TopicscoveredThenatureoflightandcolourColourdetectioninthehumaneyeThephysicalbasisoffluorescenceFluorescentprobesanddyesDyesthatbindorganellesChemicalDyesFluorescentproteinsPhotobleachingandQuenchingTheNatureofLightTheenergyoflightiscontainedindiscreteunitsorquantaknownasphotonsLightisaformofelectromagneticradiationPhotonshavethepropertyofbothparticlesandwavesForsimplicity,usuallyonlytheelectricalcomponentisdrawnLightasawave:Thenatureoflightandcolour-1TheElectromagneticSpectrumWavelengths400nm-750nmarevisibletothehumaneyeThenatureoflightandcolour-2TheHumanEye

Sensitivity Peaksensitivityisat555nm(yellow-green) Inbrightlight,3ordersofmagnitude Aftertimetoaccommodate,10ordersofmagnitude!Resolution ~0.1mmforanobject25mmfromtheeyeComposedofRodandConecellsCandetectdifferencesinlightintensityandwavelength(colour)Colourdetectioninthehumaneye-1Rodcellphotoreceptorscomprise95%ofphotoreceptorsintheretinaactiveindimlightbutprovidenocoloursensepeaksensitivityat510nm(blue-green)containRhodopsinBrightlighttemporarilybleachesRhodopsin (20-30minrecoverytime)BesthighvisualsensitivityinadarkenedroomRetinalColourdetectioninthehumaneye-2Conecellphotoreceptorscompriseonly~5%ofphotoreceptorsintheretinacontainednearlyexclusivelyinfovea(0.5mmspot)3types:red,greenandblueActionspectradifferforthedifferentconecellsColourdetectioninthehumaneye-3Positiveandnegativecolours

Positivecoloursaregeneratedbycombiningdifferentcolourwavelengths-->Yellowperceivedbystimulatingredandgreenconesindividuallywith2differentwavelengthsNegativecoloursaregeneratedbythesubtraction(absorption)oflightofaspecificwavelengthfromlightcomposedofamixtureofwavelengths-->YellowperceivedbecauseasinglewavelengthstimulatesbothredandgreenconesColourdetectioninthehumaneye-4FluorescenceOccursfollowingexcitationofafluorescentmoleculeuponabsorptionofaphotonEnergyisreleasedaslightasthemoleculedecaystoitsgroundstateThephysicalbasisoffluorescence-1absorptionEmissionTypicalfluorochrome:100,000cyclespersecondfor0.1-1secondsexcitationenergyloss(rapid10-9-10-12s)excitedstatesgroundstateemittedlight

(longerwavelength)JablonskidiagramFluorochrome“amoleculethatiscapableoffluorescing”ExcitationandEmissionSpectraStoke’sshiftForFITC(fluorescein-5-isothiocyanate)coupledtoIgGwavelengthThephysicalbasisoffluorescence-2FiltersetemissionexcitationdichromaticmirrorFITCfilterset(Chroma)LightinTodetector(eyepiece/ camera)toobjectiveEmissionintensitydependsontheexcitationwavelengthThephysicalbasisoffluorescence-3PropertiesoffluorophoresStokesshift-differencebetweenexcitationandemissionmaxima(largeadvantageous)

Molarextinctioncoefficient-potentialofafluorophoretoabsorbphotons

Quantumefficiency(QE)offluorescenceemission-fractionofabsorbedphotonsthatarere-emitted

Quantumyield-howmanyphotonsemittedbyafluorophorebeforeitisirreversiblydamagedQuenching-quantumyield(butnotemissionspectrum)alteredbyinteractionswithothermolecules

Photobleaching-permanentlossoffluorescencebyphoton-inducedchemicaldamageFluorescentprobesanddyes-1ChoiceofFluorophorewilldependontheapplicationProteinlocalization(ImmunofluorescencemicroscopyorGFP-tagging).organellemarking(e.g.DAPItolabelnucleus)proteindynamics(FRAP)proteininteractions(FRET)ionconcentration(usingratiometricdyes)enzymereactions(“caged”fluorescentcompounds)cellviability(viability-dependentpermeabilization)Fluorescentprobesanddyes-2SomeapplicationsoffluorescencemicroscopyFluorochromesinmicroscopyBiologicallyactivefluorescentcompounds-binddirectlytocellularstructuresChemicaldyes-mostneedtobecoupledtoamacromoleculetobeusefulinmicroscopy

Fluorescentproteins-canbefusedgeneticallytoaproteinofinterestFluorescentprobesanddyes-3DyesthatbindcellularstructuresororganellesDAPICrystalstructureofDAPIboundtoDNASporulatingBacillussubtilisFM4-64andDAPIDyesthatbindorganelles-1Chemicalconjugationoffluorescentdyestochemicalsthatbindcellular structuresRhodamine-coupledPhalloidin(PhalloidinisamushroomtoxinthatbindstoF-actin)Dyesthatbindorganelles-2ImmunofluorescencemicroscopyfluorophoreSecondaryantibodyPrimaryantibodyUseantibodiesraisedagainstyourproteinofinterestOR…ChemicalDyes-1mouseanti-mouserabbitanti-rabbitEpitopetagsinFluorescencemicroscopyCommonepitopes=Myc,HAGeneX6xHAFuseproteinofinteresttoanepitope“tag”Buycommercially-availableantibodiestotheepitopeanduseasprimaryantibodyforIFAdvantage: Fast(donotneedtoraiseantibodies)Disadvantages: Proteinfusionmaynotbefullyfunctional ProblemsofspecificityofantibodiestotagChemicalDyes-2FluorophoresformicroscopyFluorescein(IgG-coupled)(FITC)520nm-greenTexasRed(IgG-coupled)601nm-redTetramethylrhodamine(dextrancoupled)(TRITC)573nm-redFluoresceinandRhodaminederivativesCoupledwithIsothiocyanates-allowsattachmentviaaminogroupsinproteinsChemicalDyes-3ImproveddyesCyDyes(Cyaninedye-based)Amersham-PharmaciaIncAlexafluor

(molecularprobes/invitrogen)(brighter,morestable)ChemicalDyes-4QdotnanocrystalsExtremelyphotostable(molecularprobes/invitrogen)DifferentwavelengthsachievedbyvaryingsizeofcrystalSmallsemi-conductorsChemicalDyes-5cadmium/seleniumZincsulphideMulticolourlabelingcansimultaneouslyimagemultiplefluorophorese.gtolocalizemultipleproteinsinthesamecellneedtoisolatethesignalfromeachfluorophoreindividuallyChoosefluorophoreswithminimumemissionoverlapChoosefiltersetsthatminimize“bleedthrough”intoanotherchannelsuitablenotsuitableChemicalDyes-6FluorescentproteinsGreenFluorescentprotein(GFP)isolatedfromthejellyfishAequoreavictoriaMyproteinGFPShortflexiblelinkerFusionproteinAdvantages: canuseinlivecells fixingartefactsavoided dynamicsDisadvantages: photobleaching foldingenvironmentdependent functionalityoffusionproteinFluorescentproteins-1MutagenisationofGFP -->morestable -->spectrallyshiftedvariantsOtherfluorescentproteinsfromotherorganismse.g.DsRedfromDiscosoma(26%homologywithGFP)GFPvariantsShanerNC,SteinbachPA,TsienRY(2019)Aguidetochoosingfluorescentproteins.NatMethods.2(12):905-9.GFP(wt)

395/475

509

GreenFluorescentProteinsEGFP

484

507

AcGFP

480

505

TurboGFP

482

502

Emerald

487

509

AzamiGreen

492

505

ZsGreen

493

505

BlueFluorescentProteinsEBFP

383

445

Sapphire

399

511

T-Sapphire

399

511

CyanFluorescentProteinsECFP

439

476

mCFP

433

475

Cerulean

433

475

CyPet

435

477

AmCyan1

458

489

Midori-IshiCyan

472

mTFP1(Teal)

462

492

OrangeandRedFluorescent

ProteinsKusabiraOrange

548

mOrange

548

562

dTomato

554

581

dTomato-Tandem

554

DsRed

558

583

DsRed2

563

582DsRed-Express(T1)

555

DsRed-Monomer

556

mTangerine

568

585

mStrawberry

574

596

AsRed2

576

592

mRFP1

584

607

JRed

584

610

mCherry

587

610

HcRed1

588

618

mRaspberry

598

625

HcRed-Tandem

590

mPlum

590

649

YellowFluorescentProteinsEYFP

514

527

Topaz

514

527

Venus

515

528

mCitrine

516

529

YPet

517

530

PhiYFP

525

537

ZsYellow1

529

539

mBanana

540

553Fluorescentproteins-2PhotobleachingPhotobleaching:afluorophorepermanentlylosestheabilitytofluoresceduetophoton-inducedchemicaldamageandcovalentmodification.Largelyduetothegenerationoffreeoxygenradicalsthatattackandpermanentlydestroythelight-emittingpropertiesofthefluorochrome.Absorption(10-15sec)Fluorescence(10-9-10-12sec)(nSec-pSec)Internalconversion(heat)Phosphorescence(102-10-2sec)(100Sec-0.01Sec)*Tripletstate*Tripletstate-VERYREACTIVEmayinteractwithanothermoleculetoproduceirreversiblecovalentmodifications(photobleaching)groundstateexcitedstatePhotobleachingandQuenching-1HowtoreducephotobleachingchemicalreactivityofthefluorophoreintensityandwavelengthoftheexcitationlightintracellularchemicalenvironmentPhotobleachinginfluencedby:Reducephotobleachingby:choiceoffluorophorelimitexposuretime(butwillreduceemission)useofantifadereagentsPhotobleachingandQuenching-2AntifadeReagentsActbyscavengingreactionoxygenspeciesCommonAntifadeReagentsDIY(buyfromSigma)p-phenylenediamine n-propylgallateDABCOProprietySlowFade MolecularProbes(Invitrogen)ProLongAntifadekit MolecularProbes(Invitrogen)Vectashield VectorlaboratoriesPhotobleachingandQuenching-3FRAP(Fluoresencerecoveryafterphotobleaching)PhotobleachingandQuenching-4phenomenonofphotobleachingisexploitedinFRAPFRAP-learnhowdynamicaproteinisbymonitoringrecoveryoffluoresenceafterphotobleachingbleachTimetakentorecoverQuenchingPhotobleachingandQuenching-5

Quenching-reducedfluoresenceintensityasaresultofthepresenceofoxidizingagentsorthepresenceofsaltsofheavymetalsorhalogencompoundsQuenchingreducesemissionQuenchingsometimesresultsfromthetransferofenergytoother“acceptormolecules”closetotheexcitedfluorophore=ResonanceenergytransferResonanceenergytransferhasbeenexploitedtomeasuretheproximityoftwomoleculesinatechniquecalledFRET(Fluorese