseasilyfromthepside(anode)tothenside(cathode),but
notinthereversedirection.Chargecarrierselectronsandholesflow
intothejunctionfromelectrodeswithdifferentvoltages.Whenan
electronmeetsahole,itfallsintoalowerenergylevel,andreleases
energyintheformofaphoton(Figure29).Thiseffectiscalled
electroluminescenceandthecolorofthelightisdeterminedbytheband
gapenergyofthematerialsformingthepnjunction.AnLEDisusually
smallinarea(lessthan1mm2),andintegratedopticalcomponentsare
usedtoshapeitsradiationpatternandassistinreflectiontoimprovethe
output(Figure29).

Figure29.Workingprinciple(left),andschematicsofanLED
(right).[FromWikipedia:LED]
LEDspresentmanyadvantagesoverotherlightsources,includinglower
energyconsumption,longerlifetime,smallersize,fasterswitchingand
greaterreliability.LEDdevelopmentbeganwithinfraredandreddevices
madewithgalliumarsenide(seeTable3).Advancesinmaterialscience
havemadepossibletheproductionofLEDshavingbandgapswith
energiescorrespondingtonearinfrared,visibleorultravioletlight.LEDs
madefromsiliconorgermaniumarenoteffective,sinceelectronsand
holesrecombinebyanonradiativetransition,producingnolight
emission.
DiodeLaser:Likemanyothersemiconductordevices,adiodelaseris
formedbydopingaverythinlayeronthesurfaceofacrystallinewafer.
Thecrystalisdopedtoproduceanntyperegionandaptyperegion,
oneabovetheother,resultinginapnjunction.Chargeinjection
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distinguishesdiodelasersfromallother,opticallypumpedlasers.
Thereforediodelasersarecalled"injectionlasers".Theinitialmodeof
actionisnotdifferentfromthatofanLED.Electronholerecombination
belowalaserthresholdmayresultinspontaneousemission,asinan
LED.Spontaneousemissionisnecessarytoinitiatelaseroscillation.In
theabsenceofstimulatedemission,electronsandholesmaycoexistin
proximitytooneanotherforacertaintimebeforetheyrecombine.This
characteristictimeistermedthe"recombinationtime"(abouta
nanosecondfortypicaldiodelasermaterials).Thenanearbyphotonwith
energyequaltotherecombinationenergycancauserecombinationby
stimulatedemission.Thisgeneratesanotherphotonofthesame
frequency,travelinginthesamedirection,withthesamepolarizationand
phaseasthefirstphoton.Thismeansthatstimulatedemissioncauses
gaininanopticalwaveintheinjectionregion.Thegainincreasesasthe
numberofelectronsandholesinjectedacrossthejunctionincreases.
Asinotherlasers,thegainregionissurroundedwithanopticalcavityto
formalaser.Inthesimplestformofadiodelaser,anopticalwaveguide
ismadeonthecrystallinewafer,suchthatthelightisconfinedtoa
relativelynarrowline.Thetwoendsofthecrystalarecleavedtoform
perfectlysmooth,paralleledges,forminganopticalresonator.Photons
emittedintoamodeofthewaveguidewilltravelalongthewaveguide,
andbereflectedseveraltimesfromeachendface(mirror)beforethey
areemitted.Asalightwavepassesthroughthecavity,itisamplifiedby
stimulatedemission,butlightisalsolostduetoabsorption,andby
incompletereflectionfromtheendmirrors.Finally,ifthereismore
amplificationthanloss,thediodebeginstoperformlaseraction.Dueto
diffraction,thebeamdivergesrapidlyinverticalandlateraldirections
afterleavingthechip.Alensmustbeusedinordertoformacollimated
beamlikethatproducedbyalaserpointer.
InFigure30,adiagramofadoubleheterostructurediodelaseris
shown.Inthisdevice,alayeroflowbandgapmaterialissandwiched
betweentwohighbandgaplayers.Onecommonlyusedpairofmaterials
isgalliumarsenide(GaAs)withaluminumdopedgalliumarsenide
(AlGaAs).Eachofthejunctionsbetweendifferentbandgapmaterialsis
calledaheterostructure,hencethename"doubleheterostructure"
laserorDHlaser.TheadvantageofaDHlaseristhattheactiveregion
wherefreeelectronsandholesexistsimultaneously,isconfinedtothe
thinmiddlelayerwithlowerbandgapenergy.Thismeansthatmany
moreoftheelectronholepairscancontributetoamplification.In
addition,lightisreflectedfromtheheterojunctionhence,thelightis
confinedtotheregionwheretheamplificationtakesplace.

Figure30.Diagramoffrontviewofadoubleheterostructure
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laserdiode(nottoscale).MaterialAis,forinstance,AlGaAs
(bandgap~2.8eV).MaterialBis,forinstance,GaAs(band
gap1.43eV).[FromWikipedia:DiodeLaser]
Therelativelylowcostofmassproduceddiodelasersmakesthem
attractiveforawiderangeofapplicationsindiverseareas.Thesmallsize
ofdiodelasersisalsoadvantageous.Diodelasersareusedinfiberoptics
communication,opticaldatarecordingandbarcodereaders.Redand
greenlasersarecommonaslaserpointers.Bothlowandhighpower
diodelasersareusedextensivelyintheprintingindustry,bothfor
scanningofimagesandforveryhighresolutionprintingplate
manufacturing.InfraredandreddiodelasersarecommoninCDandDVD
players.Diodelasersarealsoindispensablelightsourcesinconfocal
fluorescencemicroscopyandtimeresolvedfluorescencespectroscopy.
Highpowerdiodelasersareusedforpumpingother,solidstatelasers
likeTiSapphire.Medicalapplicationsinclude,amongothers,
photodynamictherapytophotoactivateporphyrinderivativesasanti
canceragents.
SolarCells:Asolarcellisadevicethatconvertstheenergyofsunlight
directlyintoelectricitybythephotovoltaiceffect.IncontrasttoLEDsand
diodelasersthatproducelight,solarcellsuselighttoproduceelectricity.
Anassemblyofsolarcellsmakesasolarpanel.Constructionofsolarcells
hasalonghistory,startinginthe1880s,andresultinginsemiconductor
(silicon)deviceswithasunlightenergyconversionefficiencyofaround
6%inthe1950s.Highlyeffectiveheterostructuresolarcellswere
createdfrom1970on,untilconversionefficiencieshavebeenreached
closeto40%.Acurrentchallengetobringdownthecostofsolarenergy
istoincreasethephotovoltaicefficiency.
Solarcellsareessentiallypnjunctionsunderillumination.Light
generateselectronsandholesonbothsidesofthejunction,inthentype
emitterandintheptypebase.Chargesbuilduponeithersideofthe
junctionandcreateanelectricfield.Thegeneratedelectrons(fromthe
base)andholes(fromtheemitter)thendiffusetothejunctionandare
sweptawaybytheelectricfield,thusproducingelectriccurrentacross
thedevice.Theelectriccurrentsoftheelectronsandholesreinforceeach
other,sincetheseparticlescarryoppositecharges.Thepnjunction,
therefore,separatesthecarrierswithoppositecharge,andtransforms
thegenerationcurrentbetweenthebandsintoanelectriccurrentacross
thepnjunction.(Figure31).

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Figure31.Photovoltaiceffectonpnjunctions.Panels1and2
areexplainedinFigure26.Inpanel3theelectricfieldsare
plotted.EF istheFermilevel.Inpanel4theactualcurrentis
generatedbylight.Notethatelectronsandholesmovein
oppositedirections.[FromWikipedia:SolarCells]
Ohmicmetalsemiconductorcontactsaremadetobothntypeandptype
sidesofthesolarcell(Figure32).

Figure32.Basicstructureofasiliconbasedsolarcell,andits
workingmechanism.Theelectrodesareconnectedtoan
externalresistanceload.[FromWikipedia:SolarCells]
Electronsthatarecreatedonthentypesideofthejunctionmaytravel
throughthewire,powertheload,andcontinuethroughthewireuntil
theyreachtheptypesemiconductormetalcontact(aluminum).Here
theyrecombinewithahole.Theholewaseithercreatedasanelectron
holepairontheptypesideofthesolarcellorsweptacrossthejunction
afterbeingcreatedatthenside.Thevoltagemeasuredisequaltothe
differenceinenergylevelsoftheminoritycarriers,i.e.,electronsinthe
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ntypeportionandholesintheptypeportion(V0inpanel4ofFigure
31).
SuggestedReading
Birks,J.B.,PhotophysicsofAromaticMolecules,WileyInterscience,
London,1970.
Cantor,C.R.andSchimmel,P.R.,BiophysicalChemistry,PartII:
TechniquesfortheStudyofBiologicalStructureandFunction,W.H.
Freeman,SanFrancisco,1980.
Engelborghs,Y.andVisser,A.J.W.G.Visser(eds.),Fluorescence
SpectroscopyandMicroscopy:MethodsandProtocols,Methodsin
MolecularBiology,vol.1076,SpringerScience+BusinessMedia,LLC,
2014.
Jameson,D.M.,IntroductiontoFluorescence,CRCPress,2014.
Lakowicz,J.R.,PrinciplesofFluorescenceSpectroscopy,3rded.,Springer,
NewYork,2006.
Saleh,B.E.A.andTeich,M.C.,FundamentalsofPhotonics,2nded.,John
Wiley&Sons,Hoboken,NewJersey,2007.
Steinfeld,J.I.,MoleculesandRadiation.AnIntroductiontoModern
MolecularSpectroscopy,Harper&RowPublishers,NewYork,1974.
Valeur,B.andBerberanSantos,M.N.,MolecularFluorescence,2nded.,
WileyVCH,Weinheim,2012.
Manytopicstreatedinthismodulecanbefoundin:Wikipedia.
Averyusefulreviewoncellularapplicationsofquantumdotscanbe
foundin:Michalet,X.,Pinaud,F.F.,Bentolila,L.A.,Tsay,J.M.,Doose,S.,
Li,J.J.,Sundaresan,G.,Wu,A.M.,Gambhir,S.S.andWeiss,S.,Quantum
DotsforLiveCells,inVivoImaging,andDiagnostics,Science307,538
544,2005.
Fluorescencemicroscopy,andhistorycanbefoundin:Microscopy.
Awideselectionoffluorescentprobescanbefoundin:MolecularProbes.
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