Superresolution microscopy of the volume phase transition of pNIPAM microgels

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ContentslistsavailableatScienceDirect

Colloids

and

Surfaces

A:

Physicochemical

and

Engineering

Aspects

jo u r n al ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f a

Superresolution

microscopy

of

the

volume

phase

transition

of

pNIPAM

microgels

Gaurasundar

M. Conley

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_,

_Soﬁ

_Nöjd

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_,

_Marco

_Braibanti

a

_,

_Peter

_{Schurtenberger}

b

_,

Frank

Scheffold

a,∗

a_Department_of_Physics,_University_of_Fribourg,_Chemin_du_Musée_3,₁₇₀₀_Fribourg,_Switzerland b_Physical_Chemistry,_Department_of_Chemistry,_Lund_University,₂₂₁₀₀_Lund,_Sweden

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•Superresolutionmicroscopyapplied tostudythestructureofmicrogel col-loids.

•Microgel radial density proﬁles revealedinrealspace.

•Workset’sthestagefortheuseof superresolutionmicroscopyin mate-rialsresearch.

•dSTORM may allow for nanoscale characterization of distinct poly-mericsubunitsin3D.

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Articlehistory:

Received19January2016

Receivedinrevisedform3March2016 Accepted4March2016

Availableonline22March2016 Keywords:

Superresolutionmicroscopy dSTORM

Microgels pNIPAM

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HierarchicalpolymerstructuressuchaspNIPAMmicrogelshavebeenextensivelystudiedfortheirability toundergostructuralandphysicaltransformationsthatcanbecontrolledbyexternalstimulisuchas temperature,pHorsolventcomposition.However,adirectthree-dimensionalvisualizationofindividual particlesin-situhassofarbeenhinderedbyinsufﬁcientresolution,withopticalmicroscopy,orcontrast, withelectronmicroscopy.Inrecentyearssuperresolutionmicroscopytechniqueshaveemergedthatcan providenanoscopicopticalresolution.Herewereportonthein-situsuperresolutionmicroscopyof dye-labelledsubmicronsizedpNIPAMmicrogelsrevealingtheinternaldensityproﬁleduringswellingand collapseofindividualparticles.UsingdirectSTochasticOpticalReconstructionMicroscopy(dSTORM)we demonstratealateralopticalresolutionof30nmandanaxialresolutionof60nm.

1. Introduction

Polymermicrogelsareahybridbetweenacolloidanda poly-merandthecombinationofthesetwoclassesofmaterialsoffers

∗ Correspondingauthor.

E-mailaddress:Frank.Scheffold@unifr.ch(F.Scheffold).

anumberofadvantages[1].Thewell-deﬁnedshapeandsizeofa colloidprovidescontroloverthemicrostructurallengthscalesand responsetimeswhilethepolymericnatureoffersphysico-chemical controlparametersthatcanbesensitivetoexternalstimuli.The mostwidelystudiedmaterialofthiskindisbasedonthepolymer poly(N-isopropylacrylamide)(pNIPAM)[1].Itcanbecross-linked duringsynthesistoobtainmicrogelparticleswithasizethatcanbe controlledintherange100–1000nm.Thevastintereststemsfrom

http://dx.doi.org/10.1016/j.colsurfa.2016.03.010

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thefactthatthepolymeristhermosensitivewithalower-critical solutiontemperatureofapproximately32◦Cinwater[2],which isclosetophysiologicalconditions.Thevolumephasetransition ofthemicrogelscanalsobeinducedbyadditionofalcohols[3,4]

andbychangesinpHwhichoffersaplethoraofpossibilitiesfor thedesignofstimuliresponsivematerialsandforsensingand sub-stancereleaseapplications[5–12].Ithasalsobeenarguedthatthe polymercollapseisreminiscentoftheproteinfoldingmechanism

[13–15].Despitetheoverwhelminginterestadirectvisualization ofthevolumephasetransitionofindividualparticlesin-situhasso farbeenlacking.

Thesizeofmicrogelparticlesistypicallyamicrometreorless and therefore conventional light microscopy cannot resolve its internalstructure.Transmissionelectronmicroscopy(TEM)is nor-mallyonlyappliedtodriedandcollapsedmicrogels[16].Inone studycryo-TEMhasbeenappliedtoswollenpolystyrene-pNIPAM core–shell particles at a singletemperature [17]. This method, althoughcumbersomeandsufferingfromlowcontrast,mighthave potentialforthecharacterizationoftheinternaldensityproﬁleof pNIPAMmicrogels.EquallymodernsynchrotonbasedX-ray nan-otomography,witharesolutioninthe50nmrange,couldin princi-plebeused[18].ScatteringmethodsusingX-ray,neutronsandlight dohavetherequiredresolvingpowerandhavethusbeenemployed frequently[7,19,20].Howevertheyonlyprovideinformationabout radiallyaveragedpropertiesofanensembleofparticlesandthus informationonasinglepartlevelisnotaccessible.

In recent years superresolution microscopy techniques have emergedthatinprinciplecanprovidenanoscopicoptical resolu-tion[21–28].Despitetheiroverwhelmingpopularityintheﬁeldof bioimagingrelativelyfewsuccessfulexamplesfortheirapplication inmaterialssciencesareknown[29–31].Herewereportonthe in-situsuperresolutionmicroscopyofpNIPAMmicrogelsrevealingthe internaldensityproﬁleduringswellingandcollapseofindividual particlesinducedbyadditionofcontrolledamountsofmethanolto thesolvent[19].

2. Experimental

2.1. Microgelsynthesisandlabelling

The microgels are synthesized by free radical precipitation polymerizationin a batch reactor using N-isopropylacrylamide (Acros Organics, 99%), NIPAM, as the monomeric unit and N-(3-aminopropyl)methacrylamide hydrochloride (Polysciences), APMA, asa co-monomer. Theco-monomer is usedin orderto incorporatefreeaminegroupsintothemicrogelsthatarefurther usedas conjugationpoints forthe ﬂuorescentbiomarkerAlexa Fluor647.N,N-Methylenebis(acrylamide) (Sigma–Aldrich, 99%), BIS,isusedasacross-linker.NIPAMisre-crystalizedinhexaneand allotherchemicalsareusedasreceived.NIPAM(1.460g)andBIS (0.103g)aredissolvedin85gofH2O.APMA(0.0066g)isdissolved

in10gofH2O.5goftheAPMAsolutionisaddedtothereactor

andthereactionmixtureislefttode-gasunderanargon atmo-spherefor40minbeforethetemperatureisraisedto70◦C.The initiator, 2,2-Azobis(2-methylpropionamidine) dihydrochloride (Sigma–Aldrich,97%,0.0365g)isdissolvedin5gofH2Opriorto

additiontothereactionmixture.Inordertoobtainaconstantratio betweenNIPAMmonomersandtheco-monomerwecontinuously addAPMAataconstantratethroughoutthesynthesisfollowing a similar procedure suggested recently by Still and coworkers

[32].Threeminutesaftertheinitiatorisaddedtheinjectionpump is started, injecting the remaining 5g of APMA solution at an additionrateof0.5ml/min.Thereactionmixtureiskeptat70◦C for4hbeforeit is lefttocooldownover nightunder constant stirring.Thesuspensionisthencleanedbyfourcentrifugationand re-dispersion cyclesin order to remove unreactedspecies. The

resultingmicrogelshaveadegreeofcross-linkingof4.9mol%and a co-monomer concentration of 0.27mol% assuming complete consumption of allcomponents. It is knownhowever that the crosslinkerBISisconsumedfasterthanNIPAMwhichleadstoa heterogeneouscrosslinking-densityinPNIPAMmicrogels[20].The continuousadditionoftheAPAMduringthesynthesisassuresthat theconjugationpointsfortheﬂuorophoresaredistributed homo-geneously.To labelthemicrogels wemix themwithanexcess amountofﬂuorescentdyeAlexaFluor647inwaterinsideaclean Eppendorfvial.Wethenplaceitonaslowlyoscillatingplatform atroomtemperaturefor2h.Atleastthreecyclesofcentrifugation andresuspensionarethenperformedtoremoveunreacteddye. 2.2. SamplepreparationfordSTORM

Typicalexperimentslastseveralminutesthusrequiring immo-bilizationofthesample.Toachievethisweirreversiblyadsorbthe particlesbyplacingadropofmicrogelsuspensionbetweentwo glasscoverslips,spreadingitintoathinlayer,thenplacingitin theovenat55◦Cuntiltheyaredry.Thecoverslipsarepreviously treatedwithpiranhasolution(amixtureofsulfuricacid(H2SO4)

andhydrogenperoxide(H2O2))tocleanthemthoroughly.The

par-ticlesare thenresuspended intheappropriate water–methanol solutionandfoundtobeimmobileforthedurationofthe exper-iments.ThesolutionalsocontainsCysteamine(Sigma–Aldrich)at 50mMconcentrationandthepHisadjustedto8usingHCl.These stepsareessentialtoachieveblinkingofﬂuorophores[28].In prin-ciple,toachieveoptimalblinkingandthereforethehighestpossible resolutiononeshouldalsoaddoxygenscavengerssuchasGLOX

[28,33]totheimagingsolution.Wedecidedtoonlyinclude cys-teamineinordertokeepthesystemsaspureaspossible,preserving themicrogeldeswellingbehaviourattheexpenseofﬂuorophore brightnessandphotostability.

2.3. Staticanddynamiclightscattering

We use a commercial light scattering goniometer (ALV, Germany) for the characterization of the size of the microgel particlesinsuspension.Agreenlaserwavelength(=532nm)is selectedbecauseitisonlyveryweaklyabsorbedbyﬂuorophores presentinthemicrogelstructure. Weextractthedynamiclight scatteringhydrodynamicradiusRhfromastandardﬁrstcumulant

fitoftheintensitycorrelationfunctionatthreedifferentscattering angles40◦,50◦ and 60◦.The viscosityof themixture hasbeen takenfrompublishedvalues[34].Theradiusandpolydispersity measuredbymeansofstaticlightscatteringareobtainedbyfitting thescatteredintensityasafunctionofthescatteringvectorqto thefuzzyspheremodel[20].Thecontributionofback-reflected light at high scattering angles is taken into account with an additionaladjustableparameterasdescribedinRef.[35].Toavoid aggregation of the microgelsat higher temperatureswe lower theCysteaminecontentto20mMuntilthesuspensionisfound stable.ToverifythatCysteaminehasnoinfluenceonparticlesize wealsoperformmeasurementsatdifferentconcentrations,before theonsetofaggregation.Wefindthatsizedoesnotdependonthe Cysteamineconcentration.

2.4. dSTORMsuperresolutionmicroscopy

WeapplydirectSTochasticOpticalReconstructionMicroscopy (dSTORM)forsuperresolutionimagingasdescribedin[28].Tothis endweusea897NikonTiEclipseinvertedmicroscope,equipped withanEMCCDcamera(AndoriXonUltra) and atotal internal reflection fluorescence (TIRF) arm to achieve a highly inclined illuminationsituationwithlimitedfluorescencebackgroundnoise. The illuminationisprovided by a powerfulred laser(Coherent

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Genesis1Wat639nm)andaweakervioletone(Toptica120mW at405nm),bothcoupledintoasinglemodefibreintotheTIRFarm. Thelightisfocusedonthebackapertureofahighnumerical aper-tureandmagnificationobjective(NA1.49and100×magnification), collimatingthebeam.Illuminationwiththeredlaserisusedto achievesparsefluorophoreblinkingwhilethevioletlaserisused inordertotunethedensityofblinkingmolecules.Anextrazoom lensisplacedbeforethecameratoachieveafinalpixelsize corre-spondingtoanedgelengthof104nmatthegivenmagnification.A dichroicfilterwithacentralwavelengthof700nmandbandwidth of75nmisplacedinthedetectionpathway(ET700/75,Chroma).

For every image we reconstruct we acquire 60,000 frames with8–10msexposuretimeandEMgainsetto300.Weobtain 3000–5000ﬂuorophorelocalizationsperparticlewithtypicallyone totwothousandphotonsdetectedperlocalization.Thethousands of images are then analyzed using the open source software ThunderSTORMwhichdetermines,foreveryﬂuorescentspot,the position,brightnessandlocalizationprecision[36].Wekeeponly pointsthatare localizedwithaprecisionbelow 15nm.To cor-rectfor residualsmalldriftstheimagestack is subdividedinto 20 subsets, each is used to reconstruct an image, then those imagesare crosscorrelated todeterminedriftinformation. The samesoftwareisalsousedtoreconstructsuperresolutionimages

[36].Three-dimensionalimagingisperformedusingthemethod ofastigmatism[21]whichweimplementusingtheadaptiveoptics microscopeadd-onMicAO(ImagineOptic,France).WiththeMicAO inplacethepointspread functionchangesshape dependingon theaxialpositionofthefluorescentemitter.Wecalibrateforthis byimagingfluorescentbeads(100nmdiameter,Tetraspeck, Ter-mofisher)scannedthroughthefocalplane.Duetotherefractive indexmismatchbetweenthesampleandtheglassa focalshift ispresentandmustbecorrectedfor.Asshownin[37],sincethe sampleiswithin1.5␮mofthecoverslipsurface,itissufficientto rescaleallaxialpositionsbyafactorof0.57whenusingthesame optics.Wedeterminethenanoscopicresolutionfromthe distribu-tionofmeasuredlocalizationsobtainedbythesamefluorophore. Althoughthefluorophorepositionisimmobileitstillgives stochas-ticallyspreadlocalizationswhichcanbemodelledbyaGaussian

[21,27].Thefullwidthhalfmaximum(FWHM)thengivesusthe lateralresolution which wedeterminetobe30nm onaverage. Wenotethatﬁtting2Dfor thecollapsedmicrogelswitha box

profileofconstantdensityequallyyieldsaresolutionof30nm. Theaxialresolution is estimatedtoabout60nm butthis value doesnot enter ourquantitativeanalysisandthus hasnotbeen determinedwiththesameaccuracy.ForcomparisontheFWHMof thediffractionlimitedfluorescentspotsisapproximately350nm whichmeansthatinourconfigurationtheresolutionofthe cor-respondingwidefieldmicroscopyisaboutanorderofmagnitude lowerthandSTORM.

3. Resultsanddiscussion

3.1. Lightscatteringcharacterizationofmicrogelparticles

We preparedilute pNIPAM microgel particle suspensions at roomtemperature(T=22◦C)inamixtureofwaterandmethanol. Purewaterisagoodsolventforpoly(N-isopropylacrylamide)at theselectedtemperatureand themicrogels arehighlyswollen. Methanolischosenasadeswellingagentbecauseitoffersa con-venienthandletoinducethevolumephasetransitionbychanging the solvent composition. Moreover, deswelling with methanol doesnotrequiresettingaccuratelydifferenttemperaturesinthe microscopesamplechamber.Addingsomepercentileofmethanol induces deswelling until the particles are maximally collapsed foramethanolcontentof30%[3,4,38].Weﬁrstapplystatic(SLS)

Fig.1.StaticanddynamiclightscatteringcharacterizationofdilutepNIPAM micro-gelsuspensions.(a)Symbols:Staticlightscatteringcurvesfordifferentmethanol contents.Curvesareshiftedforclarity.Dashedlines:Fitwiththefuzzyspheremodel forasizepolydispersityof7%.(b)Tableofresultsobtainedforthehydrodynamic radiusRhfromDLSandR,surfextractedfromtheﬁtoftheSLSdatashownin

panel(a).

anddynamic light scattering(DLS)tocharacterizethemicrogel particles in-situ for different solvent compositions. As we add methanolwe observea shiftof theminimumof thescattering curveI(q)towardslargervaluesofqandanincreaseofthescattered intensity,Fig.1.Theseobservationsshowthatthemicrogel par-ticlesdeswell.Wecanmodelthescatteringcurvesquantitatively in the weak scattering or Rayleigh-Gans-Debye approximation consideringanisotropicdensitydistribution.Tothisendweﬁtthe experimentaldatawiththe‘fuzzysphere’modelofStiegeretal.

[20]takingintoaccountpolydispersityandinternalreﬂectionsin thelightscatteringcuvette.Themodelisderivedbyconvoluting theboxproﬁleofahomogeneoussphere,radiusR,witha Gaus-sian,standarddeviationsurf.InFourierspacetheconvolutionis

representedbyaproductandtheintensitydistributiontakesthe simple form I(q)∝

3[sin(qR)−qRcos(qR)]/exp(−(surfq)2/2)

2

. Thesimplicityofthisexpressionistoalargeextentresponsible for the success of this model although it hasbeen recognized early-onthatthemodelisnotentirelysatisfactoryasitdoesnot predictadecaytozerodensityataﬁnitedistance[1].Alternative modelsforthedensitydistributionhavebeensuggestedsuchas anantisymmetricparabolicshell[39],alinearshellproﬁle[40]or adensecorecoveredbyabrush[41,42].Thelatterdescribeswell theonsetofparticle-particleinteractionsbutuntilnowhasonly beendiscussedforabrushwithconstantdensity[41,43].

Inpracticeweﬁndtheﬁtofthefuzzyspheremodeltothelight scatteringdataexcellentasshowninFig.1.Weobservethatthe particlecoreradiusRshrinksfrom215nmto171nm.Atthesame timetheshellofabout2surf∼50nmthicknesscollapsestonearly

zero.Fromdynamiclightscattering(DLS)onthesamesampleswe obtainthehydrodynamicradiusoftheparticlesRh.Assuggestedin

previousstudiesweﬁndthatR+2surf≈Rh[20].

3.2. dSTORMimagingofmicrogelparticles

WeperformdSTORM [28]measurementsbyhomogeneously illuminating the sample at 639nm under nearly total internal

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Fig.2. dSTORMsuperresolutionmicroscopyofpNIPAMmicrogelsatT=22◦_C._(a)

Standardwideﬁeldﬂuorescencemicroscopyimageoftheswollenmicrogelparticles. (b)dSTORMimageofthesamesample.(c)dSTORMofthedeswellingofthemicrogel particlesuponadditionofmethanol.(d)3Dimageoftheswollenmicrogelparticles withouttheadditionofmethanol.Gridsizeis300nm,scalebar600nm.Theinset showsanenlargedviewofthedensitydistributioninsideanindividualparticle. dSTORMlateralresolutionis∼30nmandaxialresolutionis∼60nm.

reflectionconditions.Additionalilluminationat405nmisadded whennecessarytomaintainasufficientdensityofblinking fluo-rophores.InFig.2(b)and(c),weshowatypicaldSTORMimages.For comparisonwealsoshowanimagetakenwithconventional wide-fieldmicroscopyunderthesameconditionsinFig.2(a).Foreach dSTORMimageweacquire60,000frameswithan8–10ms expo-sure,resultinginmeasurementtimesof8–10min.Foreachframe, imagesofsinglefluorophoresarefitto2DGaussians,determining thex–yposition,intensityandlocalizationprecision.Fromthiswe determinealateralresolutionof30nm(FWHM).Severalmethods areavailabletoobtainthree-dimensionalsuperresolutionimages

[21,44–47],outofthosewechosethatofastigmatism,duetoits relativesimplicityandavailabilityofsoftwareforanalysis.With

thismethodthepointspreadfunctionisdistortedinacontrolled way,encodingaxialpositioninformationintoitsshape.Previous calibrationofthedistortionallowsforthereconstructionof three-dimensionalimages,asshowninFig.2.Withthismethodtheaxial resolutionislowerthanintheplaneandisestimatedtobe60nm, stillanorderofmagnitudebetterthanconfocalmicroscopy.Such 3Dimagingonthenanoscalemightnotbecrucialformoreorless isotropicmicrogelsbutwillbecomeofkeyimportancewhenever theparticlearchitectureismorecomplexsuchasforellipsoidal particlesorheterogeneousstructures[48–50].Inourexperiments wedonotobserveananisotropyoftheparticlesandwetherefore decidedtotakeadvantageofthehigherlateralresolutionforthe quantitativeanalysisoftheradialdensitydistributioninsidethe microgelparticle.Alldensityproﬁlesarethusmodelledbasedon the2Dprojectionofdetectedsitesontheplaneofobservation.

3.3. Microgeldensityproﬁles

InFig.3weshowthemeasuredensembleaveraged2D den-sityproﬁles2D(r)forthreedifferentsolventcompositions.Note

Fig.3. dSTORManalysisofthemicrogelradialdensityproﬁle.(a)Symbols: Mea-sured2Ddensityproﬁles(averageoveraboutonehundredparticles).Dashedlines: FitwiththefuzzyspheremodelwithacoreradiusR,ashellparametersurfanda

fixeddSTORMresolutionof30nm(FWHM).Inset:Measured2Ddensityprofilefrom widefieldfluorescencemicroscopyimagesofswollenpNIPAMmicrogelsasshown inFig.2(a).ThedashedlineiscalculatedusingR=231nm,surf=30nmtakenfrom

Table1(b)andaresolutionof350nm(FWHM).(b)3Dradialdensityproﬁles cor-respondingtotheﬁtsshownin(a).(c)DistributionofsizeR+2surfobtainedfrom

ﬁttingthedensityproﬁleofindividualparticles.Averagesize=291nm,standard deviation=9nm(MeOH=0%).(d)Tableofresultsforthemeanparticlecoreradius andshelldeterminedbydSTORM.

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thatduetotheprojectionahomogeneousspherewithradiusR appearsas2D(r)∝

(R2₋_r2_);_r_≤_R._Moreover_we_have_to_include

thefiniteresolution30nm(FWHM)inouranalysis.Nextwefit imagesoftheswollenmicrogelswhilekeepingconstant.Weapply thefuzzyspheremodelbyStiegeretal.[20]thatwehadusedto modelthestaticlightscatteringcurves,Fig.1.Theadjustablefit parametersarethecoreradiusRandthesmearingparametersurf

whichcorrespondstoabouthalfthethicknessofthefuzzyshell.In 3Dthemodelpredictsadensitydistribution_(r)_∝erfc(r₋R,_surf). Fortheﬁtweconvolutethisfunctiontotakeaccountfortheﬁnite resolution=30nm,projectittotheplaneandadjustR,surfuntil

weobtainabestﬁt(seealsosupportinginformation).Theresults areshown inFig.3(a)asdashedlines. Thecoreradiusandthe smearingparametersurfextractedfromtheﬁtareinquantitative

agreementwiththescatteringresults.Thecorrespondingradial densityprofilesin3DareshowninFig.3(b).Foracomparisonwe alsoshowameasured2Ddensityprofilesobtainedfromstandard widefieldmicroscopy,Fig.2(a).Witharesolution of350nm (FWHM)wefindthisdatainagreementwiththepredictions calcu-latedbasedonthelightscatteringresults(dashedline).Thelargely inferiorresolution howeverdoesnotpermitameaningful inde-pendentanalysisoftheradialdensityprofile.InFig.3(c),weshow theresultsfortheparticlesizeobtainedbyanalysingthedensity profilesofindividualmicrogels.Fromthisdatawecanextractthe polydispersity(standarddeviationdividedbythemean)ofthe par-ticlesize∼R+2surfandfindavalueof3.1%.Thisvalueissomewhat

smallerthantheoneobtainedfromstaticlightscattering,Fig.1.We believethisdiscrepancyisduetothefailureoftheRayleighGans Debyeapproximationinlightscatteringduetotheﬁniterefractive indexcontrastevenforthelowdensityswollenmicrogels[19].The latterresultisahintforthepowerofavisualizationoftheindividual microgels:itallowsadirectreal-spaceinterpretationofthe exper-imentalresultsandthusinmanycasescanprovidemoreaccurate information.

4. Summaryandconclusions

Insummarywecoulddemonstratethesuccessfulapplication ofdSTORMsuperresolutionmicroscopytostimuli-responsive pNI-PAMmicrogels.ForthemicrogelsstudiedthedSTORMresultsare inquantitativeagreementwithstaticanddynamiclight scatter-ing.Ourresultscanserveasabenchmarkandopenthepathway forfutureapplicationstowardsmorecomplexpolymerand micro-gelarchitectures.Anisotropic,deformedorcore–shellmicrogelsas wellasmicrogelsthataredopedordecoratedwithmetal nanoparti-clescanbestudied[49,50].Suchsystemsaredifﬁcultorimpossible tocharacterizein-situwithconventionaltechniques.Moreovera generalizationofourapproachusingmulticolordSTORM[51]can beappliedinanextstepforthenanoscalecharacterizationand co-localizationoffunctional,chemicallydistinctsubunitsofcolloidal particles.

Acknowledgements

ThisworkwassupportedbytheSwissNationalScience Foun-dationthroughprojectnumber149867,andthroughtheNational Centre of Competence in Research Bio-Inspired Materials. We acknowledge ﬁnancial support by the Adolphe Merkle Foun-dation.We thank IsabelleSpühler, Georges Brügger, Veronique Trappe and Davide Calzolari for fruitful discussions. PS grate-fullyacknowledgessupportfromtheEuropeanResearchCouncil (ERC-339678-COMPASS)andtheSwedishResearchCouncil (621-2014-4037).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfa.2016.03. 010.

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