Table Of ContentMarkus Sauer, Johan Hofkens,
and J¨org Enderlein
Handbook of Fluorescence
Spectroscopy and Imaging
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Markus Sauer, Johan Hofkens, and Jörg Enderlein
Handbook of Fluorescence
Spectroscopy and Imaging
From Single Molecules to Ensembles
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ISBN:978-3-527-31669-4
V
Contents
Preface IX
1 BasicPrinciplesofFluorescenceSpectroscopy 1
1.1 AbsorptionandEmissionofLight 1
1.2 SpectroscopicTransitionStrengths 5
1.3 Lambert–BeerLawandAbsorptionSpectroscopy 7
1.4 FluorophoreDimerizationandIsosbesticPoints 9
1.5 Franck–CondonPrinciple 12
1.6 TemperatureEffectsonAbsorptionandEmissionSpectra 15
1.7 FluorescenceandCompetingProcesses 17
1.8 StokesShift,SolventRelaxation,andSolvatochroism 20
1.9 FluorescenceQuantumYieldandLifetime 22
1.10 FluorescenceAnisotropy 27
References 29
2 FluorophoresandFluorescentLabels 31
2.1 NaturalFluorophores 31
2.2 OrganicFluorophores 35
2.3 DifferentFluorophoreClasses 38
2.4 MultichromophoricLabels 49
2.5 Nanocrystals 52
References 56
3 FluorophoreLabelingforSingle-MoleculeFluorescence
Spectroscopy(SMFS) 61
3.1 InVitroFluorescenceLabeling 61
3.2 FluorescenceLabelinginLivingCells 69
References 80
4 FluorophoreSelectionforSingle-MoleculeFluorescenceSpectroscopy
(SMFS)andPhotobleachingPathways 85
References 91
HandbookofFluorescenceSpectroscopyandImaging.M.Sauer,J.Hofkens,andJ.Enderlein
Copyright(cid:1)2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-31669-4
VI Contents
5 FluorescenceCorrelationSpectroscopy 93
5.1 Introduction 93
5.2 OpticalSet-Up 98
5.3 DataAcquisitionandEvaluation 99
5.4 MillisecondstoSeconds:DiffusionandConcentration 103
5.4.1 Single-FocusFCS 104
5.4.2 Dual-FocusFCS 111
5.5 NanosecondstoMicroseconds:Photophysics,Conformational
Fluctuations,BindingDynamics 120
5.6 PicosecondstoNanoseconds:RotationalDiffusion
andFluorescenceAntibunching 122
5.6.1 Antibunching 122
5.6.2 RotationalDiffusion 125
5.7 FluorescenceLifetimeCorrelationSpectroscopy 135
5.8 Conclusion 143
References 143
6 ExcitedStateEnergyTransfer 147
6.1 Introduction 147
6.2 Theoryof(Förster)EnergyTransfer 148
6.2.1 MechanismandMathematicalFormalismofFRET 148
6.2.2 MeasuringFRETEfficienciesThroughExcited-StateLifetimes 153
6.2.3 SpinRulesforFRET 154
6.2.4 Homo-FRETandFRET-InducedDepolarization 154
6.3 ExperimentalApproachforSingle-PairFRET-Experiments 157
6.3.1 Single-LaserExcitation 157
6.3.2 Alternating-LaserExcitation(ALEX) 160
6.4 ExamplesandApplicationsofFRET 161
6.4.1 FRETProcessesinBulkExperiments 162
6.4.1.1 FRET-BasedMolecularBiosensors 162
6.4.1.2 EnergyHoppingandTrappinginChromophore-Substituted
PolyphenyleneDendrimers 164
6.4.2 Single-MoleculeObservationofFRET 168
6.4.2.1 Light-HarvestingSystems:PhycobilisomesandAllophycocyanins 168
6.4.2.2 HairpinRibozymeDynamicsandActivity 179
6.4.2.3 Protein(Un)foldingandDynamics 180
References 183
7 PhotoinducedElectronTransfer(PET)Reactions 189
7.1 FluorescenceQuenchingbyPET 189
7.2 Single-MoleculeFluorescenceSpectroscopytoStudyPET 192
7.3 Single-MoleculeSensitiveFluorescenceSensorsBasedonPET 199
7.4 PETReporterSystem 202
7.5 MonitoringConformationalDynamicsandProteinFolding
byPET 205
Contents VII
7.6 BiologicalandDiagnosticApplications 209
References 215
8 Super-ResolutionFluorescenceImaging 219
8.1 DiffractionBarrierofOpticalMicroscopy 219
8.2 Multi-PhotonandStructuredIlluminationMicroscopy 221
8.3 StimulatedEmissionDepletion 223
8.4 Single-MoleculeBasedPhotoswitchingMicroscopy 226
8.5 BackgroundandPrinciplesofSingle-MoleculeBased
PhotoswitchingMicroscopyMethods 229
8.6 TemporalResolutionofSuper-ResolutionImagingMethods 236
References 237
9 Single-MoleculeEnzymatics 241
9.1 Introduction:WhyStudyEnzymesonaSingle-MoleculeLevel? 241
9.2 BiochemicalPrinciplesofEnzymaticActivity:theMichaelis–Menten
Model 242
9.3 ‘‘Looking’’atIndividualEnzymes 243
9.3.1 Single-EnzymeStudiesandKinetics 244
9.3.1.1 Space-Resolved,butTime-AveragedSingleEnzymeAssays 244
9.3.1.2 Single-TurnoverExperiments:Space-andTime-Resolved
EnzymeAssays 248
9.3.1.3 Results:RevisionoftheClassicalMichaelis–MentenModel 255
9.3.2 ConformationalDynamics 259
9.3.3 Single-MoleculeDNASequencing 260
9.3.4 SheddingLightonSingle-EnzymeMechanisms 262
9.3.4.1 MovementofMolecularMotorEnzymesonActinFilaments 263
9.3.4.2 Lipase-CatalyzedHydrolysisofPhospholipidBilayers 264
9.4 DataAnalysisofFluorescenceIntensityTimeTraces
ofSingle-TurnoverExperiments 264
9.4.1 ThresholdMethod 264
9.4.2 AutocorrelationAnalysis 267
9.5 Conclusions 267
References 268
Index 273
IX
Preface
Fluorescence spectroscopy and imaging have become powerful and widely used
methods in almost any laboratory around the globe for the non-invasive study of
polymers,inorganicmaterials,cells,andtissues.Withthedevelopmentofelaborate
single-molecule fluorescence techniques, energy and electron transfer methods
haveexperiencedarenaissanceandtodaytheyareusedsuccessfullytostudyprotein
folding,molecularinteractions,andthemotionandinteractionofindividualmole-
cules, even in living cells with high temporal resolution. New methods that have
beenintroducedtoincreasetheopticalresolutionofmicroscopybeyondthediffrac-
tionbarrierenableresearcherstostudycellularstructuresinfixedandlivingcells
with unprecedented resolution, which seemed impossible to achieve only a few
yearsago.Withtheongoingsuccessoffluorescencespectroscopyandmicroscopy,
fundamentalknowledgeaboutthechemicalandphotophysicalpropertiesoffluor-
ophoreslieattheheartofsingle-moleculesensitiveexperiments.
This book is intended to give interested readers the fundamental knowledge
necessary for planning and designing successful fluorescence spectroscopy and
imaging experiments with high spatiotemporal resolution. After a basic introduc-
tion to fluorescence spectroscopy, different fluorophores and their properties, we
describethedetailsofspecificfluorescencelabellingoftargetmolecules.Achapter
that explains our current understanding of fluorophore photophysics and photo-
bleaching pathways in single-molecule fluorescence experiments is followed by
chapters giving introductions to fluorescence correlation spectroscopy and also to
energyandelectrontransfer.Finally,thebookintroducesvarioussuper-resolution
imagingmethodsandgivesexamplesofhowsingle-moleculespectroscopycanbe
used for the successful study of enzyme kinetics, conformational dynamics of
biopolymers,proteinfolding,andfordiagnosticapplications.
Thebookismainlyaimedatgraduatestudentsandresearcherswhowanttobegin
usingtheadvancedfluorescencetechniquesofsingle-moleculefluorescencespec-
troscopy and imaging. However, we hope that even experts will find it a useful
resourcetoconsultfortheplanninganddiscussionoftheirexperiments.
Würzburg,Leuven,Göttingen,November2010 MarkusSauer
JohanHofkens
JörgEnderlein
HandbookofFluorescenceSpectroscopyandImaging.M.Sauer,J.Hofkens,andJ.Enderlein
Copyright(cid:1)2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-31669-4
j
1
1
Basic Principles of Fluorescence Spectroscopy
1.1
AbsorptionandEmissionofLight
Asfluorophoresplaythecentralroleinfluorescencespectroscopyandimagingwe
willstartwithaninvestigationoftheirmanifoldinteractionswithlight.Afluorophore
isacomponentthatcausesamoleculetoabsorbenergyofaspecificwavelengthand
thenre-remitenergyatadifferentbutequallyspecificwavelength.Theamountand
wavelengthoftheemittedenergydependonboththefluorophoreandthechemical
environmentofthefluorophore.Fluorophoresarealsodenotedaschromophores,
historically speaking the part or moiety of a molecule responsible for its color. In
addition,thedenotationchromophoreimpliesthatthemoleculeabsorbslightwhile
fluorophoremeansthatthemolecule,likewise,emitslight.Theumbrellatermused
inlightemissionisluminescence,whereasfluorescencedenotesallowedtransitions
withalifetimeinthenanosecondrangefromhighertolowerexcitedsingletstatesof
molecules.
Inthefollowingwewilltrytounderstandwhysomecompoundsarecoloredand
othersarenot.Therefore,wewilltakeacloserlookattherelationshipofconjugation
to color with fluorescence emission, and investigate the absorption of light at
different wavelengths in and near the visible part of the spectrum of various
compounds.Forexample,organiccompounds(i.e.,hydrocarbonsandderivatives)
without double or triple bonds absorb light at wavelengths below 160nm, corre-
sponding to a photon energy of >180kcalmol(cid:1)1 (1cal¼4.184J), or >7.8eV
(Figure 1.1), that is, significantly higher than the dissociation energy of common
carbon-to-carbonsinglebonds.
Below a wavelength of 200nm the energy of a single photon is sufficient to
ionizemolecules.Therefore,photochemicaldecompositionismostlikelytooccur
when unsaturated compounds, where all bonds are formed by s-electrons, are
irradiated with photon energies >6.2eV. Double and triple bonds also use
p-electronsinadditiontoas-bondforbonding.Incontrasttos-electrons,which
arecharacterizedbytherotationalsymmetryoftheirwavefunctionwithrespectto
thebonddirection,p-electronsarecharacterizedbyawavefunctionhavinganode
atthenucleusandrotationalsymmetryalongalinethroughthenucleus.p-bonds
HandbookofFluorescenceSpectroscopyandImaging.M.Sauer,J.Hofkens,andJ.Enderlein
Copyright(cid:1)2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:978-3-527-31669-4
j
2 1 BasicPrinciplesofFluorescenceSpectroscopy
Gamma Visible
rays X- rays UV IR Microwave Radio
Wavelength (m)
10-13 10-11 10-9 10-7 10-5 10-3 10-1 10
Wavenumber (cm-1)
1011 109 107 105 103 10 10-1 10-3
Frequency (Hz)
1021 1019 1017 1015 1013 1011 109 107
Energy (kcal)
108 106 104 102 100 10-2 10-4 10-6
Figure1.1 Theelectromagneticspectrum.
are usually weaker than s-bonds because their (negatively charged) electron
density is further from the positive charge of the nucleus, which requires more
energy. From the perspective of quantum mechanics, this bond weakness is
explained by significantly less overlap between the component p-orbitals due to
their parallel orientation. These less strongly bound electrons can be excited by
photonswithlowerenergy.Iftwodoublebondsareseparatedbyasinglebond,the
double bonds are termed conjugated. Conjugation of double bonds further
induces a red-shift in the absorption (a so-called bathochromic shift). All fluor-
ophores that have a high absorption in the visible part of the spectrum possess
several conjugated double bonds.
Above200nmonlythetwolowestenergytransitions,thatis,n ! p(cid:2)andp ! p(cid:2),
are achieved as a result of the energy available from the photons. When sample
moleculesareexposedtolighthavinganenergythatmatchesapossibleelectronic
transition within the molecule, some of the light energy will be absorbed as the
electronispromotedtoahigherenergyorbital.Asasimplerule,energeticallyfavored
electronpromotionwillbefromthehighestoccupiedmolecularorbital(HOMO),
usually the singlet ground state, S , to the lowest unoccupied molecular orbital
0
(LUMO),andtheresultingspeciesiscalledthesingletexcitedstateS .Absorption
1
bandsinthevisibleregionofthespectrumcorrespondtotransitionsfromtheground
stateofamoleculetoanexcitedstatethatis40–80kcalmol(cid:1)1abovethegroundstate.
Asmentionedpreviously,insaturatedhydrocarbonsinparticular,thelowestelec-
tronicstatesaremorethan80kcalmol(cid:1)1abovethegroundstate,andthereforethey
donotabsorblightinthevisibleregionofspectrum.Suchsubstancesarenotcolored.
Compoundsthatabsorbinthevisibleregionofthespectrum(thesecompoundshave
color)generallyhavesomeweaklyboundordelocalizedelectrons.Inthesesystems,
theenergydifferencebetweenthelowestLUMOandtheHOMOcorrespondstothe
energiesofquantainthevisibleregion.
