Quantification of biomolecule dynamics and interactions in living zebrafish embryos by fluorescence correlation spectroscopy

QUANTIFICATIONOFBIOMOLECULEDYNAMICSAND
INTERACTIONSINLIVINGZEBRAFISHEMBRYOSBY
FLUORESCENCECORRELATIONSPECTROSCOPY
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SHIXIANKE
(B.Sc.,USTC,P.R.CHINA)
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ATHESISSUBMITTED
FORTHEDEGREEOFDOCTOROFPHILOSOPHY
DEARTMENTOFCHEMISTRY
NATIONALUNIVERSITYOFSINGAPORE
2009
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I
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This work is a result of collaboration between the Biophysical Fluorescence
LaboratoryatDepartmentofChemistry,NationalUniversityofSingapore(NUS)and
theFishDevelopmentBiologyLaboratoryatInstituteofMolecularandCellBiology
(IMCB),underthe supervisionofAssociateProfessorThorsten Wohland(NUS) and
AssociateProfessorVladimirKorzh(IMCB),betweenJuly2004andNovember2008.
Theresultshavebeenpartlypublishedin:
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Shi, X., Teo, L. S., Pan, X., Chong, S. W., Kraut, R., Korzh, V., & Wohland, T., 2009,
Probingeventswithsinglemoleculesensitivityinzebrafishand Drosophilaembryos

byfluorescencecorrelationspectroscopy,Dev.Dyn.,238(12),3156‐67
Shi,X.,Foo,Y.H.,Sudhaharan,T.,Chong, S.W.,Korzh,V.,Ahmed,S.,&Wohland,T.,
2009, Determination of dissociation constants in living zebrafish embryos with
singlewavelengthfluorescencecross‐correlationspectroscopy,Biophys.J.,(97)678‐
686
Shi.X.,andWohland,T.,Fluorescencecorrelationspectroscopy,2010,inNanoscopy
and Multidimensional Fluorescence Microscopy, edited by Diaspro, A., Taylor and
Francis
Pan, X., Shi, X., Korzh, V., Yu, H., & Wohland, T., 2009, Line scan fluorescence
correlationspectroscopyfor3Dmicrofluidicflowvelocitymeasurements,J.Biome.
Opt.,(14)024049
Pan, X., Yu, H., Shi, X., Korzh, V., & Wohland, T., 2007, Characterization of flow
direction in microchannels and zebrafish blood  vessels by scanning fluorescence
correlationspectroscopy”J.Biome.Opt.,(12)014034
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II
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Acknowledgements
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As a foreign student, I can still vividly remember the feeling of loneliness and
helplessness when I first came to Singapore and NUS. Without the help of many
people, a life would be difficult for the past five years, let alone a doctoral thesis.
Takingthisopportunity,Iwouldliketoexpressmydeepestgratitudetothemall.
I am heartily thankful to my supervisor Associate Professor Thorsten Wohland for
introducingmethisexcitingresearchprojectandguidingmeallthewaywithgreat
patience. His passion for scientific research deeply inspired me and his German‐
style seriousness towards work gradually influenced me. This thesis would not be
possiblewithouthisenlighteningadvicesandhearteningencouragements.
I would like to thank my co‐supervisor Associate Professor Vladimir Korzh for

offeringme theopportunity tojoinhis family‐likeresearchgroup andshowing me
theexcitingworldofdevelopmentalbiology.Hiskindsupportwasalwaysavailable
through these years and his profound knowledge of zebrafish research provided
numerousnewideastothiscross‐disciplinaryproject.
I would like to show my gratitude to Associate Professor Sohail Ahmed and
Associate Professor Rachel Kraut for the great collaboration. Their warm help and
supportmadecrucialcontributiontothiswork.
I am grateful to all my colleagues from the Biophysical Fluorescence Laboratory in
NUS: Liu Ping for helping me with the biological sample handling and FCS
measurementsincellcultures;PanXiaotaoforhelpingme withtheFCSalignments
and the two photon excitation instrument setup;  Guo Lin and Foo Yong Hwee for
helpfuldiscussionsandcollaboration;YuLanlan,HwangLingChin,Liu Jun,HarJarYi,
KannanBalakrishnan,MannaManojKumar,TeoLinShinandJagadishSankaranfor
theirfriendshipsandsupport.
I am also grateful to all my colleagues from the Fish Development Biology
Laboratory
 in IMCB: in particular, Chong Shang‐Wei for guidance of basic biology
and zebrafish research; Cathleen Teh, Poon Kar Lai and William Go for technical
assistance,helpfuldiscussionandtheirfriendships.
Lastbutnotleast,Iwouldliketothankmyparentsfortheirunconditionalloveand
care. I would
like to thank my beautiful wife Zhang Guifeng for her continuous
support,loveandthehappiestmomentsshebringstomylife.
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III
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TableofContents
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Acknowledgements II
TableofContents III

Summary VI
ListofTables VIII
ListofFigures IX
ListofSymbolsandacronyms XI
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Chapter1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1
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Chapter2TheoryandMethods∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
2.1FluorescenceCorrelationSpectroscopy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
2.1.1TheAutocorrelationAnalysis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
2.1.2TranslationalDiffusion∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14
2.1.3FCSinstrumentation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙21
2.1.4DataFitting∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙25
2.2SingleWavelengthFluorescenceCross‐CorrelationSpectroscopy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27
2.2.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27
2.2.2TheoryofSW‐FCCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29
2.2.3BindingQuantification∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙33
2.2.4SW‐FCCSInstrumentation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙34
2.3PreparationofZebrafishEmbryosforImagingandSW‐FCCSMeasurements37
2.3.1ZebrafishEmbryoPreparation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙37
2.3.2ImagingandFCS/SW‐FCCSMeasurementsofZebrafishEmbryos∙∙∙∙∙∙∙∙∙∙∙40
2.4Preparationofbiologicalsamples∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙42
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Chapter3ZebrafishembryoasamodelforFCSmeasurements∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44
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3.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44
3.2GeneExpressioninZebrafishEmbryos∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙46
3.3AutofluorescenceStudy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙49
IV


3.3.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙49
3.3.2Autofluorescencedistributioninembryobody∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙50
3.3.3AutofluorescenceSpectrum∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙53
3.3.4AutoflurescenceIntensity∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙54
3.4PenetrationDepthStudy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙57
3.4.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙57
3.4.2Penetrationdepthofconfocalmicroscopy∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙58
3.4.3PenetrationdepthofFCSusingOPE∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙60
3.4.4PenetrationdepthofFCSusingTPE∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙62

Chapter4ProbeSingleMoleculeEventsinLivingZebrafishEmbryoswithFCS∙∙∙∙∙∙∙69
4.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙69
4.2BloodFlowMeasurementsinLivingZebrafishEmbryo∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙71
4.2.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙71
4.2.2FCSTheoryofFlowMeasurement∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙72
4.2.3FlowVelocityMeasurementbyFCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙74
4.3ProteinTranslationalDiffusionMeasurementsinLivingZebrafishEmbryo∙∙∙78
4.3.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙78
4.3.2ProteinTranslationalDiffusionMeasurementsinCytoplasmand
Nucleoplasm∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙79
4.3.3ProteinTranslationalDiffusionMeasurementsinMotorNeuronCellsand
MuscleFiberCells∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙82
4.3.4ProteinTranslationalDiffusionMeasurementsofCxcr4b‐EGFPon
Membrane∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙86

4.3.5DataAnalysisUsingAnomalousSubdiffusionModel∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙89

Chapter5DeterminationofDissociationConstantsinLivingZebrafishEmbryoswith
SW‐FCCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙92
5.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙92

5.2SystemCalibration∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙94
5.2.1Determinationofcps,background,andcorrectionfactors∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙94
5.2.2DeterminationoftheEffectiveVolume∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙96
V

5.2.3InstrumentCalibration∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙97
5.3ControlMeasurements∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙99
5.3.1MixtureofmRFPandEGFPasNegativeControl∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙99
5.3.2mRFP‐EGFPTandemConstructasPositiveControl∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙101
5.4InteractionofCdc42andIQGAP1∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙105
5.4.1Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙105
5.4.2InteractionofCdc42
G12V
andIQGAP1∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙108
5.4.3InteractionofCdc42
T17N
andIQGAP1∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙111
5.4.4ComparisonofResultsfromZebrafishEmbryoandCHOcells∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙115
5.4.5Summary∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙117

Chapter6ConclusionandOutlook∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙120
6.1Conclusion∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙120
6.2Outlook∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙125

References∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙131


VI

Summary



Fluorescence correlation spectroscopy (FCS) and fluorescence cross‐correlation
spectroscopy (FCCS) are widely used biophysical techniques to determine
biomolecule concentrations, photophysical dynamics of fluorophores, diffusion
coefficientsofDNAsandproteins,anddissociationconstantsofinteractingparticles.
In this work, we extended the application of FCS and single wavelength
fluorescencecross‐correlationspectroscopy(SW‐FCCS),avariantofFCCSdeveloped
in our lab, to a multicellular living organism. We chose zebrafish embryo for this
purposeasitstransparenttissueaidedtheinvestigationsofcellsdeepbeneathskin.
Wefirstexaminedhowandtowhatextentzebrafishembryoscanbestudiedusing
FCS. Then the applicability of FCS to study molecular processes in embryo was
demonstrated by the determination of blood flow velocities with high spatial
resolution and the determination of diffusion coefficients of cytoplasmic and
membrane‐boundenhanced green fluorescence protein (EGFP) labeled proteins in
differentsubcellularcompartmentsaswellasindifferentcelltypes.Lastly,weshow
that protein‐protein interactions can be directly quantified in muscle fiber cells in
living zebrafish embryo with SW‐FCCS. This thesis is organized in the following
chapters:
1. Chapter 1 introduces the motivation to study protein dynamics and
interactionsinlivingorg anisms.Itprovidesa
literaturereviewonthehistory
and development of FCS/SW‐FCCS, as well as the application of FCS/SW‐
FCCSinstudyingbiomoleculedynamicsandinteractions.
2. Chapter 2 describes the theories and experimental setups of FCS and SW‐
FCCS.Thepreparationofbiologicalsamplesandthepreparationofzebrafish
embryoforimaging
andFCSmeasurementarealsoillustratedanddiscussed
inthischapter.
3. Chapter3examineshowandtowhatextentzebrafishembryocanbeused

as a  model for the study of molecular processes. Firstly, the approachesto
express foreign genes in zebrafish embryos are discussed and compared
with that in cell cultures. Secondly, the autofluorescence in living zebrafish
embryos, in particular the autofluorescence distribution and emission
spectra, is examined in order to minimize background interference. Lastly,
the working distance of FCS measurements in zebrafish tissues is studied
withbothonephotonexcitationandtwophotonexcitation.
VII

4. Chapter 4 presents the studies of molecular processes in living zebrafish
embryos with FCS. We first show that systolic and diastolic blood flow
velocitiescanbenoninvasively determinedwithhighspatialresolutioneven
intheabsenceofredbloodcells.Wethenshowthatdiffusioncoefficientsof
cytoplasmic and membrane‐bound proteins can be accurately determined.
We measure the diffusion coefficients of EGFP in cytoplasm and
nucleoplasm,aswellasinmotorneuroncellsandmusclefibercells.Wealso
determine the diffusion coefficients of Cxcr4b‐EGFP, an EGFP labeled G
protein coupled receptor (GPCR), on the plasma membrane of the muscle
fibercells.WefinallyanalyzetheFCSdatawiththeanomaloussubdiffusion
modelandstudythemolecularcrowdednessofcellsinlivingembryos.
5. Chapter5describesthedirectquantificationofprotein‐proteininteractions
in living zebrafish embryos with SW‐FCCS. The SW‐FCCS instrument is
calibrated using Rhodamine 6G and the effective volume is calculated
accordingly. Positive (mRFP‐EGFP tandem construct) and negative
(individuallyexpressedmRFPandEGFP)controlsaremeasuredfirsttoprobe
theupperandlowerlimitsofSW‐FCCSmeasurementsinembryos.Thenthe
interactions of Cdc42, a small Rho‐GTPase, and IQGAP1, an actin‐binding
scaffolding protein, are studied and the dissociation constants are
determined.Finally,theresultsobtainedinzebrafishembryosarecompared
tothatinChinesehamsterovarycellcultures.

6. Chapter 6 concludes the finding in this work and envisions the future
developmentofFCS/SW‐FCCSinembryos.




VIII

ListofTables

Table4.1:Bloodflowvelocitiesofdorsalaortaandcardinalvein.∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙76
Table4.2:Translationaldiffusionmeasurementsinzebrafishembryos∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙91
Table5.1:Molecularbrightnessobtainedfromcalibration.∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙96
Table5.2:DataobtainedfrommusclefibercellsinembryoandCHOcell∙∙∙∙∙∙∙∙∙∙∙∙∙118
Table6.1:Fluorescentpropertiesofsomefluorophores∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙127




IX

ListofFigures

Fig.2.1:Characteristicsoffluorescencecorrelationfunctions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
Fig.2.2:AtypicalopticalsetupofconfocalFCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24
Fig.2.3:ExcitationandemissionspectraofEGFPandmRFP∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29
Fig.2.4:TheoryofFCCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31
Fig.2.5:AtypicalopticalsetupofSW‐FCCS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙36
Fig.2.6:Zebrafishembryopreparation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙39
Fig.2.7:Identificationofsinglecellandsubcellularcompartmentinzebrafish

embryo∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙41
Fig.3.1:Autofluorescencedistributioninzebrafishembryobody∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙52
Fig.3.2:Autofluorescencespectrum∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙54
Fig.3.3:Fluorescenceintensitychangesagainstdepthinconfocalmicroscopy∙∙∙∙∙∙59
Fig.3.4:FCSpenetrationdepthstudyusingonephotonexcitation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙62
Fig.3.5:CalibrationofFCSusingtwophotonexcitation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙65
Fig.3.6:FCSpenetrationdepthstudyusingtwophotonexcitation∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙68
Fig.4.1:FCSbloodflowmeasurementinlivingzebrafishembryos∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙75
Fig.4.2:AtypicalFCSmeasurementofbloodflowintheheartofzebrafishembryo
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙77

Fig.4.3:Diffusiontimemeasurementswithinonecell∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙82
Fig.4.4:Diffusiontimemeasurementsindifferentcelltypes∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙85
Fig.4.5:DiffusiontimemeasurementsofCxcr4b‐EGFP∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙88
Fig.5.1:SystemcalibrationusingRhodamine6G∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙98
Fig.5.2:SW‐FCCScontrolmeasurementsinlivingzebrafishembryos∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙103
X

Fig.5.3:Scatteringplotof
g
r
CC

vs
g
r
C
forbothpositiveandnegativecontrols∙104
Fig.5.4:Fiveprotein‐interactingdomainofIQGAP1∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙106
Fig.5.5:InteractionofIQGAP1withCdc42andF‐actin∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙108

Fig.5.6:SW‐FCCSmeasurementsofCdc42andIQGAP1∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙111
Fig.5.7:DeterminationofK
D
fortheinteractingproteinpairofCdc42andIQGAP1
∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙114
Fig.5.8:SW‐FCCSresultsobtainedinCHOcellculture∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙119
Fig.6.1:Excitationandemissionspectraoftwofluorophorepairs∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙128

XI

ListofSymbolsandAcronyms



anomalityfactor


mediumviscosity


correlationtime
d


diffusiontime
f


flowtimeforamoleculethroughtheobservationvolume
trip



tripletstaterelaxationtime
0


theradialdistancewheretheexcitationintensityreaches1/e
2
ofits
valueatthecenteroftheobservationvolume

2


Chisquare,usedtodescribegoodness‐of‐fit
ACF autocorrelationfunction
APD avalanchephotodiode
C Concentration
CCD charge‐coupleddevice
CCF cross‐correlationfunction
Cdc42 celldivisioncycle42
CHD Calponinhomologydomain
CHO Chinesehamsterovary
CLSM confocallaserscanningmicroscopy
CMV Cytomegalovirus
cps countratepermoleculepersecond
Cxcr4a/b chemokine(C‐X‐Cmotif)receptor4a/b
D diffusioncoefficient
DNA deoxyribonucleicacid
dpf dayspostfertilization

EGFP enhancedgreenfluorescenceprotein
EGFR epidermalgrowthfactorreceptor
XII

EMCCD electronmultiplyingcharge‐coupleddevice
EtBr ethidiumbromide
F(t) fluorescenceintensityattimet
FCM fluorescencecorrelationmicroscopy
FCS fluorescencecorrelationspectroscopy
FCCS fluorescencecross‐correlationspectroscopy
FLIM fluorescencelifetimeimagingmicroscopy
Flu Fluorescein
FP fluorescenceprotein
FRAP fluorescencerecoveryafterphotobleaching
FRET fluorescenceresonanceenergytransfer
F
trip
 thefractionoftheparticlesthathaveenteredthetripletstate
x
g ,
y
g
,
z
g 
basictermofcorrelationfunctionineachdimension
(0)G

correlationfunctionamplitude
()G



correlationfunction
G


theconvergencevalueof
()G

forinfinitetime
GDP guanosinediphosphate
GEF guaninenucleotideexchangefactor
GPCR G‐proteincoupledreceptor
GRD GAP‐relateddomain
GTP guanosinetriphosphate
IQGAP1 IQmotifcontainingGTPaseactivatingprotein1
K Boltzmann’sconstant
K

geometric ratio of axial to radial distance of the observatoion
volume,where
00
/Kz



K
D
 dissociationconstant
hpf hourspostfertilization

M molecularmass
MO morpholinooligos
mRFP monomericredfluorescenceprotein
N numberofmolecules
XIII

N
A
 Avogadro’snumber
NA numericalaperture
NIR nearinfrared
N‐WASP neural‐Wiskott‐Aldrichsyndromeprotein
OPE one‐photonexcitation
PBS phosphatebuffersolution
PCH photoncountinghistogram
PCR polymerasechainreaction
PIV particleimagingvelocity
PMT photomultipliertubes
PSF pointspreadfunction
PTU 0.003%1‐phenyl‐2‐thioureain10%Hank’ssaline
QD quantumdots
R6G rhodamine6G
SD standarddeviation
SW‐FCCS singlewavelengthfluorescencecross‐correlationspectroscopy
SPT singleparticletracking
T absolutetemperature
TIR totalinternalreflection
TMR tetramethylrhodamine
TPE two‐photonexcitation
UV Ultraviolet

V
eff
 effectiveobservationvolume
z
0
 theaxialdistancewheretheexcitationintensityreaches1/e
2
ofits
valueatthecenteroftheobservationvolume

Chapter1
Introduction

The end of the 20
th
 and the beginning of the 21
st
 century witnessed exciting
developmentsinthelifesciencesandtheemergenceofnovelquestionswithinthe
field.Inparticular,theadvancesinmolecularandcellbiologybroughttheneedto
understandcellbehaviorbasedonveryfundamentalmolecularprocesses.However,
conventional ensemble or bulk measurements cannot address this issue as single
moleculeparametersand their distributionaremasked by theensemble averages
andstandard deviations.Under ensemblemeasurement,questionswhether there
isoneormultiplemolecularspecies,e.g.proteinsofmultipleconformations,cannot
be answered. Thus new tools and strategies that can detect single molecules and
distinguish a single molecule among heterogeneous populations are needed. The
firstsingle moleculedetectionwasachievedin1976usingfluorescencemicroscopy
(Hirschfeld, 1976). Fluorescence‐based techniques are advantageous in terms of
specificity,sensitivity andversatility. They are non‐destructive to the samples and

thuscanbeappliedtolivingcellsinreal‐time.Bylabellingtheobjectofinterestwith
a fluorophore and illuminating a small observation volume with a tightly focused
laser beam, single molecule detection can be achieved even in the presence of
1

cellular autofluorescence (Yu et al, 2006). In addition, fluorescence intensity is
directly proportional to the number of fluorophores, providing the basis for
quantitative analysis. Consequently, the field of  fluorescence microscopy and
spectroscopygrewatanacceleratingpaceandisstillgrowingstronglywithanever
increasing number of new techniques and methods being published (Haustein &
Schwille,2007;Hwang&Wohland,2007;Kolin&Wiseman,2007;Liuetal,2008a;
Thompsonetal,2002).
Florescencecorrelationspectroscopy(FCS),onegroupofthefluorescencemethods,
analyzes fluorescence intensity fluctuations from a confined observation volume
with single molecule sensitivity but at the same time based on fast statistical
treatment of the recorded data. Any  process within the volume which causes
variationsinthefluorescenceintensityandhappensonatimescaleslowerthanthe
recording speed will leave characteristic fluctuations in the intensity trace. By
performing either a Fourier transformation or an autocorrelation analysis,
parameters such as local concentrations, molecular mobility and photophysical
dynamicscanbedeterminedforthefluorescentlylabeledmolecules.FCSwasfirst
introducedbyMagde,ElsonandWebbinthe1970s(Elson&Magde,1974;Magde
et al, 1972). In its first appearance,Magde and others successfully  monitored the
bindingreactionbetweenethidiumbromide(EtBr)anddoublestrandedDNAusing
FCS. The selection of EtBr and DNA simplified the situation as the fluorescence
quantumyieldofEtBrincreases20timesuponinsertionintotheDNA.However,the
early FCS measurements suffered from poor signal‐to‐noise ratio due to technical
limitations and the applicability of FCS was quite limited. In the following two
decades, FCS was held back in favour of photobleaching methods for diffusion
2


coefficientmeasurements.TherenaissanceofFCScamein1993,whenRigleretal.
introduced a confocal illumination scheme to improve the signal‐to‐noise ratio of
FCSmeasurements(Rigleretal,1993b).Theuseofatightly focusedlaserbeamand
a small pinhole in this setup generated a very small observation volume on the
rangeofonefemtoliter(10
‐15
L).Thisensuredthataminimumnumberofmolecules
weredetected,andthusthefluctuationscausedbysinglemoleculescanbeeasily
distinguished, which in return guaranteed good signal‐to‐noise ratio. Since then,
FCSbecameanincreasinglypopulartechniquetostudymoleculardynamicsandthe
applicabilitywasextended.Inthefollowingyears,FCS has been usedtomeasure
translational diffusion (Rigler et al, 1993b),singlet‐triplet interactions of
fluorophores (Widengren & Rigler, 1995),photophysical properties of chemical
dyes (Widengren & Schwilles, 2000) and fluorescent proteins (Haupts et al, 1998;
Maederet al, 2007;Schwilleetal,2000),chemicalreactions(Widengren&Rigler,
1998), pH values in subcellular compartments (Llopis et al, 1998), hydrodynamic
flowprofile inmicrochannelstructures (Gosch etal,2000) withmoreapplications
comingasIwrite.
FCS can also be used to measure inter‐molecular binding, e.g. receptor‐ligand
interactions(Raueretal,1996;VanCraenenbroeck&Engelborghs,1999;Wohland
etal,1999;Wrussetal,2007;Zema
novaetal,2004).Itisbasedonthetheorythat
relativechangesinmassuponbindingleadtoareductioninthediffusioncoefficient.
However,inordertodistinguishtwocomponents(beforeandafterbinding)inFCS, 
theirdiffusioncoefficientsmustdif
ferbyatleastafactorof1.6(Mesethetal,1999).
BasedontheStokes‐Einsteinrelation(D
‐1
~M

1/3
),themassmustdifferbyatleasta
factor of 4. Dimerization is therefore difficult to resolve. In addition, FCS cannot
3

resolve specific binding in a multi‐component system, and protein‐protein
interactions in living cells are generally not assessable due  to the complex
environmentandalargenumberofpotentiallyinteractingcomponents.Therefore
in1994,theconceptofmultiplecolorsfluorescencecross‐correlationspectroscopy
(FCCS)wasintroducedtospecificallystudymolecularbinding(Eigen&Rigler,1994).
In dual‐color FCCS, both binding partners of interest are labelled with distinct
fluorescent dyes. The two labels are simultaneously excited and fluorescence
signalsarecollectedinseparatechannels.Asidefromtheautocorrelationofsignals
fromeachchannel,thesignalsfrombothchannelsarecross‐correlated.Thebinding
induced concurrent movement of the two labels therefore produces a positive
signal in the cross‐correlation analysis. Dual‐color FCCS was first experimentally
realized by Schwille et al. to measure nucleic acid hybridizations (Schwille et al,
1997), and the potential ofFCCS to effectively measure biomolecular interactions
wasdemonstratedinthefollowingyearsbothinvitro(Camachoetal,2004;Foldes‐
Papp&Rigler,2001;Kettlingetal,1998;Kornetal,2003)andin vivo(Baciaetal,
2002; Baudendistel et al, 2005; Muto et al, 2006; Saito et al, 2004). Since this
technique is independent of distance and orientation of the fluorophore, FCCS
repres
ents an attractive alternative to Fluorescence Resonance Energy Transfer
(FRET)measurementswhicharetypicallyusedtostudymolecularinteractions(Liu
etal,2008a).
Inthe realm ofbiological research,biochemical techniqueswerefirstly employed,
which allowed the separation and purification of cellular components. Is
olated
components or proteins can thereby be used in in vitro experiments to model

biochemical reactions and molecular interactions. This information was then
4

combined and interweaved to reproduce the complex cellular processes involving
multiplecomponentsandstructures.Biochemicalexperimentsprovideasimplified
andcontrollable platform, butitis alsocrucialtobe abletoobserve andquantify
biologicalprocessesdirectlyinlivingcells,asthesubcellularlocalization,subcellular
compartmentalizationandlocalconcentrationalsoplayimportantrolesindefining
biological processes. Fluorescence‐based biophysical methods, also known as F‐
techniques,i.e.FRAP,FRET,FLIM,andFCS/FCCS,therebycametotheforefrontin
this research. FCS and FCCS are well suited for intracellular applications. Theyare
non‐invasive in nature and highly sensitive. The spatial resolution of FCS/FCCS is
defined by the size of the confocal volume, usually less than one micrometer in
dimensionandcanbefurtherreducedtothenanometerscale(Eggelingetal,2009).
Intypicalbiologicalsamples,biomolecularconcentrationsrangefrom1nMto1µM,
which results in about 1 – 1000 particles in the observation volume, a range just
measurablebyFCS.Therefore,FCScanbedirectlyusedtostudyproteindynamics
attheirphysiologicalexpressionlevels.Atthesametime,FCSprovidesawiderange
oftemporal informationfrom microseconds to seconds. This allowsmeasurement
ofabroaderspectrumofbiomolecules,whosediffusionbeha
viourcanbeextremely
diverse in living biological samples. Furthermore, new instrumentations that
combine FCS with imaging techniques, e. g. confocal laser scanning microscopy
(CLSM),alsoexpandtheapplicabilityofFCSinbiological samples.Thecombination,
alsoknownasfluorescencecorrelationmicroscopy(FCM,Brock&Jovin,1998;Pa
n
etal, 2007a; Terry etal, 1995), allows the user to obtainan image of the sample
first before identifying a position on the image where subsequent FCS
measurements can be performed. This is especially useful in intracellular
5


applications.Thetypicalvolumeofaeukaryoticcellis10
‐12
L,whichisthreeorders
of magnitude larger than the observation volume of FCS. Using FCM, protein
dynamics can be specifically investigated in subcellular compartments. Owing to
thoseadvantages,thesolution‐basedtechniquesawmoreandmoreapplicationsin 
thefieldofcellbiology(Baciaetal,2006;Hwang&Wohland,2007;Kimetal,2007;
Liuetal,2008b;Schwille,2001).
However, up to now, most intracellular measurements using FCS and FCCS are
performedinPetridish‐based cellculturesystems.Cellculturesareengineeredas
isolated individual cells that can be artificially cultivated. Since their introduction,
2Dcellcultureshavegreatlyenhancedourunderstandingofcellularbehaviourand
molecular actions and interactions. The commonly used 2D cell cultures have the
advantageofeasygeneticmanipulation anddirectaccessibilitytobiochemicaland
biophysical analysis. The highly controlled and simplified cellular environment has
made possible single molecule detection within the complex matrix of cells.
Nowadays, Petri‐dish based cell culture systems have become a standard tool of
research in cell and molecular biology, and cell culture based drug screening is
regularlyperformed in the pharmaceuticalindustry. Nevertheless, 2D cellcultures
cannotfullyreflectthenaturalenvironmentofcellspresentinlivingorganisms.The
flatglasssubstrateandtheartifi
cialmediumbufferaresignificantlydifferentfroma
realphysiologicalenvironment.Theabsenceofextracellularmatrixandvariouscell‐
cellcommunicationfunctionsalsomakestheinformationharvestednotpredictive
in drug development. Numerous studies have pointed out the insufficiency of 2D
cell culture as a biological res
earch model. Mooney et al. showed that even
genetically normal primary cells placed in cell culture quickly lose their
6


differentiated gene expression pattern and phenotype (Mooney et al, 1992). By
culturingcellsina3Dstructure,Weaveretal.demonstratedthatmalignantbreast
tumourcellscanreverttotheiroriginalstatewhenanantibodyagainstβ‐integrinis
addedtothesystem(Weaveretal,1997),whilethisresultcannotbereproducedin
2D cell culture. In another reportby Anderset al., a receptor responsible for cell
infectionwasfoundtohavesimilarandhighlevelofexpressioninbothnormaland
malignant cells in 2D cell culture, but in 3D only malignant  cells carried large
number of the receptors (Anders et al, 2003). All these findings suggest that the
physiological relevance of findings made in 2D culture remains unclear and
questions of developmental biology cannot be addressed in this simplified and
biasedmodel.Therefore,itisdesirabletoextendFCSandFCCSmeasurementsinto
opticallyaccessiblesmalllivingorganisms,e.g.nematodes(Caenorhabditiselegans),
fruitflies(Drosophilamelanogaster),medaka(Oryziaslatipes)andzebrafish(Danio
rerio)togatherphysiologicallyrelevantdata.
Up till now,  FCS application in living animals is stilllimited due tothe thick tissue
induced light scatterings. In one example, Nagao and others reported diffusion
coefficientmeasurementsofGFPlabeledgranulesinmedakaprimordialgermcells
using FCS and fluorescence recovery after photobleaching (FRAP) (Nagao et al,
2008).To avoidthedeeptissue penetration,  themedakaembryos were dissected
andcellsofinterestwererevealed.Incontrast,workingwithmuchsmalleranimals
Petrasek and others applied scanning FCS to study the localization and
redistributionofGFPlabelledNMY‐2andPAR‐2proteinsduringtheasymmetricfirst
division of C. elegans embryos (Petrasek et al, 2008). Working with transparent
animalshelpstoalleviatethisproblemtoo.WehaverecentlyshownthatFCScanbe
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usedefficientlytomeasurebloodflowvelocitiesinlivimgzebrafishembryos(Korzh
etal,2008;Panetal,2007b).
Inthisthesis,theapplicabilityofFCSandFCCSisstudiedindetailinlivingzebrafish

embryo. Zebrafish, as a model of vertebrate development, has been introduced
onlyrelativelyrecently.Butitisfastcatchingupasmanymethodologiesdeveloped
inDrosophilaandothermodelsaretransferredtozebrafishresearch(Chakrabartiet
al, 1983; Streisinger et al, 1981; Stuart etal, 1988). Asa model species it is more
complex, evolutionarily closer to humans and amenable to standard genetic and
molecular tools. Numerous human diseases, both genetic and acquired, can be
introducedandstudiedinzebrafish,whichmadeita modelvertebrateofchoicefor
drug discovery and large‐scale studies of genetics, development and regeneration
(Fetchoetal,2008;Korzh,2007;Lieschke&Currie,2007;Strahle&Korzh,2004).In 
addition, zebrafish are small, easyto grow and sexually reproductive within three
months after fertilization. Zebrafish embryos are fertilized and develop externally 
andtheembryosandearlylarvaareopticallytransparent,allowinginvestigationof
cell‐biologicaleventsdeepwithinthetissue.In this work, we explore the limitation
of FCS and FCCS and their use in zebrafish embryos an
d demonstrate several
applicationsofFCSinlivingzebrafishembryos,showingthatsinglemolecule‐based
studiesinlivingorganismarepossible:
1. The autofluorescence expression pattern of zebrafish embryo was studied
firsttominimizebackgroundinterference.Theautofluorescencedistribution
wasexaminedintheembryobody,andtheautofluorescencespectrumand
intensitywasinvestigated.
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2. ThepenetrationdepthofFCSintheembryotissuewasexploredwithboth
one‐photonexcitation(OPE)andtwo‐photonexcitation(TPE).
3. Thebloodflowvelocitiesindifferentvesselsweremeasured.
4. The diffusion coefficients of cytoplasmic and membrane‐bound EGFP and
EGFP labelled proteins were measured in different subcellular
compartmentsanddifferentcelltypes.
5. Thedissociationconstants(K

D
)oftheinteractingprotein pairofCdc42and
IQGAP1 was determined using single wavelength fluorescence cross‐
correlationspectroscopy(SW‐FCCS).
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Chapter2
TheoryandMethods

2.1FluorescenceCorrelationSpectroscopy
2.1.1TheAutocorrelationAnalysis
Acorrelationbetweentwovariablesaandbdescribesthedependenceofthesetwo
variables.Inpracticaltermsthismeansthatifweknowthevalueofaorbwecan
make some prediction of the value of b or  a, respectively. The concept of
correlationisveryusefulsinceitallowssomepredictionsaboutthesecondvariable
fromtheobservationofthefirstone.Nowifwehavetwovariablesaandbwhich
are correlated, then the values of the two variables will change with a similar
pattern. Consequently, we expect that a multiplication of the two variables with
eachotherwillreinforcethispattern.Ifwecalculatenowtheaveragevaluesofa,b
andtheproductofa∙b,weexpect(Berne&Pecora,2000):
ab a b
 (2.1)
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
where denotes the average value. We can simplify the description of
correlationsbynormalizing
ab

with

ab
:
ab
g
ab


 (2.2)
Thevalueofgisnow1foruncorrelatedvariablesand>1forcorrelatedvariables.
If we have one variable a whose values change over a duration of time, and we
correlate the values of the same variable at different time point, e.g. and
()at
(at )


, where τ describes the difference in time, we then have a so called
“autocorrelation” analysis. The correlation between and
()at (at )


will only be
foundifthevalueofthevariablepersistslongerthanthetimeτ,i.e. changes
onatimescalelargerthanτ.Theautocorrelationfunction(ACF)iswrittenas:
(at)
() ( )
()
() ( )
at at
G
at at







 (2.3)
Itdescribestheself‐similarityofasignal(valuesofthevariable)intime.Therefore,
theACFisanindicationinhowfarwecanmakepredictionsintothefutureofthe
values of a variable. If the variable has long correlation in time we can make
predictionsfarahead;ifthecorrelat
iontimeisshortwecanpredictthefutureonly
overshorttimespans.Ifthecorrelationisstrongwecanmakegoodpredictions;if
thecorrelationisweakourpredictionwillbelessaccurate.
InFCSmeasurements,thefluorescenceintensitiesovertime aremeasuredin
a confined observation volume  where fluorescent probes undergo different
()Ft
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