BasicPrinciplesofFluorescence

Champaign,2012

BenBarbieri

WhatisFluorescence?

FLUORESCENCE isthelightemittedbyanatomormoleculeafterafinite durationsubsequenttotheabsorptionofphotons. Specifically,theemitted lightarisesfromthetransitionoftheexcitedspeciesfromitsfirstexcited electronicsingletleveltoitsgroundelectroniclevel.

WhatisFluorescence?

Thedevelopmentofhighlysophisticatedfluorescentprobechemistries,new lasersandmicroscopyapproachesandsitedirectedmutagenesishasledto manynovelapplicationsoffluorescenceinthechemical,physicalandlife sciences.Fluorescencemethodologiesarenowwidelyusedinthe biochemicalandbiophysicalareas,inclinicalchemistryanddiagnosticsand incellbiologyandmolecularbiology.

CommonFluorophores

Fluorescein

RhodamineB

Quinine

Tryptophan

POPOP

WhyFluorescence?

Itprovides:informationonthemolecularenvironment.
informationondynamicprocessesonthenanosecondtimescale

WhyFluorescence?

temperature

electric fields

viscosity ions

Fluorescent Probe

pH

polarity

WhyFluorescence?

Inrecentyears,duetothe availabilityofmultiphoton lasersandGFPprobes, fluorescencehasbeen successfullyusedtomonitor processesatthecellularlevel withdetailedspatial resolution.

WhyFluorescence?

Also,fluorescenceisveryverysensitive! Applicationstosolutionswithsubnanomolarconcentrationsis fairlycommon.Withproperinstrumentation

Singlemoleculedetection
hasbecomealmostroutine

SystemsaccessiblebyFluorescenceTechniques
Molecularstructure Cellstudies

Liveanimals

Microarrays

Instrumentation

Spectrofluorometers
(courtesy of ISS)

Confocal microscopes
(courtesy of Carl Zeiss)

Instrumentation

FlowCytometry
(courtesyofICyt)

GenomeSequencing
(courtesyofPacificBiosciences)

Instrumentation

ImmunoassayChemistryAnalyzer
(courtesyofAbbottDiagnostics)

MicrowellPlateReaders
(courtesyofMolecularDevices)

Thefirstrecordsoffluorescence

NicolsMonardes, aSpanish physicianandbotanistpublishes in1565theHistoriamedicinal delascosasquesetraende nuestrasIndiasOccidentales in whichhedescribesthebluish opalescenceofthewater infusionfromthewoodofa smallMexicantree. Whenmadeintocupsandfilled withwater,apeculiarbluetinge wasobserved.

Whatisthebluecolor?

AnearlyLatintranslation(1574)ofMonardeswork bytheinfluentialFlemishbotanistCharlesde Lcluse (15261609),inwhichthewoodsnameis givenasLignumNephriticum(kidneywood),helped toextendawarenessofitsstrangeoptical propertiesinEurope.Thiswoodwasverypopularin XVI XVIIEurope,becauseofitsmedicinalvirtues fortreatingkidneyailments. AnEnglishman,JohnFrampton,translatedMonardesdescriptionin 1577as..white woodde which gives a blewe color whenplacedin waterthatwasgoodfor them that doeth not pisse liberally and for the
pains of the Raines of the stone..

FluorescenceFluorescence

RobertBoyle(1664)wasinspiredbyMonardes reportandinvestigatedthissystemmorefully. Hediscoveredthataftermanyinfusionsthewood lostitspowertogivecolortothewaterand concludedthattherewassomeessentialsaltin thewoodresponsiblefortheeffect.Healso discoveredthatadditionofacidabolishedthecolor andthatadditionofalkalibroughtitback.

Epipolicdispersion

JohnHerschel(1845)madethefirstobservationof fluorescencefromquininesulfate hetermedthis phenomenonepipolicdispersion.

Stokesexperiment

Stokesusedaprismtodispersethesolarspectrumandilluminate asolutionofquinine.Henotedthattherewasnoeffectuntilthe solutionwasplacedintheultravioletregionofthespectrum.

Understandingthephenomenon

GeorgeGabrielStokes(1852)publishedhismassivetreatiseOnthe ChangeofRefrangibilityofLight morethan100pages.Heinitially usedthetermdispersivereflectiontodescribethephenomenon presentedbyquininesulphate.

Stokesshift

ThisobservationsledStokestoproclaimthatfluorescenceisof longerwavelengththantheexcitinglight,whichledtothis displacementbeingcalledtheStokesShift. Healsoseemstohavebeenthefirsttopropose,in1864,theuseof fluorescenceasananalyticaltool,inalecture"Ontheapplicationof theopticalpropertiestodetectionanddiscriminationoforganic substances.

ModernFluorescence

1905E.NicholsandE.Merrit:firstexcitationspectrumofadye 1919SternandVolmer:fluorescencequenching 1923S.J.VavilovandW.L.Levshin:fluorescencepolarizationofdyes 1924S.J.Vavilov:firstdeterminationoffluorescenceyield 1925F.Perrin:theoryoffluorescencepolarization 1926E.Gaviola:firstdirectmeasurementofnanosecondlifetime 1935A.Jablonski:diagram 1948T.Frster:QMtheoryofdipoledipoleinteraction

WhataretheParametersmeasuredbyFluorescence?

1.Thefluorescenceemissionspectrum 2.Theexcitationspectrumofthefluorescence 3.Thequantumyield 4.Thefluorescencelifetime 5.Thepolarization(anisotropy)oftheemission

PerrinJablonskidiagram

PerrinJablonskidiagram

Theexcitationspectrum
Inrecordinganexcitationspectrum,oneobservestheintensityofemissionata fixedwavelengthwhilescanningtheexcitation

Theexcitationspectrumshould matchtheabsorptionspectrum: companiesprovidetechnical correctionstothedata

Theemissionspectrum
Inrecordinganemissionspectrum,onekeepstheexcitationat afixedwavelengthandwhilescanningtheemission.

Somerulesaboutspectra
1)Thefluorescencespectrumliesatlongerwavelengthsthanthe absorption(Stokesshift) 2)Thefluorescencespectrumis,toagoodapproximation,amirrorimage oftheabsorptionbandofleastfrequency. 3)Thefluorescencespectrumisinvariant,remainingthesameindependent oftheexcitationwavelength

Quantumyield

ThefluorescenceQYisthefractionofexcitedmoleculesthatreturntothe groundstatewithemissionofphotons.

kR QY = k R + k NR
Anotherwayofthinkingaboutthisparameteris:

no. of photons emitted QY = no. of photons absorbed

Listofquantumyields[fromMolecularFluorescencebyB.Valeur]

Lifetime

Absorptionandemissionprocessesareconceptsthatinvolveapopulationof N1 isthepopulationoftheexcitedlevel S1 ,the molecules.Ingeneral,if populationisdescribedbytherelation:

d N1 = (k R + k NR ) N1 + f (t ) dt
t

N1 = N1 (0) e
S
isthelifetimeofexcitedstate

S =

1 k R + k NR

S1

If a population of fluorophores is excited at time t=0, after a time the number of molecules in is decreased to 1/e or to about 36.8%

Quantumyieldandlifetime

ThefluorescenceQYisthefractionofexcitedmoleculesthatreturntothe groundstatewithemissionofphotons.

kR QY = k R + k NR

S = R

Canthelifetimebecalculated?
Knowledgeofafluorophoresexcitedstatelifetimeiscrucialforquantitative interpretationsofnumerousfluorescencemeasurementssuchasquenching, polarizationandFRET. Inmostcasesofinterest,itisvirtuallyimpossibletopredictapriori theexcited statelifetimeofafluorescentmolecule.Theradiativelefetime,i.e.,thelifetime oneexpectsintheabsenceofanyexcitedstatedeactivationprocesses canbe approximatedbytheStricklerBergequation(J.Chem.Phys.37:814,1962).

8 i230cn 2 F ( F ) d F = 3 R N F F ( F ) d F 1

( A ) d A A

is the fluorescence emission the extinction coefficient the wavenumber

Lifetimesofsomearomatichydrocarbonsinethanol

Naphtalene

2.7ns

Anthracene

5.1ns

Perylene

4.3ns

Pyrene

410ns

Lifetimeandtheenvironment
Thelifetimeandquantumyieldforagivenfluorophoreareoftendramatically affectedbyitsenvironment. ANSinwateris~100picosecondsbutcanbe8 10nsboundtoproteins Ethidium bromideis1.8nsinwater,22ns boundtoDNAand27nsboundtotRNA

Thelifetimeoftryptophaninproteinsrangesfrom ~0.1nsto~10ns

Twowaystomeasurelifetime
Time domain Frequency domain

I (t ) = I 0 e

P =

tan

M =

1 1 2 m

TimeCorrelatedSinglePhotonCounting

(courtesy of Becker and Hickl)

AnalogFrequencyDomain
f
frequency synthesizer 1 light source sample

phase lock

frequency synthesizer 2

f + f
light detector

ISSoffersbothmethodologies

ChronosFD and ChronosBH

ISSoffersbothmethodologies

UVMeasurements

280 nm pulsed LED WG 380 LP filter

= 3.0 ns

300 nm cw LED WG 320 LP filter

= 3.1 ns

Picosecondsstandard
473nmpulsedlaserdiode 515nmLPfilter

= 77 ps

471nmcwlaserdiode OG530LPfilter

= 78 ps

TimeResolvedAnisotropy
447nmpulsedlaserdiode KV505LPfilter;T=20C

= 2.30 ns
= 2.6 ns R0 = 0.38

473nmcwlaserdiode WG499LPfilter;T=27C

= 2.33 ns
= 2.0 ns R0 = 0.38

DataAnalysis
Timedomain

=
2

k =1

[ N (tk ) N c (tk )]

2 k

k =1

[ N (tk ) N c (tk )]
N (tk )

Frequencydomain
N N M M c 1 2 c = + j =1 M j =1

In FRET the Acceptor Shortens the Donors Lifetime

AssayscanbedesignedwithLifetimeReadout

A
Longer Shorter

A D

InFRETtheAcceptorShortensthe DonorsLifetime

CompetitiveEnergyTransferImmunoassay

Antibody AntigenComplex
donor-labelled antibody antigen acceptor-labelled antigen

FluorescentDonor NonfluorescentAcceptor NoNeedtoSeparateDandASignals LifetimeindependentofVolume, Color Quenching,etc

(Dbound) < (Dfree)

BiosensorSystem

ELECTRONIC INTERFACE

INSTRUM USER INTERFACE DISPLAY CONTROL

ANALYTE SIEVE

SENSOR

vandeVen et al. 2002; 5th Intl Weber Symposium

DetectionofPicomolarfreeCU(II)Inseawater

TheRVKnorristheresearch vesselownedbytheU.S. Navyandoperatedbythe WoodsHoleOceanographic Institution.

ChronosFD 660nm laser diode 250m-long fiber

Courtesy of Dr. Richard Thompson

DetectionofPicomolarfreeCU(II)Inseawater

ChronosFD 660nm laser diode 250m-long fiber

Courtesy of Dr. Richard Thompson

FluorescencePolarization(Anisotropy)
Lightisanelectromagneticwave. Theelectricfield E andthemagneticfield B oscillateperpendicularlytothe directionofpropagation.

Electric field component

Direction of Propagation

Magnetic Field Component

NaturalLight

Unpolarized Light

Polarization

Unpolarized Light

Polarizer

Plane Polarized Light

(1) dichroicdevices,whichoperatebyeffectivelyabsorbingoneplaneofpolarization (e.g.,PolaroidtypeHsheetsbasedonstretchedpolyvinylalcoholimpregnatedwith iodine) (2) CaCO3 crystalpolarizers whichdifferentiallydispersethetwoplanesofpolarization

Photoselection
Photoselection:Whenapopulationoffluorophoresisilluminatedbyalinearly polarizedincidentlight,thefluorophoreswiththetransitionmomentsorientedin adirectionclosetothatoftheelectricfieldarepreferentiallyexcited.

no absorption

S0 S1
max absorption

S0 S 2

cos 2

Polarization
Z

Light source

Detector

Anisotropy

P=

I I I + I

r=

I I I + 2I

r=

2P 3 P

1 P 1

0.5 r 1

r = i f i ri

Insolutiontheselimits(e.g.,+/1)arenotrealized. Consider,asshownbelow,fluorophoresattheoriginof ourcoordinatesystem

Anisotropy
LetusconsiderapopulationofNmoleculesexcitedattime0bya shortpulseoflightpolarizedalongz.Attimet,theemission transitionmomentsME haveacertainangulardistribution. Therelationbetweentheemissionanisotropyandtheangular distributionoftheemissiontransitionmomentsis:
E

ME

2 3 < cos E (t )> 1 r= = I + 2I 2

I I

Parallelabsorptionandemissionmoments: FundamentalAnisotropy

Theabsorptionandemissionmomentsareparallel(excitationtothe firstsingletstate.Inthiscase:

3 < cos > = 5

2

2 r0 = = 0.4 5

Thisisthetheoreticalanisotropyintheabsenceofanymotion.The experimentalvalue,calledlimitinganisotropy,isalwaysalittlesmaller thanthetheoreticalvalue.

Nonparallelabsorptionandemissionmoments

Thissituationoccurswhentheexcitationbringsthefluorophoretoanexcited statehigherthanS1

3 < cos 2 A > 1 3 < cos 2 > 1 2 3 < cos 2 > 1 = r0 = 2 2 5 2

Where istheanglebetweentheabsorptionandemissionmoments

0.2 r0 0.4

Anisotropy

EffectofBrownianmotion
3 <cos2 (t )> 1 r (t ) = r0 2
Isotropic molecules r (t ) = r0 exp ( 6 D t ) a. Timeresolved b. Steadystatepolarizationmeasurements.Forsingle decay: 1 r = r (t ) exp (6 D t ) dt 0

RT with D = 6 V

1 1 1 = (1 + 6 D ) = 1 + (Perrinequation) r0 C r r0

EffectofBrownianmotion

Anisotropicrotators Hinderedrotators

r (t ) = r0 i exp ( t ci )
r (t ) = ( r0 r ) exp (t c ) + r
i

Someapplicationsofpolarization
(fromMolecularFluorescencebyB.Valeur)

Spectroscopy Polymers

Separationofexcitedstates Localviscosity Molecularorientation Chaindynamics Antigenantibodyreactions immunoassays Proteinsinteractions,denaturation DNAproteininteractions Nucleicacids Biologicalmembranes(fluidity,additives,..) Micellarsystems(microviscosity,..)

Immunology

Molecularbiology

Acknowledgements

Iamgreatlyindebtedforthispresentationto:
Prof.EnricoGratton UniversityofCaliforniaatIrvine Prof.DavidJamesonUniversityofHawaii

andtomyColleagues:
Dr.EugenePovrozin Dr.ShiChuLiao Mr.MarkParsons

PLEASEVISITUS W W W. I S S . C O M