FLOURESENCE

OUTLINE

q FLUORESENCEq QUANTUMYIELDOFFLUORESCENCEq FLUORESCENCELIFETIMEq FLUORESCENCEINNATUREq APPLICATIONSOFFLUORESCENCEq GREENFLUORESCENTPROTEIN

FLUORESENCEq  Fluorescenceistheemissionoflightbyasubstancethathas

absorbedlightorotherelectromagneFcradiaFon.q  TheemiHedlighthasalongerwavelength-thereforelower

energythantheabsorbedradiaFon.q  Themoststrikingexampleoffluorescenceoccurswhen:

q  TheabsorbedradiaFonisinultravioletregionofspectrum-whiletheemiHedlightisinthevisibleregion-whichgivesthefluorescentsubstanceadisFnctcolor.

q  FluorescentmaterialsceasetoglowimmediatelywhentheradiaFon

sourcestops.

Fluorophores:aremoleculesthatabsorblightorEMradiaFonandemitlightDifferentfluorophoresabsorbdifferentwavelengthsofoflight–EachfluorophorehasspecificexcitaFon(absorpFon)spectrumItalsohasaspecificemissionspectrum.

FLUORESENCEFluorescenceoccurswhenanorbitalelectronofamoleculeoratom-relaxestoitsgroundstatebyemiVngaphotonfromanexcitedsingletstate.Withh-Plank’sconstantandν-frequencyoflightS0isthegroundstateofflourecentmoleculeS1isthefirstexcitedstate

ThespecificfrequenciesofexcitaFonandemiHedlightaredependentontheparFcularsystem.

ExcitaFon

Fluorescence(emission)

FLUORESENCE

q  AmoleculeinS1canrelaxbyvariouscompeFngpathways:

Non-Radia)ve : In this relaxaFon theexcitaFonenergy isdissipatedasheatorvibraFonstothesolvent

Phosphorescence:Excitedorganicmoleculescanalsorelaxviaconversion

toatripletstate-whichmaysubsequentlyrelaxviaphosphorescence - or

byasecondarynon-radiaFverelaxaFonstep.

Fluorescence quenching: RelaxaFon from S1 can also occur through

interacFonwithasecondmoleculethroughfluorescencequenching-

Quenchingreferstoanyprocesswhichdecreasesthefluorescenceintensity

ofagivensubstance

Atripletisaquantumstateofasystemwithaspinof1.

FLUORESENCE

q Stokesshi>:isthedifferencebetweenposiFonsofthebandmaximaoftheabsorpFonandemissionspectraofthe

sameelectronictransiFon.

q RESONANCEFLUORESCENCE:IftheemiHedradiaFonhave

thesamewavelengthastheabsorbedradiaFon.

FLUORESENCE

QUANTUMYEILDOFFLUORESCENCE

q  Quantumyieldgivestheefficiencyofthefluorescenceprocess.

q  ItisdefinedastheraFoofthenumberofphotonsemiHedtothe

numberofphotonsabsorbed.

q  Themaximumfluorescencequantumyieldis1.0(100%)-each

photonabsorbedresultsinaphotonemiHed.

q  Compoundswithquantumyieldsof0.10aresFllconsideredquite

fluorescent.

QUANTUMYEILDOFFLUORESCENCE

q  Theremanyprocessesthateffectsthequantumyield

q Dynamiccollisionalquenching

q Resonanceenergytransfer:amechanismdescribingenergy

transferbetweentwolight-sensiFvemolecules

q Internalconversion-isnon-radiaFveandoccursbetweenrotaFonalandvibraFonallevelsofmolecule

q Intersystemcrossing-betweensinglettotripletstatesand

viceversa

FLUORESCENCELIFETIMEq The fluorescence lifeFme refers to the average Fme the

moleculestaysinitsexcitedstatebeforeemiVngaphoton

q  Fluorescencetypicallyfollowsfirst-orderkineFcs:

[S1]istheconcentraFonofexcitedstatemoleculesatFmet

[S1]0istheiniFalconcentraFon

ГisthedecayrateortheinverseofthefluorescencelifeFme

FLUORESCENCELIFETIMEq  VariousradiaFveandnon-radiaFveprocessescande-populate

theexcitedstate.q  Insuchcasethetotaldecayrateisthesumoverallrates:ГtotisthetotaldecayrateГradtheradiaFvedecayrateГnradthenon-radiaFvedecayrateIftherateofspontaneousemission-oranyoftheotherratesare

fast-thelifeFmeisshort.

FLUORESCENCELIFETIME

q  Forcommonlyusedfluorescentcompounds-typicalexcited

state decay Fmes for photon emissions with energies from

the UV to near infrared are within the range of 0.5 to 20

nanoseconds

q  The fluorescence lifeFme is an important parameter for

pracFcal applicaFons of fluorescence such as fluorescence

resonance energy transfer and Fluorescence-life)me

imagingmicroscopy.

FLOURESENCE

q Aeeranelectronabsorbsahighenergy photon the system is

excited electronical ly and

vibraFonally.

q  T h e s y s t e m r e l a x e s

vibraFonally, and eventually

fl u o r e s c e s a t a l o n g e r

wavelength.

RULESq  ThereareseveralgeneralrulesthatdealwithfluorescenceKasha–Vavilovrule:

§  TheKasha–Vavilov ruledictates that thequantumyieldof fluorescence is independent of the wavelength ofexciFngradiaFon.

§  The Kasha–Vavilov rule does not always apply and isviolatedinmanysimplemolecules

§  ThefluorescencespectrumshowsveryliHledependenceonthewavelengthofexciFngradiaFon.

Mirrorimagerule:

§  FormanyfluorophorestheabsorpFonspectrumisamirror

imageoftheemissionspectrum

FLUORESCENCEINNATUREq  Therearemanynaturalcompoundsthatexhibitfluorescence-and

theyhaveanumberofapplicaFons

q Biofluorescence

q AquaFcbiofluorescence

q AbioFcfluorescence

q  Biofluorescence - is theabsorpFonofelectromagneFcwavelengths

from the visible light spectrum by fluorescent proteins in a living

organism-andthere-emissionofthatlightatalowerenergylevel.

q  Thiscausesthelightthatisre-emiHedtobeadifferentcolorthanthe

lightthatisabsorbed

Biofluorescence q SFmulaFnglightexcitesanelectron,raisingenergytoan

unstablelevel.q  Thisinstabilityisunfavorable-sotheenergizedelectronis

returnedtoastablestatealmostasimmediatelyasitbecomesunstable

q Thisreturntostabilitycorrespondswiththereleaseofexcessenergyintheformoffluorescentlight.

q  ThisemissionoflightisonlyobservablewhenthesFmulantlightissFllprovidinglighttotheorganism/object.

Biofluorescenceq Chromatophores:arepigment-containingandlight-reflecFng

cells-orgroupsofcells

q  Fluorescentchromatophore:PigmentcellsthatexhibitfluorescenceandfuncFonsomaFcallyaresimilartoregularchromatophores

q Fluorescentchromatophores:canbefoundintheskin-e.g.in

fish-justbelowtheepidermis-amongstotherchromatophores.

AQUATICBIOFLOURESCENCEq  Waterabsorbs lightof longwavelengths– so less light from

thesewavelengthsreflectsbacktoreachtheeyeq  Therefore-warmcolors fromthevisual lightspectrumappear

lessvibrantatincreasingdepths.q  Water scaHers light of shorter wavelengths- meaning cooler

colorsdominatethevisualfieldinthephoFczone.q  Lightintensitydecreases10foldwithevery75mofdepth_so

atdepthsof75m-lightis10%asintenseasitisonthesurface-andisonly1%asintenseat150masitisonthesurface.

ThephoFczoneisthedepthofwaterwherealmostallofthephotosynthesisoccursandabout90%ofallmarinelifelivesinthiszone

AQUATICBIOFLOURESCENCEq As any type of fluorescence depends on the presence of

external sourcesof light -biologically funcFonalfluorescenceisfoundinthephoFczone-wherethere isnotonlyenoughlight to cause biofluorescence - but enough light for otherorganismstodetectit.

q  ThevisualfieldinthephoFczoneisnaturallyblue,socolors

of fluorescence can be detected as bright reds, oranges,yellows,andgreens.

ABIOTICFLUORESCENCEq  Gemstones,minerals,mayhaveadisFncFvefluorescenceor

mayfluorescedifferentlyundershort-waveultraviolet,long-waveultraviolet,visiblelight,orX-rays.

q  Crudeoil(petroleum)fluorescesinarangeofcolors,fromdull-brownforheavyoilsandtarsthroughtobright-yellowishandbluish-whiteforverylightoilsandcondensates.

q  OrganicsoluFonssuchanthraceneorsFlbene,dissolvedinbenzeneortoluene,fluorescewithultravioletorgammarayirradiaFon.

q  FluorescenceisobservedintheatmospherewhentheairisunderenergeFcelectronbombardment.

fluorescentminerals

ABIOTICFLUORESCENCEq  Incasessuchasthenaturalaurora,high-alFtudenuclear

explosions,androcket-borneelectrongunexperiments,themoleculesandionsformedhaveafluorescentresponsetolight.

q  VitaminB2fluorescesyellowq  Tonicwaterfluorescesblueduetothepresenceofquinine.q  Highlighterinkisoeenfluorescentduetothepresenceof

pyranineq  Banknotes,postagestampsandcreditcardsoeenhave

fluorescentsecurityfeatures.

APPLICATIONSOFFLUORESCENCEq  LighFng

q Thecommonfluorescentlampreliesonfluorescence_containsacoaFngofafluorescentmaterial-calledthephosphor-whichabsorbstheultravioletandre-emitsvisiblelight.

q FluorescentlighFngismoreenergy-efficientthanincandescentlighFngelements.

APPLICATIONSOFFLUORESCENCESPECTROSCOPYq  Usuallythesetupofafluorescenceassayinvolvesalightsource–

whichemitmanydifferentwavelengthsoflight.q Asinglewavelengthisrequiredforproperanalysis–lightispassed

throughanexcitaFonmono-chromator-andthenthatchosenwavelengthispassedthroughthesamplecell

q AeerabsorpFonandre-emissionoftheenergy-manywavelengthsmayemergeduetoStokesshie.

q Toseparateandanalyzethem-thefluorescentradiaFonispassedthroughanemissionmonochromator-andobservedselecFvelybyadetector

APPLICATIONSOFFLUORESCENCEq  Biochemistryandmedicine

q  Fluorescenceinthelifesciencesisusedgenerallyasanon-destrucFveway of tracking or analysis of biological molecules bymeans of thefluorescent emission at a specific frequency where there is nobackground from the excitaFon light - as relaFvely few cellularcomponentsarenaturallyfluorescent

q  A protein or other component can be – labelled with an extrinsicfluorophore-afluorescentdyethatcanbeasmallmolecule,protein,orquantumdot

APPLICATIONSOFFLUORESCENCEq  Microscopy

q When scanning the fluorescent intensity across a planeone has fluorescence microscopy of Fssues, cells, orsubcellularstructures_

q That can be done by labeling an anFbody with afluorophore and allowing the anFbody to find its targetanFgenwithinthesample.

q  LabellingmulFpleanFbodieswithdifferentfluorophoresallows visualizaFon of mulFple targets within a singleimage(mulFplechannels).

APPLICATIONSOFFLUORESCENCEFLIM:FluorescenceLifeFmeImagingMicroscopy_canbeusedtodetectcertain bio-molecular interacFons that manifest themselves byinfluencingfluorescencelifeFmes.Cell and molecular biology: detecFon of colocalizaFon usingfluorescence-labelledanFbodiesforselecFvedetecFonoftheanFgensofinterestusingspecializedsoeware,suchasCoLocalizerPro.

FluorescentpaintlitbyUVtubes

GREENFLUORESCENTPROTEINq  Thegreenfluorescentprotein(GFP)isaproteincomposedof

238aminoacidresiduesthatexhibitsbrightgreenfluorescencewhenexposedtolightinthebluetoultravioletrange

q  Althoughmanyothermarineorganismshavesimilargreenfluorescentproteins,GFPtradiFonallyreferstotheproteinfirstisolatedfromthejellyfishAequoreavictoria

q  TheGFPfromA.victoriahasamajorexcitaFonpeakatawavelengthof395nmandaminoroneat475nm.

q  Itsemissionpeakisat509nm,whichisinthelowergreenporFonofthevisiblespectrum

q  Thefluorescencequantumyield(QY)ofGFPis0.79.

GREENFLUORESCENTPROTEIN

Aequoreavictoria

GREENFLUORESCENTPROTEINq  GFPmakesanexcellenttoolinmanyformsofbiologydueto

itsabilitytoforminternalchromophoreq  Incellandmolecularbiology-theGFPgeneisfrequently

usedasareporterofexpressionq  Ithasbeenusedinmodifiedformstomakebiosensors

q ManyanimalshavebeencreatedthatexpressGFP

GREENFLUORESCENTPROTEINq  GFPcanbeintroducedintoanimalsorotherspeciesthrough

transgenictechniquesandmaintainedintheirgenomeandthatoftheiroffspring.

q  ScienFstsRogerY.Tsien,OsamuShimomura,andMarFnChalfiewereawardedthe2008NobelPrizeinChemistryon10October2008fortheirdiscoveryanddevelopmentofthegreenfluorescentprotein.

GREENFLUORESCENTPROTEINq  GFPiscomposedof238aminoacidsq  Eachmonomercomposedofacentralα-helixsurroundedby

anelevenstrandedcylinderofanF-parallelβ-sheetsq  Cylinderhasadiameterofabout30Aandisabout40Alongq  Fluorophorelocatedoncentralhelix

TheAcBveSite

GREENFLUORESCENTPROTEIN

q The Fluoropore Active Site

q  Ser65-Tyr66-Gly67

q  Deprotonated phenolate

of Tyr66 is cause of

fluorescence

q  Forster Cycle (1949-

Theodor Forster)

q  Proton transfer to His148

GREENFLUORESCENTPROTEIN

q Fluorophore formation

q  One limitation of GFP is its slow rate of fluorescence

acquisition in vivo

q  Renaturation most likely by a parallel pathway

q  Oxidation of Fluoropore (2-4 hours)

q  Two step process

GREENFLUORESCENTPROTEIN

GREENFLUORESCENTPROTEIN

GREENFLUORESCENTPROTEIN

ADVANTAGESOFGFPq  ThebiggestadvantageofGFPisthatitcanbeheritable_

dependingonhowitwasintroduced-allowingforconFnuedstudyofcellsandFssues.

q  VisualizingGFPisnoninvasive-requiringonlyilluminaFonwithbluelight.

q  GFPalonedoesnotinterferewithbiologicalprocesses,butwhenfusedtoproteinsofinterest-carefuldesignoflinkersisrequiredtomaintainthefuncFonoftheproteinofinterest.

APPLICATIONSOFGFPq  Fluorescencemicroscopy

q TheavailabilityofGFPanditsderivaFveshasthoroughlyredefinedfluorescencemicroscopyandtheway it isusedincellbiologyandotherbiologicaldisciplines

q  While most small fluorescent molecules such as FITC(fluorescein isothiocyanate)arestronglyphototoxicwhenused in live cells, fluorescent proteins such as GFP areusuallymuchlessharmfulwhenilluminatedinlivingcells.

q Thishastriggeredthedevelopmentofhighlyautomatedlive-cell fluorescencemicroscopy systems_which canbeused to observe cells over Fme expressing one or moreproteinstaggedwithfluorescentproteins.

APPLICATIONSOFGFP

MiceexpressingGFPunderUVlight(leJ&right),comparedtonormalmouse(center)

APPLICATIONSOFGFPq  Transgenicpets

q  Alba_agreen-fluorescentrabbit_wascreatedbyaFrenchlaboratoryusingGFPforpurposesofartandsocialcommentary

q  TheUScompanyYorktownTechnologiesmarketstoaquariumshopsgreenfluorescentzebrafish(GloFish)thatwereiniFallydevelopedtodetectpolluFoninwaterways.

q  NeonPets-aUS-basedcompanyhasmarketedgreenfluorescentmicetothepetindustryasNeonMice.

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