Atomic force microscopy for the investigation of molecular and cellular behavior

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Micron89(2016)60–76

ContentslistsavailableatScienceDirect

Micron

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n

Review

Atomic

force

microscopy

for

the

investigation

of

molecular

and

cellular

behavior

Alper

D. Ozkan,

Ahmet

E. Topal,

Aykutlu

Dana,

Mustafa

O. Guler,

Ayse

B. Tekinay

∗

BilkentUniversity,UNAM-InstituteofMaterialsScienceandNanotechnology,Ankara,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received4May2016 Accepted27July2016 Availableonline29July2016 Keywords:

Atomicforcemicroscopy Biomacromolecules Mechanicalcharacterization Cells

a

b

s

t

r

a

c

t

Thepresentreviewdetailsthemethodsusedforthemeasurementofcellsandtheirexudatesusingatomic forcemicroscopy(AFM)andoutlinesthegeneralconclusionsdrawnbythemechanicalcharacterizationof biologicalmaterialsthroughthismethod.AFMisamaterialcharacterizationtechniquethatcanbe oper-atedinliquidconditions,allowingitsusefortheinvestigationofthemechanicalpropertiesofbiological materialsintheirnativeenvironments.AFMhasbeenusedforthemechanicalinvestigationofproteins, nucleicacids,biofilms,secretions,membranebilayers,tissuesandbacterialoreukaryoticcells;however, comparisonbetweenstudiesisdifficultduetovariancesbetweentipsizesandmorphologies,sample fixationandimmobilizationstrategies,conditionsofmeasurementandthemechanicalparametersused forthequantificationofbiomaterialresponse.AlthoughstandardprotocolsfortheAFMinvestigation ofbiologicalmaterialsarelimitedandminordifferencesinmeasurementconditionsmaycreatelarge discrepancies,themethodisnonethelesshighlyeffectiveforcomparativelyevaluatingthemechanical integrityofbiomaterialsandcanbeusedforthereal-timeacquisitionofelasticitydatafollowingthe introductionofachemicalormechanicalstimulus.Whileitiscurrentlyoflimiteddiagnosticvalue,the techniqueisalsousefulforbasicresearchincancerbiologyandthecharacterizationofdiseaseprogression andwoundhealingprocesses.

Contents

1. Introduction...60

2. Effectofprobemorphology,compositionandsurfacechemistry...61

3. Atomicforcemicroscopyofunicellularorganisms...62

3.1. Bacterialcellsurfaces...62

3.2. Bacterialsecretions,exudatesandbioﬁlms ... 64

4. Atomicforcemicroscopyofmammaliancellsandtissues...64

4.1. Cancerdiagnosisandcharacterization ... 67

4.2. Diagnosisofotherdiseases ... 69

4.3. Stemcelldifferentiation...70

4.4. Extracellularsecretionsandtissuemicroenvironments ... 71

5. Futuredirections...71

References...73

1. Introduction

Bothuni-andmulticellularorganismscoordinatetheirbehavior usinganetworkofchemical,electricalandmechanicalsignals,and employavarietyofsensorymechanismstoperceiveandrespond

∗ Correspondingauthor.

E-mailaddress:atekinay@bilkent.edu.tr(A.B.Tekinay).

tointernalorexternalregulatorycues(Riccaetal.,2013;Johnson, 2013).Inunicellularorganisms,suchsignalsmayassistin feed-ing,attractingconspeciﬁcs,synchronizingreproductivecyclesor initiatingdefensemechanismsinahostileenvironment(Dufour andLevesque,2013);whilemulticellularlifeutilizescell signal-ingnetworkstoregulatecellrecruitment,adhesion,differentiation, proliferationanddeath(WattandHuck,2013;Ravichandran,2003; Zoranovicetal.,2013;JaenischandBird,2003;OwensandWise, 1997).Asthelattercategoryofprocessesareintegraltosustain complexlife,thecharacterizationofregulatorysignalsisofgreat http://dx.doi.org/10.1016/j.micron.2016.07.011

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A.D.Ozkanetal./Micron89(2016)60–76 61 importancetothemedicalandbiologicalsciences,andmuchwork

hasbeenperformedtoelucidatethelinksbetween environmen-talcuesandcellularprocesses(Andoetal.,2013;Carvalhoetal., 2013;Dorobantuetal.,2012).However,whilethechemicaland biological environment of cells are relatively well-deﬁned, the mechanicalpropertiesofcellsandtheirimmediateenvironment areinvestigatedonlytoalesserdegree;partlybecauseofthehigh complexityandvariabilityofthemechanicalinteractionsexhibited bycells andpartly duetolimitationsassociated withthe high-resolutionmechanicalprobingofcellsurfacesandinteriors(Cohen andKalfon-Cohen,2013).Nonetheless,considerableefforthasbeen spenttoestablishhowcellsperceiveandactuponthephysical char-acteristicsofnearbysubstrates(Shaoetal.,1996),andtodetermine howthemechanicalpropertiesofcellsandtissuesarealteredin responsetodiseasestateorenvironmentalfactors,usingmaterial characterizationtoolssuchasmagnetictwistingcytometry,optical tweezers,microneedleprobes,scanningacousticmicroscopyand atomicforcemicroscopy(NeumanandNagy,2008).

Atomicforcemicroscopy(AFM)isacharacterizationtoolthat measures the topology and material properties of surfaces by recordingthedeflectionofametallicprobe(or“tip”)asitmoves overthetargetsurface.AFMcanbeoperatedunderthree princi-palmodes:Incontactmode,thetipisdraggeddirectlyoverthe surfaceanddeflectsawayduetoarepulsiveCoulombic interac-tion,while in non-contact modeit is held at a short(typically <100nm)distanceoverthesampleandoscillatesatafrequency thatdependsontheattractivevanderWaalsforcesactingupon it.Intappingorintermittentcontactmode,thetipiskept oscil-latingabovethesample,andtheoscillationfrequencychangesas thetipapproachesthesurfaceatregularintervals(Giessibl,2003). Contactand intermittentmodesareparticularlysuitableforthe probingofbiologicalsamples,duetotheirapplicabilityinliquid media(Danino,2008).Despiteconsiderablelossesinresolution,a liquidsampleenvironmentallowscellularimaginginanative(or pseudo-native)environmentand,moreimportantly,permitsthe directinvestigationofmechanicalchangesonalivecellsurfacein responsetoanintroducedstimulus(Liuetal.,2005).Time-lapse elastographstakeninthisfashionhavebeenutilizedforadiverse arrayofapplications,includingtovisualizetheformationof amy-loid(Harperetal.,1997)orcollagen(Revenkoetal.,1994)fibers underdifferentenvironmentalconditions,determinehow mem-braneintegrityisalteredin thepresenceofantibiotics(Fantner etal.,2010a),orrecordtheproductionanddissolutionof cytoskele-talelementsduringcellmovement(RotschandRadmacher,2000). Inaddition,itispossibletoutilizetheAFMtipasastimulusto elicitaresponsefromthetargetcell,andtheprobeitselfcanbe functionalizedwithligandmoleculestodeterminetheaffinityof thecellmembranetoaparticularbiologicalmoiety.

DuetotheversatilityandpotentialapplicationareasofAFM, thetechniquehasattractedsubstantialinterestinbiomechanical research,andhasbeenusedinthecharacterizationofagreat vari-etyoftissues,cellsandsub-cellularstructuresinbothlivecondition andfollowingﬁxinganddrying.Thepresentreviewaimstocover thosestudiesthatfocusonthedifferencesinmechanicalproperties associatedwithpathologicalconditionsorchangesin environmen-talcues,andemphasizestheimportanceofthemechanicalUmwelt inmodulatingthebehaviorofbothsingle-celledandmulticellular systems.

2. Effectofprobemorphology,compositionandsurface chemistry

BeforediscussingtheAFMimagingofbiologicalmaterials,the importanceofAFMtipchoiceshouldbeunderlined.The diame-ters,materials,morphologiesandcantileverlengthsofcommercial

AFMprobesshowconsiderablevariance,andoptimalperformance requirestheuseofaprobeconductivetothetaskathand.The com-positionofthesamplematerialshouldbetakenintoconsideration tochoosethespringconstantoftheAFMprobe,assoftermaterials, suchascells,maybedamagedoverrepeatedcontactwiththeAFM tip(Costa,2003).Inaddition,dependingontheareatobescanned, itmaybedesirabletoincreaseordecreasethetipdiameter.Larger tipsareassociatedwithlowerresolution,butcanbeutilizedtoscan largersampleareaswithoutcompromisingtipintegrity,assharper tipsmayexperiencesigniﬁcantwearoverlongscanningdistances, suchaswhenscanningcells.Ontheotherhand,sharpertipsare capableofresolvingsmallerfeaturestoagreaterextent,whichis invaluablewhenmeasuringproteinsandothernanoscale biolog-icalmaterials.Consequently,differencesinmaterialstiffnessthat areevidentundernanoscaleinvestigationmaybeunmeasurable usingmicroscaletips(Stolzetal.,2009a).Ifadhesiondataistobe collected,thematerialandmorphologyoftheAFMtip(alongside substrateproperties)alsodeterminesthesuitablemodelforusein elasticitycalculations(Fig.1).

AFMprobescanalsobefunctionalizedinordertocharacterize theinteractionbetweentwospecifictypesofbiologicalmoieties, suchasbetweenareceptoranditsligand.Thistypeofinteraction isbestexemplifiedbybiotinandavidin,usedbyColtonetal.in theirhallmarkpapertoillustratethepossibilityofusingAFMto directlyevaluatethestrengthofmolecularinteractions(Leeetal., 1994).Mechanicalpropertiesofawidevarietyofproteinshavenow beenelucidated,includingtheinteractionsbetweenantibodiesand theircorrespondingantigens(Allenetal.,1997),actinandmyosin (Koderaetal.,2010),osteopontinandintegrin(Leeetal.,2007),and variouscelladhesionproteoglycans(Dammeretal.,1995).Such proteinscaneitherbecovalentlytetheredtothetargetmaterial (Ebneretal.,2007;Kamruzzahanetal.,2006)orattachedbydrying theproteinsampleonthesurface(Florinetal.,1995).Inaddition, themechanicalstrengthoftheconstituentdomainsofasingle pro-teincanbeevaluatedbyattachingthatproteintoasurfaceand usingtheAFMtiptostretchit(Lietal.,2003).Thisresultsinthe gradualunfoldingoftheprotein,andtheunwindingofeachdomain isassociatedwithamomentarydropinforce.Tensile characteris-ticsoftheimmunoglobulinandfibronectinIIIdomainsoftitinwere investigatedusingthismethod(Riefetal.,1998),andtheabilityof thebacterialribonucleasebarnasetowithstandforcewaslikewise evaluatedbyincorporatingthisproteinintoachimericconstruct consistingoffourTII27and threebarnasesubunits(Bestetal., 2001).

DNAandRNAcanalsobeimmobilizedandcharacterizedina similarmanner,andthemechanicalinvestigationofDNAmolecules ofvaryinglengthsandconfigurationshasbeenperformedusing AFM(Maoetal.,1999; Hansmaetal.,1995).In additiontothe determinationofcovalentbondstrengthinssDNAordsDNA,itis alsopossibletoevaluatethestrengthofthebondsbetween com-plementarystrandsinashortdsDNApiece,ortodeterminethe forcesnecessarytostretchanintactDNAmolecule(Hansmaetal., 1996).High-resolutionAFMimagingcanalsobeusedto character-izethephysicalstructureofaDNAhelix(Fig.2),andmorecomplex DNAarchitecturesandDNA–proteininteractionscanbevisualized andcharacterizedusingatomicforcemicroscopy.Yanevaetal.,for example,confirmedthatDNA-dependentproteinkinase(DNA-PK) canbindtoDNAwithouttheassistanceofKuproteins,andthatthe lattershowsatime-dependentpreferenceforstrandends,by visu-alizingDNA-KuandDNA-DNA-PKinteractionsusingAFM(Yaneva etal.,1997).Theaffinitybetweencellsandspecificproteinscanalso beassessedbyindentingthecellofinterestwithanAFMtip func-tionalizedwiththeproteinofinterest(Hanetal.,1995).Gaubetal. reportedamethodtodistinguishbetweenindividualredbloodcell originsinamixtureofA-andO-grouperythrocytes,usinganAFM tipfunctionalizedwithHelixpomatialectin(Grandboisetal.,2000).

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62 A.D.Ozkanetal./Micron89(2016)60–76

Fig.1. Comparisonofmicro-andnanoindentationfortheidentiﬁcationofchangesassociatedwithaginginthecartilageofC57BL/6mice.Microindentationresultscould detectnodifferencebetween1,10and19-montholdindividuals,whilenanoindentationwasabletodeterminethatthecartilageelasticityofnon-arthriticmicechanges withage.ReplicatedwithpermissionfromStolzetal.(2009b).

Fig.2. AFMtopographyofaplasmid,showingthegeneralappearanceoftheDNA helixundervaryingpeakforces(a–d).Minorandmajorgroovescanbeobserved inAFMimages(a–d,insets),andtheDNAstructurecanbecompressedunderhigh peakforces(e,f).Whitearrowdenotesadislocationinaplasmidloopcreatedby highloadforces.ReplicatedwithpermissionfromPyneetal.(2014).

Thislectindisplaysstrongafﬁnitytoglycolipidsthatarepresentin themembranesofA-groupbutnotO-grouperythrocytes,resulting inhigherruptureforcesassociatedwiththeformer.Thedifferences

inadhesiveforcesarethenutilizedtocreateamapwhereindividual A-andO-groupcellscanbeidentiﬁed.

3. Atomicforcemicroscopyofunicellularorganisms

ArepresentativeselectionofAFMstudiesonthestiffness char-acterizationofbacteria,yeastsandcellularsecretionsisprovided inTable1.Itisreadilyevidentthatseveralmeansofsample prepa-ration are available for measurement, and that values suchas tip-sampleadhesion,F-dcurveslopesandcellularspringconstants canallbeusedtocomparethemechanicalintegritiesofbiological samples;consequently,onlythesestudiesdetailingthefullrange ofmeasurementconditionswereincludedintothetable.Bothair andliquidimaginghavebeenperformedformechanical investi-gations;however,biologicalmaterialsareoftenviscoelasticand maydisplaylargechangesinelasticbehaviordependingon envi-ronmentalhumidity.Assuch,samplesinairtendtohavemuch largerYoung’smodulicomparedtosamplesimagedinliquids(e.g. a10-folddifferencewasobservedinbetweentheelasticmoduliof air-driedandrehydratedmurinesacculifromEscherichiacoli(Yao etal.,1999)).Given thedifferences inmeasurementtechniques andsamplepreparationmethods,aswellasthenaturalvariance inthematerialpropertiesofbacterialcellsandtheirsecretions,it isusuallypreferabletocompareresultswithinstudiesratherthan assumingagivenstiffnessvaluewillapplyunderother experimen-talconditions.

3.1. Bacterialcellsurfaces

Unlikemanyvertebratecelllines,bacterialcellsarenot depen-dentonahighlyspeciﬁcsetofenvironmentalconditionstosurvive, andcantolerateextendedAFMimagingsessionswithout detrimen-taleffects(Ramanetal.,2011;Franciusetal.,2008).Theireaseof procurement,non-demandinggrowthconditionsandthefactthat manylaboratoryspecieseitherare,orserveasmodelsfor,common pathogensmakebacteriapopulartargetsforAFMimaging. Bacte-riamustbeimmobilizedpriortoimaginginliquidmedia,astheir mobilityotherwisemakesitimpossibletoimage,andeven ses-silebacteriacanbelaterallypushedbytheAFMtip(Doktyczetal., 2003).Immobilizationcanbeperformedbydryingandrehydrating, electrostaticbindingtoapositivelychargedsurface(e.g.gelatinor

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A.D. Ozkan et al. / Micron 89 (2016) 60–76 63 Table1

Mechanicalcharacterizationofmembranes,secretionsandsingle-celledorganismsbyAFM.

Sample Tipproperties Imagingconditions Elasticproperties Reference

Sulfate-reducingbacteria Siliconnitride,k=0.06N/m(nominal) Contactmode,air,sampleonmica (Adhesion)−3.9to−4.3nNatcellsurface,−5.1 to−5.9nNatcell-substrateboundary,−6.5to −6.8nNatcell–cellboundary

Fangetal.(2000)

EnteroaggregativeEscherichiacoli, wild-typeanddispersinmutant

Silicon,k=2.8N/m(nominal) Contactmode,liquid(distilledwater)andair, sampleongelatin-treatedmica

(F-dslope)0.133forwild-typestrainonagar, 0.069forwild-typestraininbroth,0.81for dispersinmutantonagar,0.78fordispersin mutantinbroth

Beckmannetal.(2006)

Bacillussubtilis,Micrococcusluteus, Pseudomonasputida,twostrainsof Escherichiacoli

Siliconnitride,k=0.32N/m(nominal) Contactmode,liquid(HMbuffer),sampleon APTEScoverslip

(Springconstant)variesbetween0.16±0.01to 0.41±0.01,higherinGram-positivecells

Volleetal.(2008)

Pseudomonasaeruginosa Siliconnitrideandsiliconnitridewithsilicon oxidetips,k=0.07±0.01(calibratedbythe thermalmethod)

Contactmode,liquid(milliQwater),sampleon poly-l-lysine-coatedglass

(Springconstant)0.044±0.002N/mfor unﬁxed,0.11±0.03N/mfor

glutaraldehyde-ﬁxedcells;creepdeformation behavioralsoinvestigated

Vadillo-Rodriguezetal.(2008)

Klebsiellaterrigena Siliconnitride,k=0.06N/m(nominal) Contactmode,liquid(potassiumphosphate bufferatpH6.8),sampleonpolycarbonate membraneﬁlter/poly-l-lysine-coated glass/immobilizedontipbygluteraldehyde ﬁxation

(Adhesion)−0.26±0.05nNformembrane ﬁlter,−0.5±0.2forpoly-l-lysine,−35±2nN forgluteraldehydeﬁxation,otheradhesion parametersalsomeasured

Vadillo-Rodriguesetal.(2004)

TwoStreptococcussalivariusstrains Siliconnitride,k=0.03N/m(nominal) Contactmode,liquid(deionizedwateror0.1M KClsolution),sampleonpolycarbonate membraneﬁlter

(Adhesionandrepulsion)Fibrillatedstrain showsalargerrepulsionrange,interpretedto reﬂectthelayerofﬁbrils;retractionresultsin threeadhesionforcespotentially

correspondingtothreedifferentlengthsof ﬁbrilsobservedbyelectronmicroscopy

vanderMeietal.(2000)

Desulfovibriodesulfuricans, Pseudomonassp.andan unidentiﬁedlocalmarineisolate

Siliconnitride,k=0.12±0.02N/m(calibrated bythethermalmethod)

Contactmode,liquid(artiﬁcialseawater), samplecoatedontipandbroughtinto interactionwithmetals

(Adhesion)Allthreeisolatesadhereto aluminumbetterthanmildsteel,stainless steel316andcopper;Desulfovibrioand Pseudomonasadherebetterthanthemarine isolate.

Shengetal.(2007)

B.mycoides Silicon,k=0.064N/mand0.4N/m Contactmode(constantheight),liquid (0.145MNaCl),samplecoatedontipand broughtintointeractionwithglass

(Adhesion)7.4±3.7nNofadhesionto hydrophilicglasssurface,49.5±14.42nNof adhesiontohydrophobic-coatedglasssurface

Bowenetal.(2002)

Marinebacterialdepositions Silicon,k=45.7N/m(calibratedbytheadded massmethod)

Tappingmode,air,sampledepositedon ﬂuoridatedandnon-ﬂuoridatedpolyurethane

(Young’smodulus)between1.5and2.2GPa Bakkeretal.(2003)

Bacterialdepositions,suspectedto beextracellularpolymeric substances

Siliconnitride,kvariesfrom0.03to0.5N/m (nominal)

Contactmode,liquid(MilliQwater),sample depositedonpolystrene

(Adhesion)Forcesof0.8±0.2nNobservedover barepolystyrene,asopposedto0.2±0.2nN aftercellattachment

vanderAaandDufrene(2002)

P.aeruginosapili Siliconnitride,0.008±0.004N/m Contactmode,liquid(water),sampleattached topoly-l-lysine-coatedtipsandbroughtinto interactionwithmicasurface

(Adhesion)Ruptureforcesof95pNduring retraction.

Touhamietal.(2006)

E.colibioﬁlms Siliconnitride,k=0.07-0.4N/m Contactmode,air,sampledepositedonglass (Adhesion)Pull-offforcesof122.65pNfor cell-tipinteractionand51.79pNforglass-tip interaction

Ohetal.(2007)

Bacterialcelluloseﬁbers Siliconnitridek=1.03±0.05N/m(calibrated bythethermalmethod,nominalkof0.5N/m notusedduetolargediscrepancy)

Contactmode,air,sampleonsilicon nitride-coatedsilicongrating

(Young’smodulus)78±17GPa Guhadosetal.(2005)

E.coliandE.colispheroplasts Siliconnitride,k=0.1and0.01N/m(nominal, actualspringconstantscalibratedbythe thermalmethod)

Contactmode,liquid(TBS2buffer),sampleon APTES/Glutmica

(Springconstant)0.194N/mforintactcells, 0.571N/mforﬁxedspheroplasts

Sullivanetal.(2007)

Saccharomycescerevisiae Siliconnitride,k=0.008±0.4N/m(calibrated bythethermalmethod)

Contactmode,liquid(milliQwater),sampleon polycarbonatemembraneﬁlter

(Young’smodulus)6.1±2.4MPaonbudscar, 0.6±0.4MPaonsurroundingcellwall

Touhamietal.(2003)

Aspergillusnidulans Siliconnitride,k=0.47±0.06N/m(calibrated bythethermalmethod)

Contactmode,liquid(PBS),sampleon poly-l-lysine-coatedglass

(SpringconstantandYoung’smodulus) 0.29±0.02N/mand110±10MPafor wild-typehyphaeincompletemedium, decreasesformutantstrainlackingachitin synthesisgene,aswellasinthepresenceof 0.6MKCl.

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64 A.D.Ozkanetal./Micron89(2016)60–76 poly-l-lysine),entrapmentinadhesiveproteins,covalentbinding

toamino-orcarboxyl-functionalizedsurfacesorbyphysical con-ﬁnementinmicrowellsorporousmembranes(Doktyczetal.,2003; Suoetal.,2008).Itshouldbekeptinmindthattheimmobilization methodmayalterthesurfacepropertiesoftheentrappedcells,e.g. bydirectlyalteringthebacterialsurfacechemistryortriggeringa defensemechanismagainsttheenvironmentalstressesassociated withtheimmobilizationtechnique.Duetothesmallsizesof bacte-ria,itisalsopossibletofunctionalizeAFMtipswithbacterialcells, whichcanthenbeusedtotesttheinteractionbetweenthe bac-teriumandmaterialsurfaces.Tingetal.,forexample,usedsuchtips toshowthattheGram-negativebacteriaMassiliatimonaeand Pseu-domonasaeruginosaadherebettertostainlesssteelsurfacesthan doestheGram-positiveBacillussubtilis(Harimawanetal.,2011).

AFMstudiesofbacteriafrequentlyfocusonthemechanismsby whichcertainmoleculesinhibitthegrowthofpathogens. Many antibiotics(e.g.beta-lactamantibiotics,polymyxinsand glycopep-tideantibiotics)actbyinhibitingthesynthesisofbacterialcellwalls ormembranes,andtherebyalterthemembraneintegrityofthe affectedbacteria.Otherantibiotic-mediatedeffects,suchas mem-branethinning(Meckeetal.,2005)orporeformation(Mulleretal., 1999),canalsobeobservedbyAFM,andtheeffectsofless con-ventionalantibiotics,suchasplantextracts(Perryetal.,2009)and peptidesequences(daSilvaandTeschke,2005;Meinckenetal., 2005),canalsobeassessed(Fig.3).TheantimicrobialpeptidePGLa hasbeendemonstrated tolowerthestiffness ofEscherichiacoli membranesandcreatemicelle-likestructuresaroundcell mem-branespriortotheireventualrupture,whilegarlicextractwasalso associatedwithmembranedisruption.Chitosanand chitooligosac-charides(COSs)werealsotestedfortheirantimicrobialeffecton E.coliandStaphylococcusaureus,andthethickpeptidoglycanwallof S.aureuswasfoundtoallowthisbacteriumtobetterretainits over-allmorphology,despiteexperiencingasigniﬁcantdecreaseincell rigidity(Fernandesetal.,2009).Whiletheeffectsofantibacterial moleculesmaybeapparentevenunderdryimaging,itisalso possi-bletoquantifytheresultingchangesinatime-dependentmanner bycharacterizingthemechanicalpropertiesofbacterialcellsin liq-uidbeforeandafterintroducingtheantibioticinquestionintothe medium(Fantneretal.,2010a).Thistechniquehastheadvantageof ensuringthatthechangesobservedarefullyduetotheantibiotic,as opposedtoacombinationofitandthedryingprocess,asbacterial cellwallsareviscoelasticandmaygreatlyaltertheirmechanical propertiesinresponsetorelativehumidity(ThwaitesandSurana, 1991).

Avarietyofsubcellularelementscanalsobeidentifiedon mem-branesurfacesusingAFM.Bothbacterialandeukaryoticcellscan beusedintheseefforts,andlipidbilayerscanbesubstitutedas simplifiedmodelsofcellmembranes(Buttetal.,1990;Kuznetsov andMcPherson,2011).Thestructureandfunctionofmembrane proteins(Buttetal.,1990;Fotiadisetal.,2003;Yuanetal.,2002), porecomplexes(Stoffleretal.,1999),gapjunctions(LalandJohn, 1994),amyloidaggregates(Connellyetal.,2012)andavarietyof membrane-componentlipids(Gyorvaryetal.,2003a),aswellas themodeofactionofcholeratoxin(Mouetal.,1995),were inves-tigatedbyatomicforcemicroscopy.Ofparticularinterestarethe recentdevelopmentsinAFM-basedrapid,high-resolutionimaging methods,whichhavegrantedsubstantialinsightintothenature ofsmallsurfaceelements.Processessuchastheself-assemblyof bacterialS-layerproteins(Gyorvaryetal., 2003b), protein fold-ing(Mulleretal.,2002),misfolding(Oberhauseretal.,1999)and crystallization(Reviakineetal.,1998)events,themotionof “walk-ing”proteins(Preineretal.,2014)anddrug-membraneinteractions (Berquandetal.,2004)havebeenthesubjectofreal-timeimaging studies.

3.2. Bacterialsecretions,exudatesandbioﬁlms

Itiswell-knownthatcellsinmulticellularorganismsenhance theirsurvivalandcoordinationthroughtheproductionofan extra-cellularmatrix;however, this property isnot unique tohigher eukaryotes. Bacteria also secrete proteins, polysaccharides and quorumsensingmoleculesthatrelayinformationbetween con-speciﬁccellsandserveasabufferagainstenvironmentalstresses. Inaddition,thesurfaceattachmentof bacteriaisalsomediated byextracellulardepositions,theadhesioncapacitiesofwhichcan bemeasuredthroughAFM. Colanicacidproduction andsurface lipopolysaccharidelengths,forexample,werepreviously demon-stratedtodeterminethestrengthofattachmentofE.colicells,as colanicacid-overproducingmutantswerefoundtoexhibitstronger attachmentwhileshortersurfacelipolysaccharideswere associ-atedwithalackofadhesivecapacity(Razatosetal.,1998).

BacterialbiofilmsarealsoofspecialinterestwithregardstoAFM characterization.Biofilmsareextracellularpolysaccharidesthatare secretedtofacilitatetheattachmentofbacteria(orotherunicellular organisms)toasurface,andprotecttheadheringcellsagainst hos-tileenvironmentalfactorssuchasantibiotics,detergentsandheavy metals(Daviesetal.,1998).Biofilmsareundesirableelementsin manysettings,andsuitablemeanstoinhibittheirformationis nec-essarytopreventpotentialhealthhazardsinfood,agriculturaland medicalindustries.Assuch,themechanicalpropertiesofbiofilms, as wellasthe mechanismsby which antifoulingmolecules act againstbiofilmproduction,havebeeninvestigatedbyusingAFM and othermechanical characterizationtechniques(Beech etal., 2002;ArnoldandBailey,2000).Corrosiondamageandadhesive capacity of biofilms on a variety of metal surfaces have been detailed intheliterature: Holdenet al.reportthat unsaturated andliquid-grownbiofilmsofPseudomonasputidarespond differ-entlytodrying,suggestingthatthebiofilmcompositionisaltered foroptimalgrowthindryandwetenvironments(Auerbachetal., 2000).Inanotherreport,Tayetal.detailtheeffectofsilverions onStaphylococcusepidermidisbiofilmsandproposeamechanism throughwhichsilverdestabilizesthebiofilmstructurebybinding to the electron donor groups provided by the biofilm compo-nents,therebyweakeningthehydrogenbondsthatholdthebiofilm matrixtogether(Chawetal.,2005).

4. Atomicforcemicroscopyofmammaliancellsandtissues AselectionofAFMstudiesonthestiffnesscharacterizationof eukaryoticcellsandtissuesisprovidedinTable2.AFMof mam-maliancellsandtissuesisoftenundertakenfordiseasediagnosis orstemcelldifferentiationstudies,asdiseasedtissuesoftendisplay mechanicalcharacteristicsdistinctfromtheirhealthycounterparts and stem cells are widely known to alter their differentiation pathwaysand mechanicalcharacteristicsdependingonexternal stimuli.Consequently,AFMofcancerandstemcellscanprovide greaterinsightintothepathwaysrequiredforeventssuchas metas-tasisandlineagecommitment.However,measurementofthese samplesismoredifficultthansingle-cellularorganismsor non-livingbiologicalmaterialssuchasbiofilmsandothersecretions,as mammaliancellsrequireaspecificsetofenvironmentalconditions tosurviveandenvironmentalstresscanhaveamajorimpacton theirmechanicalproperties.Inaddition,whilefixativetreatment canbeusedpriortoimaging,fixationmaygreatlyalterthe mechan-icalpropertiesofeukaryoticcells,andlivecellimagingisgenerally requiredtoacquirereliableelasticitydata(Fig.4).Nonetheless, cellcultureconditionscanbereplicatedtosomeextentwithinthe liquidcellofanAFM,andalargenumberofsuccessful investiga-tionshavebeenmaderegardingthemechanicalcharacterofliving

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A.D. Ozkan et al. / Micron 89 (2016) 60–76 65 Table2

MechanicalcharacterizationofmammaliancellsandtissuesbyAFM.

NIH3T3ﬁbroblasts Siliconnitride,k=0.018N/m (calibratedbythermalmethod)

Contactmode,liquid(DMEM containingd-glucose(1000mg/L)and 10%fetalbovineserum,fresh,warmed Ringer’ssolutionusedasmedium replenishment),sampleon ﬁbronectin-coatedglass

(Young’smodulus)4–100kPaoverthecellsurface,lower aroundthenucleusthanintheperiphery

Hagaetal.(2000)

Breastcancerlines(MCF-10A andMCF7)

Siliconnitride,k=0.01N/m(nominal) modiﬁedwitha4.5␮mdiameter polystyrenebead

Contactmode,liquid(culture medium),sampleonglass

(Young’smodulus)0.2–1.2kParange,malignantcellline (MCF7)1.4–1.8timessofterthanbenigncellline (MCF-10A)

Lietal.(2008)

Osteoblasts,mesenchymal stemcellsandosteosarcoma cells

Siliconnitride,k∼20N/m(calibrated usingthermalmethod)

Contactmode,liquid(25mMHEPES), ﬁxedcellsonpolystyrene,glassor collagen-coatedglass

(Young’smodulus)0.7±0.1to2.6±0.7kParange,lower Young’smodulusforMG63osteosarcomacellsoncollagen

Dochevaetal.(2008)

Zonalarticularchondrocytes Sphericalgold-coatedborosilicatebead (5␮mdiameter),k∼0.065N/m (calibratedbythermalmethod)

Contactmode,liquid(DMEM),sample onpoly-l-lysine-coatedglass

(Young’smodulus)Instantaneousmoduliat0.55±0.23kPa forsuperficial,0.29±0.14kPaformiddle/deepcells; relaxedmoduliat0.31±0.15kPaforsuperficial, 0.17±0.09kPaformiddle/deepcells;apparentviscosities at1.15±0.66kPasforsuperficial,0.61±0.69kPasfor middle/deepcells

Darlingetal.(2006)

Cardiacmuscle,skeletal muscleandendothelialcells

Siliconnitride,k=0.03to0.05N/m (calibratedusingthermalmethod)

Contactmode,liquid(growth medium),sampleonglassslide

(Young’smodulus)Moduliof100.3±10.7kPaforcardiac muscle,24.7±3.5kPaforskeletalmuscleand1.4±0.1to 6.8±0.4kPadependingontheregiontestedforepithelial cells

Mathuretal.(2001)

Cardiacmyocytes Siliconnitride,k=0.06N/m(nominal) Contactmode,liquid(culture medium),sampleonlaminin-coated petridish

(Young’smodulus)35.1±0.7kPaforcardiomyocytesfrom 4-montholdrats,42.5±1.0kPaforcardiomyocytesfrom 30-montholdrats.

Lieberetal.,(2004a)

LLC-PK1andMDCKkidney epithelialcelllines

Siliconnitride,k=0.12N/m(nominal) Contactmode,liquid(artiﬁcialurine) (Young’smodulus)1.5±0.8MPaforLLC-PK1cells, 5±1.5MPaforMDCKcells,oxalatetreatmentdecreases Young’smodulusto1.2±MPaforLLC-PK1cells(other stiffnessparametersalsomeasured)

Rabinovichetal.(2005)

Neuronalgrowthcones Siliconnitride,k=0.006N/m(nominal) Contactanddynamicmodes,liquid (L15/ASWmedium),sampleon poly-l-lysine-coatedglass

(Young’smodulus)3–7kPafortheCdomain,7–23kPafor theTdomain,10–40kPaforthePdomain

Xiongetal.(2009)

Healthyandpathological erythrocytes

Siliconnitride,k=0.03(nominal) Contact,liquid(PBS),sampleon poly-l-lysine-coatedglassandﬁxedby glutaraldehyde

(Young’smodulus)Moduliof26±7kPaforhealthy erythrocytes,43±21kPaforhereditaryspherocytosis, 40±24kPaforthalassemiaand90±20kPaforG6PD deﬁciencysamples

Dulinskaetal.(2006)

Liverendothelialcells Siliconnitride,k=0.032N/m Contact,liquid(serum-freeendothelial cellmedium),cellsoncollagen-coated petridisheswithandwithout glutaraldehydeﬁxation

(Young’smodulus)Moduliof2kPaforlivingcellsandover 100kPaforﬁxedcells

Braetetal.(1998)

Oralsquamouscellcarcinoma, normalandmalignantlines

Siliconnitridetip,k=0.01to0.1N/m; APTES-modiﬁedsiliconoxidesphere tip,k∼0.5N/m(calibratedusingSader method)

Contact,air,samplepre-ﬁxedwith2% PFAandﬁxedwith3.7%PFA

(Young’smodulus)Medianvaluesof6.75MPafor“normal” and4.36MPaformetastaticcancercells.Elasticity measurementstakenusingsphere-modiﬁedtips.

Lasalviaetal.(2015)

PC-3prostatecancercells Siliconnitridetip,k=0.012N/m (calibratedusingthermalmethod)

Contact,liquid(culturemedium), samplestreatedwithanticancerdrugs orDMSOcontrolfor24hpriorto analysis.

(Young’smodulus)c.3kPaforuntreatedcells,increasedto c.6–12kPainadose-dependentmannerfollowingdrug treatment.Frequency-dependencyoftheelasticmodulus wasalsotestedandfoundtochangesignificantlyfor Celebrex,BAY,Totamine,TPAandVPAtreatment,butnot forDSF,MKandTaxol.Thiseffectislinkedtothefactthat theformerdrugsmayaltercrosslinkingratesof cytoskeletalfilaments,whilethelatteronlychangefiber lengthandthickness.

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66 A.D. Ozkan et al. / Micron 89 (2016) 60–76 Table2(Continued)

Normalandcancerousbladder epitheliumcells

Siliconnitridetip,k=0.011to0.018 (calibratedusingthermalmethod)

Contact,liquid(culturemedium), sampleonglass

(Adhesionenergy)averageof8.17×10−16_J_for_normal,

26.95×10−16_J_for_cancer_cells

(Young’smodulus)averageof27.57kPafornormal, 2.46kPaforcancercells

Canettaetal.(2014)

Porcinearticularcartilage Siliconnitride,k=0.06N/m(nominal) andborosilicateglassbeadswith r=2.5␮m,k=0.06and13N/m (nominal)

Contact,liquid(PBS),tissueson poly-l-lysine-coatedglass

(Dynamicelasticmodulus)Ontheorderof2.6MPafor borosilicateglassbeads,about100-foldlowerforsharp tips

Stolzetal.(2004)

Articularcartilageofnormal andarthritic(Col9a1−/−

knockout)mice

Siliconnitride,k=0.06N/m(nominal) andmicrospheres,k=10and12N/m

Contact,liquid(PBS),bulktissuesglued onaroundTeﬂondisk

(Dynamicelasticmodulus)1.3±0.4MPaformicrospheres (nochangerecordedbetweenages);22.3±1.5kPa, 36.8±1.5kPaand50.9±4.7kPaforsharptipsin1-, 10-and19-montholdnormalmice;22.3±1.5kPa,25.5±kPa and27.7±1.1kPainthenon-thickened,intermediateand heavilythickenedcollagenﬁbersof1-montholdarthritic mice.

Stolzetal.(2009b)

Aorticintimaofrats Notlisted,tipmountedonacustom platformforinvivoAFMimaging

Anaesthetizedlivinganimals (Young’smodulus)0.4–0.5MParangeforbloodvessels withoutdruginﬂuence,raisedtoc.1.0MPainthepresence ofnitroglycerinanddecreasedbacktoc.0.3MPainthe presenceofnorepinephrine

Maoetal.(2009)

Anteriorhumancornealstroma Phosphorus-dopedsilicon,k=25and 33N/m(calibratedbyanoptical method,asdescribedbySaderetal.)

Contact,liquid(15%dextran),bulk tissueplacedonTeﬂoncellwithout attachment

(Young’smodulus)Between1.14and2.63MPa,consistent acrosstheindentationdepths(between1.0and2.7␮m)

Lombardoetal.(2012),Sader etal.(1999)

Humancornealbasement membrane

Borosilicateglass,k=0.06N/m (nominal)

Contact,liquid(PBS),a3×3mmtissue piecedissectedandgluedontoawell onsteeldisk

(Young’smodulus)2–15KPa,meanof7.5±4.2kPaaverage fortheanteriorbasementmembrane;20–80KPa, 50±17.8kPaaveragefortheDescemet’smembrane

Lastetal.(2009)

Monkeylenses Gold-coatedtip,k=0.01N/m (nominal),calibratedbythe relationshipbetweenappliedvoltage andcantileverdeﬂection

Contact,liquid(BSS),lensesdissected andplacedonTeﬂonslide

(Young’smodulus)1.720±0.88kPa Ziebarthetal.(2007)

Humanbone Various Various (Young’smodulus)16.6±1.1to27.1±1.7fordryadult

tibiae(lowerforchildren),13.4±2.0to22.7±3.1fordry adultvertebrae(lowerforwetsamples),16.58±0.32to 26.6±2.1fordryadultfemoralmidshaft(lowerforwet samplesandinthefemoralneck),otherbone measurementsandtissuehardnessesalsonoted

Thurner(2009)

Bovineoculartendonﬁbers Siliconnitridetip,k=0.02N/m (calibratedusingthermalmethod)

Contact,air(samplingchamberkeptat 100%humidity),samplegluedonglass petridish

(Young’smodulus)60±2.69MPaforlateralrectus, 59.69±5.34MPaforinferiorrectus,56.92±1.91MPafor medialrectus,59.66±2.64MPaforsuperiorrectus, 57.7±1.36MPaforinferiorobliqueand59.15±2.03for superiorobliquetendons.Differencesbetweentendon elasticitiesarenotstatisticallysigniﬁcant.

Yooetal.(2014)

Breasttissuesections Borosilicateglasstip,k=0.06N/m (nominal),individualtipscalibrated usingthermalmethod

Contact,liquid(PBSsuppliedwith proteaseinhibitorsandpropidium iodide),samplessectionedby cryomicrotomy

(Young’smodulus)c.400Painhealthyandnon-invasive tumorregions,4-foldincreaseinaveragestiffnessin invasivetumorfront.Higheraveragestiffnessinthe invasivefrontiscausedbyhighlystiff(>5kPa)regionsin thisarea.Aggressivebreastcancersubtypesarealsofound toexhibithigherYoung’smoduli,quantiﬁedintermsof upper10%stiffness.

Acerbietal.(2015)

Benignandaggressiveprostate tumors

Siliconnitridetip,k=0.06N/m (nominal),individualtipscalibrated usingthermalmethod

Contact,liquid(physiologicalbuffer), sampleslicedbyrazorandgluedon glass

(Young’smodulus)3.03±0.64kPaforbenign,1.727±1.22 forcanceroustissues.TheaverageYoung’smodulusfor cancersampleswithGleasonscoresinthe2–7rangewas 2.07±1.30,thisvaluewas1.39±0.48forsampleswith Gleasonscoresinthe8–10range.Inaddition,metastatic tumorshadanaveragemodulusof1.06±0.58,while non-metastaticcancertissuehadanaverageelasticity valueof1.99±1.24.Thesevaluesreﬂectthetissue microenvironmentandcontrastmacro-scaleelastography results,inwhichmoreaggressivecancersarestiffer.

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A.D.Ozkanetal./Micron89(2016)60–76 67

Fig.3. Theeffectofanantimicrobialpeptide(CM15)onE.colicellwalls,asobservedbyhigh-speedAFM.Disruptionsbegintoappearoncellsurfacesasearlyast=13sand increaseinseveritywithtime(a).Althoughsomebacteriaresisttheeffectsofthepeptide(b,c),theseindividualsneverthelessreacttoprolongedtreatment(d,att=∼30min). ReplicatedwithpermissionfromFantneretal.(2010b).

mammaliancellsandtissues,eitherculturedexvivoorcollected immediatelypriortoimaging.

4.1. Cancerdiagnosisandcharacterization

Whilemedicaladvanceshaveledtosignificantdecreasesinthe incidencesofmanycancers,cancerstillremainstobeoneofthe mostimportantdiseasesinrecent history.Itis well-established thatcancercellsexhibitmarkeddifferencesinstiffnessand elas-ticitycomparedtotheirhealthycounterparts(Crossetal.,2007); however,diagnosticapplicationsof mechanicalcharacterization methodsshouldcurrentlybeconsideredlimited.Effective diagnos-tictechniquesmustascertainthepresence(orabsence)ofdisease withhighconfidenceandusingminimalamountsofsample tis-sueandtime,anddespitetheabilityofmechanicalmeasurements toeffectivelydistinguishbetweenhealthyand tumorcells,it is unlikelythatabiopsyatanearlystageofdiseasewouldyieldtumor cellsinnumbersnecessarytoperformdiagnosisbasedpurelyon mechanicaldata.Nonetheless,oncethepresenceofatumoris con-firmed usingmore conventional methods, AFM mayserve as a valuabletoolfor itscharacterization:Laidleretal.,forexample, reportthepossibilityofutilizingAFMtodeterminewhethera sus-pectedbreast orprostatetumoris malignantonthebasisofits

elasticproperties(Lekkaetal.,2012).Inaddition,AFMcantilevers canbefunctionalizedwithantibodiesfordisease-speciﬁcmarkers andusedinthedetectionofcancerandotherdisorders(Laurent etal.,2014);however,thistechniqueeffectivelyconvertstheAFM intoabiosensororsortingsysteminsteadofrelyingonitscapacity formechanicalcharacterization.

Greaterutilityliesin thecharacterization oftumorcellsand theirinteractionsbyAFM,whichmayfurtherthecurrent under-standingofcancerbiologyand allowthedesign andevaluation of novelcancerdrugs. Discrepanciesbetweentheelasticitiesof cancerandnon-malignantcells,forexample,havebeenrecorded inhumanlung,breast,pancreas,ﬁbroblast,prostate, adenocarci-nomaandothercelllines(MullerandDufrene,2008;Crossetal., 2011).Thesechanges are suspectedtoincrease themobility of malignantcellsduringmetastasis,anddecreasesincellstiffness appear tobeprogressive, withmore malignantcells expressing lowerYoung’smoduli.Fuhrmannetal.reportedthatthedysplastic Barrett’sesophaguscelllinesarelessrigidcomparedto metaplas-ticcells,whichinturnaresofterthantheirhealthycounterparts (Fuhrmannetal.,2011).Otherchangesin cellmorphologymay alsooccurtofacilitatemetastasis,andthesetoocanbequantiﬁed byAFM.Sokolov etal.,forexample, havedeterminedthat can-ceroushumancervicalepithelial cellsdisplay twobrushlayers,

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Fig.4.Effectoffixationoncellularelasticity.Unfixed(a,d)andGA-fixed(bandefor20min;candffor60min)cellshavedistinctappearancesandelasticmoduli;withGA fixationgreatlyincreasingcellularYoung’smoduliinatime-dependentmanner(g).ReplicatedwithpermissionfromShibata-Sekietal.(2015).

incontrasttothesingle-lengthmolecularbrushofnon-malignant cervicalepithelium,which mayalsoassistin invasion attempts bythesemalignantcells(Iyeretal.,2009).Inaddition,thetumor microenvironmentalsoappearstocontributesigniﬁcantlytothe alterationsintumorelasticity:Insteadoftheexpecteddecreasein Young’smoduli,Weaveretal.observedatime-dependentincrease intumorstiffnessduringmammarytumordevelopmentinPyMT mice,andthisincreaseinrigiditywasalsoreﬂectedinepithelial cellstakenfromthetumorsite.However,cellstakenoutoftheir nativeenvironmentandgrowninvitrohadlowerYoung’s mod-ulicompared totheirinvivocounterparts,andtheinhibitionof theECM-crosslinkingenzymelysyloxidasemitigatedthegradual increaseintheelasticmodulusofmammaryglandtumors(Fig.5) (Lopez etal.,2011).Assuch,tissue culturesamplesandinvivo tumorcellsmaynotnecessarilyagreeinelasticproperties,asthe stiffnessoftheformerdependsonthecytoskeletalpropertiesof thecells,whilethatofthelatterislargelymediatedbythetissue microenvironment.

DrugresponsesofcancercellscanalsobequantifiedbyAFM. Zhangetal.observedthatHeLa,HepG2andC6cellsexperience dose-dependentmorphologicalchangesontheircellmembranes followingtreatmentwithcolchicineorcytarabine.Surface rough-nessincreasedandporesappearedonthecellmembraneafterdrug administrationandbeforeMTT-quantifiabledecreasesinviability couldbeobserved,suggestingthatthesurfacealterations repre-sentanearlyresponsetodrugpresence(Wangetal.,2009).Such differencesmaybemonitoredtoevaluatetheeffectivenessofdrug candidates.Changesassociatedwithgenedeletionorrestoration canalsobeobservedthroughAFM:Zhouetal.reportedthatthe expressionofBRMS1(restoringbreastcancermetastasis suppres-sor1)inducesincreasesincelladhesioncapacity,cellularspring constantandYoung’smodulus,whichsupportstheideathatBRMS1 expressionisassociatedwithcytoskeletalrearrangementsthatare unfavorablefor metastaticactivity.Cell morphologyand rough-nesswerealsoalteredfollowingBRMS1expression,possiblyasa consequenceofcytoskeletalmodifications(Wuetal.,2010).

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A.D.Ozkanetal./Micron89(2016)60–76 69

Fig.5. Changesassociatedwithmammarytumorformation,asquantiﬁedbyAFM.Normalmammaryducttissueislessstiffcomparedtotumortissue(a),andthetissueelastic modulusincreaseswithtumorage(b).Inaddition,theinhibitionofcollagencross-linkingbyBAPN(whichblockstheactivityoflysyloxidase)preventsthetumor-associated increaseinstiffness,suggestingthatthetumorenvironmentismodiﬁedthroughchangesintheextracellularmatrix.ReplicatedwithpermissionfromLopezetal.(2011).

4.2. Diagnosisofotherdiseases

Whilecancer-associatedchangesincellelasticityare particu-larlydrastic,canceris by nomeanstheonlycondition toalter themechanicalpropertiesofaffectedtissues.Avarietyofother conditions,includingmalaria,sicklecellanemia,hepaticfibrosis, cardiovasculardisease,renalstiffness,musculardystrophiesand bonedisorders,havealsobeenassociatedwithnotablechangesin theelasticpropertiesoftheaffectedtissues(Fig.6)(Costa,2003; Nagaoetal.,2000;Lieberetal.,2004b;Bozecetal.,2005;Engler etal.,2004).Mechanicaldiagnosismethodshavebeendevisedfor somesuchconditions,andAFMcanbeutilizedtostudythe struc-turalandmechanicalpropertiesofaffectedtissues;butitmustbe notedthat,aswithcancer,AFM-baseddiagnosticmethodsforthese diseasescurrentlyappeartobemoresuitedtowardssupporting conventionaldiagnosis.However,thehigh-resolutionimagingand mechanicalprobingcapacityofAFMisidealforthedetermination ofthecausesunderlyingtheprogressionofthediseasesinquestion. SeveralreportsonAFM-baseddiseasecharacterizationexistin theliterature.Szymo ńskietal.,forexample,demonstratethatred bloodcellsbelongingtopatientswithhereditaryblooddiseases generally bear a higher Young’s modulus compared to healthy erythrocytes(Dulinskaetal.,2006).Inparticular, gluteraldehyde-fixed, poly-l-lysine-immobilized erythrocytes of patients with hereditary spherocytosis, thalassemia or G6PD deficiency were

stiffercomparedtonormalcells,whilepatientswithanisocytosis displayedtwodistinctpeaksintheirhistogramofYoung’s mod-uli,correspondingtohealthyanddiseasedpopulationsofcells.In patientswithhereditaryspherocytosis,changesincell morphol-ogywerealsoobserved.Similarly,Vatneretal.haveinvestigated theeffectsofagingoncardiacmyocytesbyAFMindentation, com-paring4month-and30month-oldratsinordertoassesswhether myocytehealthinﬂuencestheaging-associateddiastolic dysfunc-tionof theleftventricule(Lieber etal., 2004b).Myocytes were foundto signiﬁcantly increase in stiffness withage,suggesting thattheobserveddysfunctioncanbelinkedatleastinparttothe malfunctionofindividualmyocytes.Theeffectofagingoncellular Young’smoduliwasalsonotedbySokolovetal.,whohaveshown thatolderhumanepithelialcellsaremorerigidthantheiryounger counterparts,andhavefurtherdemonstratedthatanincreaseinthe densityofcytoskeletalelementsisresponsiblefortheage-related increaseinstiffness(Berdyyevaetal.,2005a,b).Theearly detec-tionofosteoarthritiswasalsoperformedonthearticularcartilage ofnormalandarthriticmice,andnanoindentation(butnot micro-scaletips)wasshowntoresolvethegradualchangesincartilage stiffnessbetweenarthriticandnon-arthriticanimalsofthesame age(Stolzetal.,2009b).

AnotherinterestingfrontierinAFM-based disease character-ization is theinvestigation of malfunctioning proteinsthat are involvedinthepathogenesisofneurodegenerativedisorderssuch

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Fig.6.Young’smodulusmeasurementsoferythrocytesfromyoungandhealthy(YHP;a,dandg);oldandhealthy(OHP;b,eandh)andoldandtype-IIdiabetic(ODP; c,fandi)individuals.ErythrocytesexhibitedhigherstiffnessinODPindividualscomparedtoeitherYHPorOHP(1.78±0.39×105_N/m2_v._1.04_±_0.19_×₁₀5_N/m2_and

1.53±0.41×105_N/m2_,_{respectively).}_In_addition,_OHP_and_ODP_erythrocytes_exhibited_higher_adhesion_compared_to_YHP_erythrocytes₍₄₂₀_±₂₅_pN_and₅₁₀_±₆₃_pN_v.

200±38pN,respectively).ReplicatedwithpermissionfromJinetal.(2010). as Alzheimer’s disease, Parkinson’s disease, Huntington’s dis-easeandamyotrophiclateralsclerosis.Thesedisordersseverely decreasethequalityoflife,areexceptionallycommonamongthe elderlyandhavenodefinitivecures,whichrendersitcrucialtogain furtherinsightintothenatureoftheircausativeagents.Assuch, theformationofamyloidplaqueshasbeeninvestigatedindetail usingavarietyofbiological,chemicalandmaterialcharacterization methods,includingseveralAFM-basedstudies.Lansburyetal.,for example,reportedontheinvitroformationofmetastableA␤ amy-loidfibrilprecursorsthatmaylaterdevelopintocompleteamyloid assemblies,andsuggestthatthehaltingofthismechanismmay preventtheonsetofAlzheimer’sdiseasebyretainingthe precur-sorfibrils(“protofibrils”)intheirbenignintermediateform(Harper etal.,1997).Inaddition,KowalewskiandHoltzmandemonstrated thatthesize,shapeandproductionkineticsofA␤aggregateswere altereddependingonthesurfaceonwhichtheaggregationoccurs (KowalewskiandHoltzman,1999)(i.e.particulateassemblieswere generatedonthehydrophilicmicasurface,while␤-sheetsformed onthehydrophobicgraphite),andsuggestedthatthe␤-sheet form-ingbehaviorofA␤ongraphitemayyieldusefulinformationonhow proteinfoldingoccursinvivo.TheabilityofA␤peptidestoform ion-channellikestructureswithoutinteractingwithother mem-branecomponentswasalsoconfirmedusingAFM,withtrimeric, tetrameric,pentamericandhexamericporestructuresbeing iden-tifiedin topographic images. BothD-and L-enantiomers of A␤ peptideswereabletoformthesechannels,suggestingthatthepore formationmechanismisnotstereospecific(Connellyetal.,2012).

4.3. Stemcelldifferentiation

Fluctuationsinmechanicalpropertiesdonotnecessarilysuggest aging-relateddeteriorationoradiseasestate,ashealthycellsmay

alsorespondtoenvironmentalsignalsbyalteringtheirmembrane integrity.Thisismostobviouslyobservedinstemcells,as differen-tiationinducesfundamentalchangesnotonlyincellmorphology andexpressionpatterns,butalsoinmembranecontentand stiff-ness(Discher,2006;Evansetal.,2009;ReillyandEngler,2010). Since themaintenance, recruitmentand differentiationof stem cellsareintimately linkedtothemechanicalpropertiesof their immediateenvironment,itisfeasibletouseAFMmeasurementsin ordertodeterminethefactorsthatdrivethedifferentiationprocess inthesecells.Suchfactorsarerelativelyclearinsomecases,such asthemesenchymalstemcelldifferentiationintomyogenic, chon-drogenicorosteogeniclineages,butthemechanicaltriggersbehind thedifferentiationofothercellsarewelllessunderstood.In addi-tiontostemcelldifferentiation,changesinthemicroenvironmental conditionsoftissuescanbeassessedusingAFMorother mechani-calcharacterizationmethods,especiallyinsituationsinvolvingthe slowrecoveryofadamagedsystem,asinthecasesofbonefracture healing.Otherprocesses,suchasthelocalremodelingand extra-cellularmatrixsecretionofcellsinatissuecultureenvironment, orthecapacityofbiomimeticmaterialstoimitatethemechanical environmentoftheirtissuemodel,alsofallwithinthepurviewof AFM.

Thedifferencesbetweenstemandderivedcellshavealsobeen investigated by AFM. Guilak et al. have conﬁrmed that chon-drocytes,osteoblastsandadipocytes,theprimarydifferentiation productsof mesenchymalstemcells, displaydifferentrigidities, anddeterminedthatundifferentiatedmesenchymalstemcellsare similartoadipocytesintheirmechanicalcharacteristics(Darling etal.,2008).Schiekeretal.reportedthatthetwosubcategories of humanmesenchymal stem cells, ﬂat cells and rapidly self-renewing cells, can also be differentiated on thebasis of their morphologicalandadhesivecharacteristics(Dochevaetal.,2008),

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A.D.Ozkanetal./Micron89(2016)60–76 71 withtheformertypeappearingtopographicallysimilartohuman

osteoblastsand displayinga highadhesiontothesurface,while thelatterexhibitedcharacteristicssimilartoMG63osteosarcoma cells anddisplayeda smoothertopography. Evenstemcellfate canbepredictedbymechanical propertiesprior tothe appear-anceofvisibleindicators.Gonzalez-CruzandDarlingreportedthat adipose-derivedstemcellscanbeclassiﬁedaccordingtotheir dif-ferentiationpotentialtoadipogenic, osteogenicorchondrogenic lineagesonthebasisoftheirmechanicalbehavior(Gonzalez-Cruz etal.,2012).Ascouldbeexpected;softer,largerand more pli-antadipose-derivedstemcellsaremorelikelytodifferentiateinto adipocytes,whilesmallerandmorerigidstemcellsareinclined towardsosteogenicor chondrogenicdifferentiation.Inaddition, theeffectofstemcellsonaninvivoenvironmentcanbedetermined followingtheirimplantation:Mesenchymalstemcellsinjectedinto thesiteofamyocardialinfarctionhavebeenrecordedtodecrease therigidityofthemyocardium,whichwasassociatedwithreduced ﬁbrosisandabetterprognosisforthepostinfarctedheart.

Theimportanceoftheextracellularmatrix(ECM)forthe main-tenance and differentiation of stem cells is both obvious and paramount(Bosnakovskietal.,2006;Suzukietal.,2003).Itis there-foreunsurprisingthatthemechanicalpropertiesofECMelements arevitalinprovidingthesignalsresponsibleforinducingstemcells todifferentiate,orforretainingthemintheirquiescentstate(Reilly andEngler,2010;Guilaketal.,2009).Greatdifferences existin theECMrigiditiesofadulttissues,from0.1kPainbrainto>30kPa inbone(Huangetal.,2012),andexcessivelysoftorrigidtissues mayresultinsuboptimaldifferentiation(seee.g.Engleretal.foran accountofaberrantmyotubeformationassociatedwith unsatisfac-torysurfacestiffness(Engleretal.,2004)).Inadditiontoadultstem cellsinmaturetissues,thereisalsoevidencethatendo-,meso-and ectodermalprogenitorsarearrangedintodistinctivegermlayers withtheassistanceofdifferencesintensilestrength(Discheretal., 2005;Puechetal.,2005):Cell-cortextensionsofendodermal pro-genitorsingastrulatingzebraﬁshembryosarehigherthanthatof themesodermalprogenitors,whichinturnarelargerthanthatof ectodermalprogenitorcells(Fig.7)(Kriegetal.,2008b).Inasimilar vein,cardiacloopinginchickenembryoswassuggestedtooccur asaresultofastiffness-mediatedasymmetryinthedeveloping cardiacjelly(Zamiretal.,2003).

4.4. Extracellularsecretionsandtissuemicroenvironments

AFMcanbeusedtomeasurethenativestiffnessoftissue micro-andnanoenvironments,althoughothermethods,suchas microin-dentation,canalsobeusedforthemicromechanicalinvestigation oflargerareas oftissues. Bone,cartilageand theoculartissues havebeenfrequentlyinvestigatedusingAFM,andage-or disease-relatedeffectshavebeenfoundtobereﬂectedintissuestiffness.As withbacterialandeukaryoticcells,themethodofsample prepara-tioncangreatlyaltertheelasticmodulusoftissues,andsampling locationlikewisehasasubstantialeffectonmechanicalproperties (Stolzetal.,2009b;Thurner,2009).Itmustalsobekeptinmind thatthemicro-andnanoscalestiffnessesofagiventissuemay dif-fergreatly,e.g.themicrostiffnessofporcinearticularcartilagewas observedtobeover100-foldgreaterthanitsnanostiffness,and theage-relatedeffectsofosteoarthritisonthearticularcartilage ofmicecouldonlybeobservedthroughnanoscaletips(Stolzetal., 2009a,2004).Assuch,caremustbetakennottocomparetheresults ofcell-ortissue-basedAFMstudiestoliteratureexamplesthatuse dissimilarconditionsofmeasurement.

Theextentofnatural heterogeneitywithina givencell pop-ulationortissue samplecan alsobeestablishedusingAFM.An AFMapparatusadaptedforuseinlivingbraintissue,forexample, hasbeenusedtodemonstratethattherat hippocampusis het-erogeneous,andthathippocampalsubregionsareassociatedwith

differentelasticmoduli(Elkinetal.,2007).Thewoundhealing pro-cesshasalsobeencharacterizedusingAFM,andtheleadingedge ofthewoundwasfoundtoexhibitaspatiallylimitedincreasein stiffnesstorecruitfibroblaststothewoundsite.Thislocalizedpeak iscreatedbycytoskeletalchangesinthecellsofthewoundedge, anddisappearsiftheexpressionoftheactinfiber-regulating pro-teinRhoAisdisabled,resultinginalackoffibroblasticrecruitment tothesiteofinjury(Waghetal.,2008).Inadditiontotissues,an individualcellcanalsobeanalyzedtoidentifydistinctregionsonits surface:Scheuringetal.havecharacterizedthemechanismsused byredbloodcellstocontortduringtheirpassagethroughblood ves-sels,utilizingamethodinwhichthefunctionofknownstructural proteinsarealteredandtheresultantchangesonthecellsurface aremeasuredtomaptheseproteinsontothemechanically het-erogeneousregionspresentontheerythrocyte(Picasetal.,2013). TheadditionofMgATPwasutilizedtoelicitchangesonthe mem-braneelementsontheextracellular(“outwards”)andcytoplasmic (“inwards”)sidesoftheerythrocyte,whichbehavedintwo dis-tinctwaysandthereforeallowedthedetailedcharacterizationof membranestructuresandmolecularcompositionsonbothsides.

In addition to characterizing the structure of previously depositedmatrixmicroenvironments,AFMcanalsodeterminehow cellularexudatesaresecretedoutsidecells,especiallywithregards tothemembranebuddingandfusioneventsthatoccurduringthe secretionprocess.Researchinthisdirectionfocusesmostlyoncells involvedinthesecretionofhormonesordigestiveﬂuids,suchas pancreaticacinarcells(Schneideretal.,1997b)orgrowthhormone (GH)-secretingcells(Choetal.,2002).Secretioninacinarcellswas foundtodependonlarge(500–2000nm)apical“pits”thatcontain 3–20smaller(100–180nm)“depressions”or“fusionpores”,the diametersofwhichincreaseduringamylasesecretion(Fig.8). Simi-larpitsanddepressionswerealsopresentinGH-secretingcells,and theexistenceofGHwithinthesestructureswasconﬁrmedusing gold-taggedGH antibodies,demonstrating that thedepressions weresecretoryinnature.Acombinationofatomicforcemicroscopy andconfocalmicroscopywasalsousedtodemonstratethatthe secretoryvesicleswellingprocessisregulatedbyaGTP-binding protein(Jenaetal.,1997).

5. Futuredirections

AFMisaversatiletechniqueandcanbeusedinthemechanical characterizationofabroadrangeofbiomaterials,rangingfrom sin-glemoleculestobulktissues.Itsnanometer-scaleresolutionallows thedirectobservationofproteins,nucleicacidsandother biologi-callyimportantmolecules,whileitsabilitytocharacterizematerial stiffnessinliquidsisacrucialadvantageforinvestigatingthe nat-uralmechanicalenvironmentofcells.However,themethodsused forthepreparationofbiologicalsamplesforAFMimagingarehighly diverseandmayalterthemechanicalpropertiesofbiomaterials. Environmentalconditionssuchastemperatureandbuffersalinity canaltertheresponseofcellstomechanicalstimuli,whileﬁxation andimmobilizationprotocolsmaydirectlychangethechemical compositionofthetreatedcells.Theseeffectsinturnmakeit difﬁ-culttocompareresultsbetweenstudiesperformedunderdifferent conditions.Whilecountermeasures,suchasliquidcellswhich sup-plytheenvironmentnormallyexperiencedbymammaliancellsin cellculture,havebeendeveloped,agreatdealofprocess optimiza-tionandstandardizationisstillnecessaryforAFMtotrulyemerge asabiologicaltool.

Nevertheless, the mechanical environment of cells remains largelyunexplored,andAFMisanidealmethodforcharacterizing thebehaviorsofcellsandtissuesunderclose-to-naturalconditions. Thetechniquewillnodoubtremainindispensableforstudyingthe mechanicalcomponentsofsignalingpathwaysand theeffectof

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Fig.7.Endo-andmesodermalcellsofthedevelopingzebraﬁshembryopreferentiallyadheretocellssharingthesamegermlayerinacadherin-dependentmanner.Adhesive forcesbetweentwocellscanbemeasuredbyattachingonecelltotheprobe(a).Endo-andmesodermalcellsexhibitstrongerhomotypicadhesioncomparedtoectodermal cells,andheterotypicadhesionisgenerallyweakerthanendoderm–endodermandmesoderm–mesoderminteractions(bandc,dusedascontroltoensurecontactstiffness didn’taccountforthedifferencesobserved).Calciumdepletionandsuppressionofcadherinblockstheobservedeffects,andcadherinexpressionishigherinendo-and mesodermalcells,suggestingthatadhesioniscadherin-dependent(e,f).ReplicatedwithpermissionfromKriegetal.(2008a).

Fig.8.Inducedsecretionofamylasefromacinarcellsofthepancreas,showing“depressions”inasingle“pit”(a)andtheirsecretoryresponses5min(b)and30min(c) aftersimulationbythesecretion-stimulatingpeptideMas7.Secretionisassociatedwithincreasesinthediametersanddepthsofthesecretorydepressions.Replicatedwith permissionfromSchneideretal.(1997a).

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A.D.Ozkanetal./Micron89(2016)60–76 73 physicalsignalsonmetabolicfunctionsundernormalanddisease

states,whichisanareathathasremainedlargelyuninvestigated untilnow,forlackofadequatemethodsofanalysis.

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