Microscopic characterization of peptide nanostructures

ContentslistsavailableatScienceDirect

Micron

jo u rn al h om epa 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

Microscopic

characterization

of

peptide

nanostructures

Rashad

Mammadov,

Ayse

B.

Tekinay,

Aykutlu

Dana,

Mustafa

O.

Guler

UNAM-InstituteofMaterialsScienceandNanotechnology,BilkentUniversity,Ankara06800,Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21April2011

Receivedinrevisedform7July2011 Accepted8July2011 Keywords: Peptide Nanofiber Imaging Nanomaterialscharacterization

a

b

s

t

r

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c

t

Peptide-basednanomaterialshavebeenutilizedforvariousapplicationsfromregenerativemedicine toelectronics since theyprovide severaladvantagesincluding easy synthesis methods,numerous routes for functionalization and biomimicry of secondary structures of proteins which leads to designofself-assemblingpeptidemoleculestoformnanostructures.Microscopiccharacterizationat nanoscaleiscriticaltounderstandprocessesdirectingpeptidemoleculestoself-assembleandidentify structure–functionrelationshipofthenanostructures.Here,fundamentalstudiesinmicroscopic charac-terizationofpeptidenanostructuresarediscussedtoprovideinsightsinwidelyusedmicroscopytools.In thisreview,wewillencompasscharacterizationstudiesofpeptidenanostructureswithmodern micro-scopes,suchasTEM,SEM,AFM,andadvancedopticalmicroscopytechniques.Wewillalsomention specimenpreparationmethodsanddescribeinterpretationoftheimages.

©2011ElsevierLtd.Allrightsreserved.

Contents

1. Introduction... 69

2. Transmissionelectronmicroscopy(TEM). ... 70

3. Scanningelectronmicroscopy(SEM)... 74

4. Atomicforcemicroscopy(AFM). ... 76

5. Opticalmicroscopy-basedtechniques... 79

6. Conclusion. ... 82

References... 82

1. Introduction

Molecularmechanismsgoverningproteinfolding and

forma-tionofsupramolecularassembliesbyproteinsinspiredscientists

todevelop peptide-basednanomaterials witha wide varietyof

applicationsfromelectronicsandnanocatalysistotissue

engineer-ingandbiosensors(Gazit,2007;GulerandStupp,2007;Ulijnand

Smith, 2008). The versatilityof application areas is due tothe

flexibilityofpeptidedesign,whichusesaminoacidswith

differ-entchemicalfunctionalities.Moreover,self-assemblyofpeptide

moleculesledtoformationofnanostructureswhichcanbe

orga-nizedtoformdifferenthigher-orderstructuressuchashydrogels

(Cuietal.,2010;Gazit,2007),surfacecoatings(Adler-Abramovich

et al., 2010), drug deliverysystems (Sarikaya et al., 2003) and

one-dimensionaltemplatesforsynthesisofnanowires(Acaretal.,

2011;RechesandGazit,2003)andnanotubes(Gazit,2007).

Pro-∗ Correspondingauthor.Tel.:+903122903552;fax:+903122664365. E-mailaddress:moguler@unam.bilkent.edu.tr(M.O.Guler).

cessesthatgovernthestructuralorganizationareprogrammedby

non-covalentinteractionsbetweentheaminoacidresidues.The

side chainsoftheamino acids,which containvariouschemical

groups,determinethetypeofintramolecularandintermolecular

interactionsanddriveformationofpeptidicnanostructures.These

interactionsincludehydrogenbonding,electrostatic,hydrophobic,

␲–␲andvanderWaalsinteractions.Eachoftheseinteractions

con-tributesindesignandsynthesisofself-assemblednanomaterials

(Toksozetal.,2010;ToksözandGuler,2009).Aminoacidsequence

determinestypesofinteractionsbetweenpeptides,which

eventu-allyleadsformationofdifferentsecondarystructures.Forexample,

someaminoacidsfavor formationof ␣-helix,whilesomefavor

␤-sheetorrandomcoilstructures.Propertiesofthefinal

nanostruc-turesareaffectedbythetypeofdominatingsecondarystructureof

thepeptides.Peptidicnanostructureswerepreviouslydiscussedin

fourdifferentgroupsas␣-helicalbased,␤-sheetbased,amphiphilic

peptidebasedand collagen-likepeptidenanostructures(Toksöz

andGuler,2009).

In regenerativemedicine,peptideassembliescanbeusedas

extracellular matrix (ECM) – mimicking materialsfor repairing

0968-4328/$–seefrontmatter©2011ElsevierLtd.Allrightsreserved. doi:10.1016/j.micron.2011.07.006

damagedtissues.Peptide-basedhydrogels havegreatadvantage

overothersyntheticmatricessincetheyallowversatilityfor

con-jugationofvariousbioactiveproperties(Dviretal.,2011).Peptide

amphiphile(PA)moleculeshavebeenusedextensivelyfor

regener-ativemedicinestudies(Webberetal.,2010).Inphysiologicalmedia,

duetopackingofhydrophobicalkyltails,theycanself-assemble

intocylindricalnanostructureswhichmakebundlesand

entangle-mentstoformhigherorderstructuresresultinginhydrogels(Cui

etal.,2010).Thesegelsarecomprisedofmeshworkofnanofibers

whicharecapableofencapsulatingupto99%water,resembling

nativeextracellularmatrices.ThePAnanofiberscanbeengineered

tocarrysignalssimilartothenativeenvironmentofcells,providing

anexcellentplatformfor inductionofcellularsignaling

mecha-nismseffectively(Stupp,2010).EpitopesderivedfromnaturalECM

proteinsorthatallowbindingtospecificbiologicalfactorswere

conjugatedtoPAnanofibersandresultingsyntheticmatriceswere

usedtoinduceangiogenesis(Rajangametal.,2006),neural(Silva

etal.,2004;Tysseling-Mattiaceetal.,2008),bone(Mataetal.,2010),

enamel(Huangetal.,2008)andcartilageregeneration(Shahetal.,

2010)andsurvivalofpancreaticislets(Chowetal.,2010).Inthese

studies,activationoftissue-specific responsewiththese

hydro-gelshasbeenshownbothinvitroandinvivo.Anti-cancerepitope

carryingPAshavebeenshowntobeinternalizedeffectivelyby

can-cercellswhileinhibitingtheirproliferation(Aulisaetal.,2009)or

destroyingthemspecifically(Standleyetal.,2010).Nanostructures

basedonpeptidemoleculeswithhydrophobicandchargedamino

acidsalsoformmatricesandmembranesinthephysiologicalmedia

(Zhangetal.,1993,1995).Thesematriceswerealsoshowntobe

promisingforregenerativemedicinestudiessuchaswound

heal-ing(Schneideretal.,2008)anddifferentiationofneuralstemcells (Gelainetal.,2006).

Peptidebasednanostructuresarealsopromisingcandidatesfor

productionofnanowiresforelectronicsapplications.Production

ofnanoelectronicdevices requiressynthesisof nanoscalewires.

Amyloid-like, _{␤-sheet-rich} peptidenanofibers were extensively

usedfor this purpose. Amyloid-like nanofiberswere covalently

attachedthroughcysteineresiduestogoldparticles,where

result-ingnanowiresshowedconductiveproperties,similartoelectrical

wires (Scheibel et al., 2003). Histidine-rich peptide nanotubes

were used as templates for synthesis of conductivity-tunable

Cunanotubesthroughbiomineralization(Banerjeeet al.,2003).

Biomineralization of titania and silica on amyloid-like peptide

nanofiberswerealsoreportedrecently(Acaretal.,2011).

Bolaam-phiphilicpeptides,developedbyMatsuiandco-workers,arealso

suitable for nanoelectronics applications. They form nanotube

structures,which capturemetals andproduceinorganiccoating

aroundnanotube(Matsuietal.,2000).Metalloporphyrincoatingon

thesenanotubeshasbeenachieved,whichcanbeusedinnanoscale

sensorsorphotonics,duetoporphyrin’shighefficiencyinelectron

andenergytransfer(MatsuiandMacCuspie,2001).

Microscopiccharacterizationgivesvaluableinformationabout

morphology and size of nanostructures. Both of these features

are important in regenerative medicine, drug delivery,

biosen-sor development and other applications, since functionality of

designednanostructuresaresignificantlyaffectedbythese

param-eters.Forexample,inregenerativemedicinestudies,researchers

try to manipulate cells by designing materials mimicking the

nativeextracellularmatrix.TheECMhascharacteristicanisotropic

nanofibrillarstructure,whichhasimportantrolesincellular

behav-ior,includingmigration,signaling,proliferationanddifferentiation

(LutolfandHubbell,2005).Forbettercellularactivity,researchers

designECMmimickingsyntheticbiomaterialswithsimilar

nanofi-brousshapeandsize.Indrugdeliveryapplications,sizeandshape

ofnano-vehicles areimportant in determiningtheirhalf-life in

physiologicalenvironmentanduptakeintocells,whicharedirectly

relatedtoefficacyofthedeliverysystem(FarokhzadandLanger,

2009).Preciseinformationaboutsizeandshapeofnanostructures

synthesized for theabove mentioned purposes is crucial, since

nanoscaleaberrationsinthesepropertiesinterferewith

function-ality.

Advancementinmicroscopytechniqueshaspavedthewayfor

developmentofnanotechnology,materialsscience,molecular

biol-ogyandothergrowingfields.Forexample,improvementsinatomic

forcemicroscopy (AFM)now allows visualizationof

nanostruc-tures insolution,which enablesanalyzingthem in theirnative

environmentandavoidingartifactimagescausedbydryingeffect

duringspecimenpreparationandstrongcapillaryactionbetween

sampleandAFMprobe(Fotiadisetal.,2002).Moreover,imaging

nanoparticlesincells,understandingmechanicalfeaturesof

bio-logical molecules suchasnucleic acids,proteins and polymers,

andgeneratingstiffnessmapsofbiologicalsurfaceswereallmade

possiblewiththeuseofmodernAFMs(Dongetal.,2009;Husale

etal.,2009;Sahinetal.,2007;Tetardetal.,2008).Dip-pen

nano-lithographytechnique,whichbroughtsignificantadvancementin

functionalizationofsurfacesatnanoscale,isanotherachievement

madethroughimprovementofAFMprovingpotentialuseof

micro-scopesforpurposesotherthanimaging(Pineretal.,1999).

Modernsensitivemicroscopestogetherwithaccuratespecimen

preparationtechniquesallowustodeterminesizeandshapeof

nanostructuresaccurately.Thereareseveralimagingtechniques

thatcanbeusedforcharacterizationofnanostructuresandeach

techniquehasadvantagesanddisadvantages(limitations),soitis

importanttounderstandtheseandchoosetherightoneforthe

desiredcharacterization.Acomparisonofthesetechniquesis

pro-videdinTable1.Inthisreview,weexaminemicroscopytechniques

usedforcharacterizationofpeptidenanostructuresinfourmain

parts:transmissionelectronmicroscopy(TEM),scanningelectron

microscopy (SEM), atomic force microscopy (AFM) and optical

microscopy-basedtechniques.Sample preparationmethods and

informationobtainedfromimagesarealsoemphasized.

2. Transmissionelectronmicroscopy(TEM)

Analysisofsizeandmorphologyofpeptidenanostructureswith

highresolutionatascaleofafewnanometersorevenbelowcan

beperformedbyTEM.Thistechniqueinvolvestransmissionofan

electronbeamthroughthespecimen,whichinteractswith

inter-nalstructuresthuscollectinginformation forimagegeneration.

Sincewavelengthofelectronbeamissmallerthanthatoflight,

TEMallowsimagingwithhighermagnificationandresolutionthan

lightmicroscopyandenablesobservationofinternalstructuresof

materials.DetailedworkingmechanismofTEMcanbefoundin

ref-erenceEgerton(2005).TEMisusedtoidentifytheverydetailsabout

nanometerscalestructuresandeventoperformatomicresolution

characterizationofmaterials,whichinvolvesnotonlystructural

characterizationbutalsoidentificationofelementalcomposition

andchemicalbonding(Urban,2008).

Thistechniqueisbasedontransmissionofelectronsthrough

specimen,whichshouldbethintogetgoodqualityimages.

Imag-ingofbulkysamplessuchashydrogelswithTEMneedsspecial

sample preparation techniques in order to preventimage

arti-facts.The easiest wayis to directlyapply gel sampleson TEM

grid(cuppergrid).Inpreviousstudies,1wt%peptideamphiphile

(PA)nanofibergelwasappliedontocuppergridsandstainedwith

phosphotungsticacid (PTA) (Guleret al., 2005).Thenanofibers

withinthe gelwere 7nmin diameter and severalhundredsof

nanometersinlength.Inanotherstudy,bolaamphiphilicpeptide

nanostructureswereinvestigatedsimilarly,withanexceptionthat

bothnegative (PTA)and positive(uranylacetate)stainingwere

performed(Claussenetal.,2003).Here,nanofiberswith5–8nm

Table1

Comparisonofmicroscopytechniques.

Properties TEM SEM AFM Opticalmicroscopy

Advantages Providesveryhigh(even atomic)resolutionand magnification Enablesvisualizationof detailedstructuressuchas subcellularstructures

Allowsimagingbulkysamples andthreedimensionalsurfaces

Allowsthreedimensional imaging

Easysamplepreparation Allowsimagingatwet conditions,enableslive specimenobservation

Fluorescentandpolarized imagingoptions Colorcanbeobserved Lessexpensive Allowsimagingoflive specimens

Disadvantages Requireslaborioussample preparation,resinembedding andsectioningforbulky samples

Duetothinandtwo dimensionalspecimen requirements,notsuitablefor imagingthreedimensional arrangementsof nanostructuresinbulky samples

Doesnotallowlivespecimen imagingduetovacuum

Limitedresolutionatnanoscale Givesinformationonlyabout surfaceofthespecimen Doesnotallowlivespecimen imagingduetovacuum

Imagegenerationisslow Sizeofobservedstructures dependsontipradius,iftip radiusisnotlowenoughwrong sizemeasurementscanbe madefornanoscalestructures

Lowresolution

Resolutionlimit 0.2–0.5nmwithconventional TEM,1 ˚AwithHR-TEM

∼10nm Dependsontipradius,couldbe smallerthan1nm

∼200nm Sampletype Nanofibers,nanoparticles,

fixedcellsandsubcellular structures

Threedimensionalstructureof hydrogels,nanofibernetworks, cellsencapsulatedin biomaterials

Nanoparticles,nanofibers, livingorfixedcellsand subcellularstructures

Fluorescentlylabeledproteins, amyloidfibrilsetc.

Samplepreparation Stainingisrequiredfororganic structures(e.g.PTAoruranyl acetate)

Lessdestructivemethodsfor threedimensionalstructures suchascriticalpointdrying, freezedryingfollowedby coatingwithafewnanometers ofAu/Pd

Airdryingorafewmin incubationissufficientfor imagingintheair

Immobilizationisrequiredfor wetimaging

Fluorescentlabelingwith antibodiesorchemicals

Samplefixation 2%glutaraldehydeandOsO4 2%glutaraldehydesolution(3%

sucroseinPBS)

2%glutaraldehyde 4%PFA,acetone,methanol

Fig.1.TEMimagestakenfromslidesofresin-embeddedPAnanofibergel.Differentstagesofmineralizationareshown.(A)After2daysofmineralization.Fiberswerestained withcalciumphosphatepremineral.(B)and(C)After5daysofmineralization,nucleationofhydroxyapatite(HA)crystalsstarted.Crystalnuclei(␣)andaggregatesofstacked crystalplates(␤)areobservableatthisstage.(D)After11daysofmineralization,advancedcrystalgrowthonPAnanofibersisobserved.Growingcrystalseed(␣),ellipsoidal aggregate(␧)andtwobundledmineralizingnanofibers(␥)arethemostnotablefeaturesobservedatthisstage.(E)and(F)Highermagnificationviewsoffeaturesobservedin (D).Formationofmineralaroundnanofiberexteriors(E)andbetweenbundlednanofibers(F).Arrowsindicatemineralgrowthatthejunctionsbetweenbundlednanofibers. ReprintedwithpermissionfromSpoerkeetal.(2009).Copyright©2009JohnWiley&Sons,Inc.

Fig.2.(A)–(D)TEMimagesofnegativelystained,helicalterthiophenepeptidelipid(TTPL)nanofibers.(A)and(B)Singlehelicalnanofibershavingwidthof9±1nmand thehelicalpitchof65±6nm.(C)Widthofdoublehelicalnanofibersincreasedto17±1,whilehelicalpitchremainedsimilar(66±5nm).(D)TripleTTPLhelicesareshown (width25±2nmandhelicalpitch66±8nm).(E)TEMimagesofD-periodiccollagen-mimeticfibers.(F)SchematicviewofD-periodicbandingpatternincollagen-mimetic fibersandrelationshipbetweenpeptideoligomersformingthesefibers.EachD-periodincludesa“gap”zoneandalarger“overlap”zone.

FiguresA-DwerereprintedwithpermissionfromTsaietal.(2008).Copyright©(2008)Elsevier.FiguresE,FwerereprintedwithpermissionfromReleetal.(2007).Copyright ©(2007),AmericanChemicalSociety.

acetate,bindingpreferentiallytoacidgroupsonnanofibers,stained

bothcoreandperipheryofbolaamphiphilicnanofiberswith

car-boxylicacidgroupsextendingfrombothsidesofbolaamphiphiles

(Claussen et al., 2003). However, only periphery of the

nanos-tructureswasstainedwhencarboxylicacidgroupswerelocated

ononly oneside ofbolaamphiphiles. Anothermethodfor

sam-plepreparation isembeddinggelsintoresin.PAnanofibergels,

whichwereembeddedintoEPONTM_{(epoxy)resinandsectioned}

withmicrotome,werevisualizedbyTEM(Hartgerinketal.,2001).

Bythismethod,itwaspossibletofollowdifferentstagesof

min-eralization,whileobservingaggregates ofstackedcrystalplates,

crystalnucleiandadvancedcrystalgrowthonPAnanofibers(Fig.1)

(Spoerkeetal.,2009).Analternativemethodisdilutinggeland

applyingdilutedsolutionontocoppergrid.Thismethodaimsto

avoidimagingofaggregatednanostructures,whileobserving

indi-vidualfeaturesonthesurface.Forthispurpose,1wt%PAgelswere

dilutedanddroppedontocarbongrids(Guleretal.,2006).Aftera

fewminutesexcesssolutionwasremovedwithfilterpaper,

nega-tivestainingwithPTAwasperformed,sampleswereairdriedand

imagedwith200kV.InanotherstudybyTsaietal.,self-supporting

gelswereformedanddiluted15-fold.Afterdryingofthesample

onTEMgrid,stainingwasperformedbyusinguranylacetate.This

methodallowedimaging helicalpeptidicnanostructures, which

haveapplicationsinnanoelectromechanicalsystems(NEMS),and

measuringimportantstructuralparameters,suchashelicalsizeand

pitch(Fig.2A–D)(Tsaietal.,2008).Single,doubleandtriple

heli-calstructureswereclearlyobservable(Tsaietal.,2008).Rajangam

etal.(2008)reportedanotherinterestingmethodforobservingthe

nanofibrousnatureofpeptidegelwherecarbongridwasdipped

into1wt%PAgelsuspensiontwicefor20sandthenstainedwith

PTA.

There are also numerous studies where TEM was used for

imaging peptidenanostructures in solutionform, which is less

challengingregardingpreparationofthinsamples.Inonestudy,

0.01wt% of peptidic thiophene molecules, which form

one-dimensionalnanostructureswithpotentialapplicationsinorganic

electronic devices, were dropped onto copper TEM grids and

dried.Sampleswerestainedwithuranylacetate for15minand

one-dimensionalnanostructureswith6–7nmwidthwereclearly

observedat100kV(Stoneetal.,2009).Dolphinetal.(2006)stained

amyloidprotofibrilswithasimilarmethodwhileusing2%uranyl

acetate.By usingsimilarmethod,peptidic fibrillarandmicellar

nanostructureswith5–20nmsizewereobserved(GulerandStupp,

2007).Releetal.(2007)designedcollagen-mimeticpeptideswith

D-periodicityand reportedthat0.5%uranylacetate stainingfor

10swassufficienttoprovidecontrastforTEMimaging(Fig.2E).

ImageresolutionwassufficienttomeasuresizeofD-periodson

carryinglight-sensitivemoietyself-assembledintodifferent

mor-phology upon light triggering and both of the nanosphere and

nanofiber forms were observed by air drying of the PA

solu-tion(0.4mM)onthesubstrateand stainingwithuranylacetate

(Muraoka et al., 2009). Anothermethodfor visualization of PA

nanofibersinvolvesinduction ofnanofiber formation ona TEM

grid.Hartgerinketal.placed10␮Lof0.01–0.02%solutionofPA

directlyonthegrid.Thegridwasthenplacedintoasealed

cham-berwithHClvaporsfor10minfornanofiberformationafterwhich

thegridswerewashedwithdeionizedwater.Samplewasstained

withphosphotungstic acidor uranyl acetate (Hartgerink et al.,

2002).In anotherstudy,1␮Lof 0.1wt%PAsolutioninaqueous

mediawasdropcastedontoacarbon-coatedcoppergrid.The

sam-plesonthegridwerestainedfor1–3mininphosphotungsticacid,

gently rinsedin water and blotted dry (Hsu et al., 2008).This

enabledobservationofnanofiberswithin5–8nmrange.

Vesicu-larstructures(50–450nm)formedbyself-assemblyofdipeptides

werevisualizedbynegativestainingwithuranylacetate(Mishra

et al.,2008).Authorsconcluded thatthere is a highdensity of

negativechargesonvesicles since there wasgrayscalecontrast

betweenvesiclesurfaceandgridbackground.Positivelycharged

vesicles showedlighterstainingthan background.Useof

nega-tivestaininghelpedtoclarifythesefinefeatures.Limetal.(2008)

observednanoribbonscomposedof␤-sheetformingand

bioac-tivepeptidesequencesdesignedforencapsulationofhydrophobic

drugsbyTEMimaging. After1mintreatmentof TEMgrid with

peptide solution, remaining solution was removed with filter

paperandpositivestainingwasperformedwithruthenium

tetrox-ide.

Sometimespre-fixationstepisnecessaryduringsample

prepa-rationtoconservedelicatestructuressuchassubcellularorganelles

and toprotect complex three-dimensionalnanostructuresfrom

effectsofsample preparation(e.g.drying).Beniashetal.(2005)

visualizedentrapmentofmammaliancellsinpeptideamphiphile

nanofibermatrixbyusingTEM.Fixationwithglutaraldehydeand

OsO4anddehydrationstepsprecededsamplepreparation,which

includedembeddinginepoxyresinandultrathinsectioning.A

sim-ilarmethodwasalsousedtovisualizepeptide-hyaluronicacidsac

structures(Capitoetal.,2008).

Metal-bindingpeptidenanofibrilsprovideagoodtemplatefor

tailored growth of metal particles on them, which is a smart

methodfornanowiresynthesis.Peptide nanofibrilswith

metal-bindingsiteshaveaffinitytodifferentmetalssuchassilver,gold

andplatinumdependingonaminoacidsequence(Kasotakisetal.,

2009).Metal-boundnanofiberswereobservedbyusingTEM

with-outanyneedforstainingtoincreasecontrast,sincemetalsprovide

contrast(Fig.3).Peptide amphiphilenanofibers areamong the

peptide-basednanostructureswhicharegoodtemplatesfor

min-eralization.Bright-fieldTEMimagingofvariousCd2+_{–PAmixtures}

revealedthatCd2+_{ionsgrowhomogenouslyonPAnanofibers(Sone}

and Stupp, 2004). Cd2+ _{ionsprovided necessary contrast}

with-outfurtherstaining.Peptideamphiphiles,which weredesigned

asMRI contrastagents, formnanofibersandnanomicelles with

higherrelaxivitytimethanknownmonomericMRcontrastagents

(Bulletal.,2005).Thiscontributionisprobablycausedbyhigher

orderstructuresformedbymonomericpeptideamphiphileswhich

chelate Gd(III)ionseffectively. Visualizationof nanofiber

struc-tureswithGd(III)ionsdidnotneedanystainingeither,sinceGd

(III)ionsthemselvesprovidecontrast.Carnyetal.reportedthat

linkeraminoacidssuchascysteine(bearingthiolgroups)allowed

ordered organization of gold nanoparticles on diphenylalanine

peptidenanotubes.Thesegoldcoatednanotubeswerealsoimaged

byTEMwithhighcontrastwithoutanyfurtherstaining(Carnyetal.,

2006).Coatingnanostructureswithappropriatemetalsisa

conve-nientwayofincreasingcontrast.Contrastofnanofibrousstructures,

withadiameterof12nm,wasincreasedbyusingplatinumcoating

afterdryingofthesample(eithergelordilutedsuspension)onTEM

grid(Smeenketal.,2005).Ryadnovetal.(2003)observed

peptide-mediated assembly of gold nanoparticlesby using TEM, where

nanoparticleswereobservedtobeseparatedwithhomogenous

dis-tances(7nm)fromeachother.Inthisstudy,nanoparticle-peptide

solutionon carbonwas driedwith filterpaper withoutfurther

stainingforsamplepreparation.Althoughgoldnanoparticleswere

clearly observed,peptidenanostructuresholdingthem together

werenotobservable.

Inordertogetridofthenegativeeffectsofdrying,QFDE-TEM

protocolcan beusedfor samplepreparation. In thistechnique,

samplesarefrozenat−195◦_{C,afterwhichtheyarefracturedin}

freeze-fracture apparatus. After etching at −95◦_{C, samples are}

coatedwithplatinium/carbonmixture.Elongatednanostructures

formedbymixingpeptideamphiphileandoligo(phenylene

ethyny-lene)wereobservedbythismethod(Bulletal.,2008).

High-resolutionfieldemissiongunTEM(HR-TEM)isanimaging

methodwhichenablesvisualizationofcrystallographicstructure

ofmaterialsatnanoscale.Resolutionthatcanbereachedwiththis

methodisbelow1 ˚A.Soneetal.usedHR-TEMtovisualizelattice

structureofCdSnanocrystalsgrownonPAnanofibers(Fig.4A).

HR-TEMwasalsousedtovisualizenegativelystained(uranylacetate)

peptidenanotubes(RechesandGazit,2003).Inthisexample,

HR-TEMprovidedindicationoftheregularstructuresofthetubewalls.

Moreover,silver-filledpeptide nanotubesfor nanowire

produc-tionwerevisualizedwithoutstaining,sinceagainsilverprovided

requiredcontrast.

Cryo-TEM isanother powerfultechnique toobserve peptide

nanostructuresintheirnativestate(Hartgerinketal., 2001).In

onestudy,hydrogels formedbyself-assemblyof␤-hairpin

pep-tidesweresnap-frozenbyliquidethaneandimagedbycryo-TEM

(Schneideretal.,2002).Cryogenicsystemholdstemperature

con-stantat−170◦_{C,preventingsublimationofthesample.Structures}

wereimagedwhileunderfocusedinordertoenhancecontrastand

nanofibrousnatureofthescaffoldwasobservedbythismethod.

Inanotherstudy,morphologicaltransformationofnanostructures

fromtwistedribbonsintohelicalribbonswasobserved(Pashuck

andStupp,2010).Pashucketal.(2010)rapidlyfrozethinlayersof

peptideamphiphilesolutionsinliquidethanetopreserve

morpho-logicalstructuresandtoavoiddryingeffect.Byusingthismethod,

nanofiberswith8–10nmdiameterswereobserved(Fig.4C).

Cryo-TEMprovidesvaluableinformationwhichcannotbeobtainedwith

techniquesbasedonsampledrying.Cuietal.(2009)observedthe

flexiblestructure ofnanobeltswhichariseastilts,flippingsand

entanglementsinmorphology(Fig.4B).Inthismethod,contrast

relatedtodifferenttiltanglesofnanobeltswasobserved(Fig.4D).

At90◦ tiltangle(nanobelt surfaceisparalleltoelectronbeam),

electronstravelthelongestdistanceinnanobeltsandthehighest

contrastisachieved.

EF-TEM is an improved electron microscopy system, which

canfilterscatteredelectronsinaspecimenaccordingtoenergy,

besidesscatterangle.Differentenergywindowscanbeusedfor

filteringprocessandselectingelectrons(KohlandReimer,2008).

Energy-basedselectionofcontrastmightallowachieving

elemen-talcontrast.Thismethodallowsimagingwithoutfurtherstaining

withsufficientcontrast.Kogisoetal.(2000)usedEF-TEMfor

exam-inationoffinestructuresinhydrogelsandproducedhigh-contrast

imageswithoutstaining.

Insummary,TEMimagingallowssuccessfulimagingofpeptide

nanostructures,whichareafewnanometersinsize,byusing

differ-entspecimenpreparationtechniquesforincreasingimagecontrast.

ModernversionsofTEMcanevenvisualizenanostructuresin

solu-tion(cryo-TEM)orcrystallographicstructuresatangstromlevel

(HR-TEM).TEMcanbeusedfordetailedmorphologicaland

struc-turalanalysisofnanostructuresatascaleofafewnanometersor

Fig.3.TEMimagesofpeptidenanofibrilsincubatedinsilver(AandB),gold(CandD)andplatinum(EandF)solution.Nonegativestainingwasperformed.Metalson nanofibrilsprovidedsufficientcontrast.

ReprintedwithpermissionfromKasotakisetal.(2009).Copyright©(2009),JohnWiley&Sons,Inc.

3. Scanningelectronmicroscopy(SEM)

Whenspecimenis too thickfor TEMimaging, using SEM is

amoreappropriatechoicetogethighresolutionimages. Unlike

TEM,SEMuseselectronsreflectedfromsurfaceofsampleas

sig-nals for image generation.Detailed information aboutworking

principleofSEMisexplainedinreferencePease(2008).SEMcan

provideinformationaboutsurfacetopography,crystalline

struc-ture,chemical compositionandelectrical behaviorofanalmost

1_␮msliceonthesurfaceofthesample(Vernon-Parry,2000).

More-over,samplepreparationtechniquesforSEMarelessdestructive

thanTEM.Therefore,itisbettertouseSEMforobservingbulky

sam-ples(Vernon-Parry,2000).ECM-mimickingbiomaterialsformedby

non-covalentinteractionsofpeptidenanofiberscanbeconsidered

asexamplesforthistypeofbulkysamples.Inordertoincrease

contrastandresolutionofimagingofthesehydrogels,specimen

preparationisacriticalstep.Foreffectivespecimenpreparation,

thefirstrequirementisdehydrationofthesamplewithout

destruc-tingthethree-dimensionalstructureofthehydrogel.Airdrying

ofhydrogels causescollapsingofthenanofiber network, which

hinders our understanding of three-dimensional structure and

porosityofthematerial(Mahleretal.,2006)(Fig.5A).Network

dehydrationbyethanoltreatmentandcriticalpointdryinghelpin

solvingthisproblem(Spoerkeetal.,2009).Thismethodenabled

observationofthree-dimensionalnanofiber networkcomprising

peptideamphiphilegels(Fig.5B)(Rajangametal.,2006;Spoerke

etal.,2009).Inthesestudies,coatingspecimenswithaverythin

layerof(3nm)gold–palladiumalloyincreasedcontrastof

nanos-tructuresandqualityofimages.Anothermethodfordehydrationof

hydrogelsisfreeze-drying.Althoughthree-dimensionalstructures

mightbedistortedthismethod,XuandKopecek(2008)achievedto

getfineimagesofmeshworkstructureformedbyself-assembled

triblockpolypeptides(Fig.5C).Thismethodincludessnap-freezing

ofhydrogelsampleswithliquidnitrogen,freeze-dryingoffrozen

hydrogelandcoatingofsampleswithgold.

Adler-Abramovichetal.(2006)visualizednanotubestructures

formedbyself-assemblyofdiphenylalanineunitsbySEM(5kV).

Forsamplepreparation,peptidesolutionwasdroppedontoaglass

coverslip,air-dried and coated withgold.Individual nanotubes

reaching80nminsizewereobservedbythis method.Ryadnov

etal.visualizedindividualpeptidenanofibers,which are50nm

Fig.4. CharacterizationofnanostructuresbythehelpofadvancedTEMmodules.(A)HR-TEMimageshowingcrystallatticestructureofmineralsgrownonPAnanofibers. (B)Cryo-TEMimageshowsmechanicalflexibilityofpeptideamphiphilenanobelts.(C)Vitroeusicecryo-TEMimagingofPAnanofibers.(D)Illustrationofcontrastgeneration mechanismincryo-TEMimages.Excellentcontrastinnanobeltimages(B)isassociatedwithdifferenttiltanglesofnanobelts.Whennanobeltistilted90◦_{,electronstravel}

maximumdistanceinnanobeltandhavethehighestpossibilitytobescattered(darkerlinesinimage).

FigureAwasreprintedwithpermissionfromSoneandStupp(2004).Copyright©(2004),AmericanChemicalSociety.FigureB,DwerereprintedwithpermissionfromCui etal.(2009).Copyright©(2009),AmericanChemicalSociety.FigureCwasreprintedwithpermissionfromPashucketal.(2010).Copyright©(2010),AmericanChemical Society.

Fig.5. SEMimagesof3Dhydrogelswithdifferentsamplepreparationmethods.(A)Air-driedFmoc–diphenylalaninegel.(B)Critical-pointdriedpeptideamphiphilenanofiber gel.(C).Freeze-driedtriblockpolypeptidehydrogel.

FigureAwasreprintedwithpermissionfromMahleretal.(2006).Copyright©2006WILEYVCHVerlagGmbH.FigureBwasreprintedwithpermissionfromSpoerkeetal. (2009).Copyright©(2009),JohnWiley&Sons,Inc.FigureCwasreprintedwithpermissionfromXuandKopecek(2008).Copyright©(2008),Springer.

carbon-coatedgridsbyairdryingandvisualizedbySEMafter

stain-ingwithuranylacetate(RyadnovandWoolfson,2003).Branching

ofnanofibers,whichself-assembledfrombranchedpeptides,could

beidentifiedthroughthismethod.

Peptide membranes, which are formed by adding peptide

solutioninhyaluronicacid(orhyaluronicacid/heparin)solution,

provideanotherexampleforhigher orderstructuresformedby

peptidenanofibers.Here,pre-fixationwith2%glutaraldehyde

solu-tion(3%sucroseinPBS)beforenetworkdehydrationandcritical

pointdryingstepsarerequiredinordertopreservemembraneand

sacstructures(Capitoetal.,2008;Chowetal.,2011).Cross-section

ofmembranescanbeobservedbycuttingsacsinhalf.PAnanofiber

–Tifoamhybridstructureswerealsopre-fixedwithglutaraldehyde

andformaldehydesolutions(Sargeantetal.,2008a).

Peptidenanoparticlesareanotherexampleofnanostructures

whichcanbeobservedbySEM.Liuetal.usedfieldemissionSEM

inordertoimageantimicrobialpeptidenanoparticleswithsizes

oflessthan150nm.Forsamplepreparation,nanoparticlesolution

wasdroppedontoasiliconwaferandair-driedatroom

tempera-ture.Driedsampleswerecoatedwithplatinumbeforeimaging(Liu

etal.,2009).

ModernSEMinstrumentsincludemoresophisticatedversions

suchas HR-SEM (high resolution SEM), E-SEM (environmental

SEM)andcryo-SEM(cryogenicSEM)whichareusedforvarious

purposes.Mahleretal.(2006)reportedthatFmoc-diphenylalanine

(FF)unitsformgelsathigherconcentrations(1wt%)which

con-sistoffibrousnetworks.HR-SEMandE-SEMwereusedtoanalyze

three-dimensionalmorphologyof thesehydrogels.Flexibility of

individualfiberscanberecognizedfromHR-SEMimages.Toimage

non-conductingsamples,E-SEMseemstobeabetterchoice,since

itdoesnotincludeanytreatments(stainingorcoating)for

sam-plepreparation.UnlikeconventionalSEM,sampleislocatedina

chamberwithhighpressureratherthanvacuum.ImagesofFFgel

obtainedbyE-SEMconfirmedexistenceofafibrillarnetworkunder

humidconditions(Mahleretal.,2006).

Inaddition,peptidenanostructurescanalsobevisualizedby

using cryo-SEM. Cryo-SEMinvolves snap-freezingof sample in

ordertoobservenanostructuresinsolutionform.Nanofibergels

formedbyFmoc-dipeptideswereobservedbycryo-SEMand

diam-etersofnanofibersweremeasured(Fig.6A).Sincethisvaluewas

wellabovethediameterofFmocdipeptidebuildingblock,authors

concludedthatobservednanofiberswerebundlesof

supramolec-ularaggregatesofdipeptides(Jayawarnaetal.,2006).

Visualization of supramolecular structures encapsulated in

membranous structures suchas liposomes can be achieved by

usingaspecialtechniqueforspecimenpreparation,called

quick-freeze/deep-etch(QFDE).Light-inducedPAnanofibersinliposomes

werevisualizedwithSEMimagingbyusingthissamplepreparation

method(Leeetal.,2008).Thistechniqueproducesreplicasfromthe

fracturedsurfaceofarapidlyfrozensample.Afterfracture,the

etch-ingprocesssublimateswaterfromthesurface,exposingstructures

otherwisehiddeninice.ThePAnanofibernetworkwasobserved

fromfracturedpartsofsurfaces(Fig.6B).

Imagingcellsinnanofibergelsisanotherissuethatwassolved

byspecial sample preparation protocolsand sophisticated SEM

techniques.To imagecellsonPAnanofiber scaffolds,fixationis

necessary.Shahetal.usedethanoldehydrationandcriticalpoint

dryingafter fixation.Mesenchymal stemcells adhered onto PA

nanofibernetworkwerevisualizedbyusingthismethod(Fig.6C)

(Shah et al., 2010). E-SEM is alsoa good instrument for

imag-ingbiologicalentitiessuchascellsentrappedinnanofibermatrix.

Chondrocytesculturedin gelwere imagedin theirnativeform

(hydrated)byusingE-SEM(Fig.6D)(Jayawarnaetal.,2006).

SEMisamicroscopytechniquewidelyusedforobservationof

thickersamplessuchasthree-dimensionalstructureofhydrogels,

cellsentrappedinnanofibermatricesorrelativelybiggerindividual

nanostructures.Thus,itshouldbeexploitedtoanalyzethebulky

natureofhigher-orderstructuresformedbypeptide

nanostruc-tures.

4. Atomicforcemicroscopy(AFM)

AFMinvolvesscanningofsurfacewithacantilever,typically

madeofsiliconnitrideforbiologicalapplications,whose

deflec-tionsarerecordedbycomputertogenerateimage(Engeletal.,

1999).MoreinformationaboutthebasicmechanismofAFMcan

befoundinreferenceAlessandriniandFacci(2005).Pyramidaltip

attheendofcantilevercanbesufficientlysharptoachieve

res-olutionoflessthan1nm.AFMhasseveraladvantagesoverother

microscopytechniques.Firstofall,samplepreparationforAFMis

easier,sinceitdoesnotneedanypretreatmentofspecimensuchas

staining,labelingorcoating(Allisonetal.,2010).Moreover,images

obtainedwithAFMcanbedemonstratedinathree-dimensional

format.Anotheradvantageisthatcellsandbiomoleculescanbe

imagedinaphysiologicallyrelevantenvironmentwithoutfreezing

oranyothertreatment(Allisonetal.,2010).

ForAFM imagingof peptidenanostructures,silicon waferor

freshlycleavedmicacanbeusedassubstrate.Bulletal.prepared

thesamplebydropcasting0.05wt%diluteaqueoussolutionof

pep-tidemoleculesontofreshlycleavedmica(Stoneetal.,2009).AFM

imagesshowedthatthesemoleculesformone-dimensional

nanos-tructureswith5–10nmwidthandheight.Similarityofwidthand

heighthasledauthorstosuggestthatnanostructureshave

cylindri-calshape.Guleretal.(2005)used10␮L0.1wt%solutionofPAand

dropcastedthesampleontosiliconwaferwhichwaspre-cleaned

byultrasonicationinwaterandisopropanol.Aggregatedviewof

nanofiberbundlescouldbeobservedfromthisimage.Inanother

study,sampleswerepreparedfrom2wt%peptideamphiphile

solu-tion,initiallygelled,andthendilutedto0.1wt%(Hsuetal.,2008).

These sampleswere later drop casted onto pre-cleaned silicon

waferand air dried. AFM wasperformedin tapping modeand

peptidenanofiberswith5nmdiameterwereclearlyobservable.

Zhouetal.visualizedFmoc-basedpeptidehydrogelsbydiluting

anddroppingontomicasurface.Waterwasremovedbycapillary

action,andsampleswerewashedandimagedwithtappingmode

AFM(Zhouetal.,2009).Interwovennetwork ofnanofibersand

bundlesobservedinthis studysuggested thatFmoc-based

pep-tidehydrogelspossessathree-dimensionalnanofibrousstructure.

Sargeantetal.coatedNiTi(nickel-titanium)surfaceswith

bioac-tivePAnanofibersforbiofunctionalizationofimplantsurfaces.AFM

imagingwasperformedbyusingtappingmodeandshowedthat

PAnanofiberscoatedtheNiTisurfaceshomogenously(Sargeant

etal.,2008b).Bulletal.(2008)usedmicasurfaceinanotherstudy

where0.1wt%solutionwasdropcastedandrestofthesolutionwas

removedandairdried.Peptidenanofibersandnanoparticleswith

nearly5nmheightwereobservedbythismethod.Inanotherstudy,

␤-amyloidpeptidesweredepositedontofreshlycleavedmica

sur-face andwashed withwater after 1.5min(Cohen etal., 2006).

Formationofseveralmicronslongandafewnanometerswide

amy-loidfibrilsafterpolymerizationphasewasobservedbythisway.

Moreover,inhibitionofpolymerizationbyindolederivativeswas

alsodeducedfromAFMimagestaken(Cohenetal.,2006).Toimage

reassemblyofbolaamphiphilicpeptidesformingnanofibersafter

theirbreakdownwithsonication,tappingmodeAFMwasused(Qiu

etal.,2008).Sampleswerewashedawayfrommicasurfaceafter

afewsecondsofincubationandairdried.AFMimagingshowed

thatlengthofpeptidenanofiberswereincreasedfrom∼300nm

to∼800nmafter25days,whichindicatesreassemblyofbroken

nanofibers.

AFM images can also provide information about secondary

structuresformedbypeptides.Itispossibletodeterminerightor

nanostruc-Fig.6.DifferentusageareasofmodernSEMtechniquesincharacterizationofpeptidenanomaterials.(A)Cryo-SEMimageofnanofibrousgelformedbyself-assemblyof Fmocdipeptide.(B)Imageofnanofibrousnetworkofpeptideamphiphiles(whitearrows)inliposome.SamplewaspreparedbyQFDEmethod.(C)Mesenchymalstemcells inPAnanofibergel.Samplewaspreparedbyfixationandcriticalpointdrying.(D)ESEMimagingofchondrocytecells(whitearrows)inFmoc-dipeptidegel.

FigureA,DwerereprintedwithpermissionfromJayawarnaetal.(2006).Copyright©2006,JohnWiley&Sons,Inc.FigureBwasreprintedwithpermissionfromLeeetal. (2008).Copyright©(2008)TheRoyalSocietyofChemistry.FigureCwasreprintedwithpermissionfromShahetal.(2010).Copyright©(2010),NationalAcademyofSciences, U.S.A..

Fig.7. Differentsupramolecularaggregatesformedbytripeptideamphiphilesdependingonthenatureofendgroup(A)determinedbyAFMimaging.Reprintedwith permissionfromLietal.(2007).Copyright©2007,JohnWiley&Sons,Inc.Straightcylindricalnanofibersformedwhenendgroupisacetate(B),howeverhelicalstructures withregularpitchformedwhenbulkierendgroupisusedsuchasdimethylacetate(CandD).

turesandmeasurepitchvaluesbetweenhelices(Lietal.,2007; Muraokaetal.,2008).Tripeptideamphiphilesarefoundtoform

helicalstructuresinorganicsolventswhenthebulkierendgroup

isused,mostlikelycausingtwistingofcylindricalnanostructures

(Fig.7A–C)(Lietal.,2007).Delicateobservationofheightprofiles

obtainedbyAFMindicatedlefthandednessofhelicalstructures

(Fig.7D).Moreover,pitchvaluesobtainedfordifferenthelical

struc-turesshowedcorrelationwithincreasedbulkinessofendgroup,

suggestingtorsionalstrainmightbethemechanismforformation

ofthesehelices(Lietal.,2007).

ContactmodeAFMisanotherchoiceforimagingpeptide

nanos-tructures.SincecontinualcontactofAFMtipwithsurfacemight

damagefeaturesonthesurface,researchersusenon-contact or

tappingmodeAFMforthispurpose.However,byusingsoftcontact

mode,itispossibletogethighresolutionimageswithout

damag-ingorganicsonthesurface.Toachievethis,itiscrucialtouseAFM

tipswithlowspringconstant(0.2N/morbelow),whichare

appro-priateforsoftcontactmodepurposes.Wehaveusedcontactmode

tovisualizedifferentpeptideamphiphilenanofibersandwereable

toobtainhighresolutionimages(Toksozetal.,2011).

Peptidenanostructurescanalsobevisualizedinsolution,which

isbettertoobservetheirnativeformbyeliminatingeffectscaused

bysampledrying. Imagingbiologicalsamplesinnativeaqueous

environmentis a keyadvantage ofAFM over othermicroscopy

techniques.One ofthe main issues in wetimaging is

immobi-lizationofspecimentothesurface. Whiletappingmodeallows

visualizingbiologicalprocesses andmolecules weaklyadsorbed

tothesurface,duetominimizationoflateralforcesinthismode,

immobilizationiscriticalforcontactmodeimaging,wherelateral

forcesofAFMtipmaydriftspecimen(Engeland Muller,2000).

Twodifferenttypesofimmobilizationarenoncovalentand

cova-lentmethods. Noncovalent approach ismore simple and based

onphysicaladsorptionofspecimenontosurface throughforces

likevanderWaalsforces,electrostaticdouble-layer(EDL)forces,

hydrationforcesandhydrophobiceffects(Wagner,1998).

Chem-icalmodificationofsurfacecanincreaseadsorptionofmolecules

ontosurface.Forexample,silanizationofsurfacecanhelp

adhe-sionofbiopolymers(Wagner,1998).Covalentlylinkingspecimen

tothesurfacebecomesimportantwhendisplacementofmolecule

onthesurfaceisacriticalproblem.Limitingissueof

immobiliza-tiontechniqueisinactivationofthebiologicalstructureorprocess

tobeobserved(EngelandMuller,2000;Wagner,1998).Surface

energy,surfacechargesandhydrophobicityarepropertiestobe

consideredforpreservationofstructuresimmobilizedtosurface.

Forexample,hydrophobicsurfacesarenotrecommendedforwet

imaging.Because,theyinterferewithAFMimagingduetoincreased

adhesionanddenaturationofproteins(Wagner,1998).

Horiietal.(2007)imagedpeptidenanofibersinaqueous

solu-tionbyusingtappingmodeAFM,whereobservedsizeofnanofibers

were greater than expected. It is acceptable to assume that

nanofibersbecame hydratedin solutionand observed sizewas

greaterthanexpectedsize.

RadiusoftheAFMtipisanimportantparameterforresolution,

sincetipconvolution(alsocalledastipimaging)shouldbeavoided

inordertounderstandexactsizeandshape ofobservedfeature

onthesurface.Forthispurpose,tipwidthshouldbesmallerthan

widthofstructuresonthesurface.Otherwise,observedimagewill

beaffectedbytipshape.Itisbettertousetipswithradiismaller

than10nmtoobservefinenanostructuresonthesurface.Genovéet

al.usedsuchAFMtipsinordertovisualizenanostructuresonthe

surface.Sampleswerepreparedwith0.01wt%peptidesolutions

andobservedbyusingtappingmodeAFM(Genovéetal.,2005).

Itisalsopossibletosubtractconvolutioneffects,byusing

mathe-maticalmodelsdevelopedforsample–tipinteraction.Byassuming

geometricshapeofstructuresonthesurface,itispossibletoconvert

observedwidthtorealwidth(Hongetal.,2003).

Atomic force microscopy imaging can yield high resolution

images that can be used to study various conformations of

biomolecules and their secondary structures. Scheuring et al.

(2003) have carried out an extensive high-resolution study on

Rhodobactersphaeroideslightharvestingcomplex2,demonstrating

the capability of force microscopy for topographic

character-ization of biomolecules with higher than 0.1nm resolution.

High-resolutionimaging of biomolecules can bedoneby using

dynamicAFM.SanPauloandGarcía(2000)haveshownthe

impor-tanceofchoosingcorrectimagingparametersinobtainingartifact

freeimagesofantibodieswithminimalsampledamage.

Surface properties of substrate used in sample preparation

mightsignificantly affectquality ofimagesobtainedwithAFM.

Jiangetal.(2007)preparedthesamplesbydippingsiliconwaferor

goldsurfaceinto0.1wt%solutionofPAandair-dryingafterslowly

withdrawingthem.Interestingly,theyobservedhigh-aspect-ratio

cylindricalnanofibersonsiliconsurface,whilesignificantlyless

lin-earfeaturesongoldsurface,withlessclearimage(Fig.8).Authors

explainedthisbysuggestingthatsurfaceroughnessofgoldsurface

probablyinterfereswithAFMimaging.Surfacepropertiesmight

affect formation of nanostructures on the surface, especially if

samplepreparationmethodisbasedonairdrying.Toeliminate

effectsofsurfaceonnanostructureformation,solutionsshouldbe

coatedontodifferentsurfaceswithvaryingchemicalproperties.

Forthis purpose,Ashkenasyet al.(2006)usedmica,highly

ori-entedpyrolyticgraphite(HOPG)andhydroxylatedsiliconoxidefor

visualizingnanotubesformedbycyclicpeptidesandimagedwith

tappingmodeAFM.Asauthorsobservedsimilarnanotube

struc-turesonallmaterials,theyconcludedthatnanotubesformedin

solution,andnotbyinteractionwithmaterialsurface.

Besidesgivingtopographical informationabouttheanalyzed

surface,AFMcanalsoprovideinformationaboutthestiffnessof

surfacenanostructuresandtheadhesionpropertiesbetween

pep-tidesandabareorfunctionalizedAFMtip.Suchnanomechanical

mappingproducesanelasticmodulusmapofthesurface,

includ-ingquantitativevaluesforeachfeatureonthesurface(Sahinand

Erina,2008).Severalestablishedtechniquesexistfor

nanomechan-icalmapping(Heubergeretal.,1995;Maivaldetal.,1991;Miyatani

etal.,1997;Radmacheretal.,1993;RosaZeiseretal.,1997).

How-ever,organicmolecules are typicallyfragile and measurements

mustbeperformedinawaythatdoesnotapplylargeforcesand

pressuresduringmapping(SanPauloandGarcía,2000).Recently,

noveltechniques that can produce nanomechanical maps with

highresolutionandminimalsampledamagehavebeenintroduced

(Dong etal.,2009;Husaleetal.,2009).While,noveltechniques

continuetogainpopularity,conventionalforcespectroscopyhas

been instrumental in nanomechanical characterization. Dagdas

etal.(2011)usedAFMtocarryoutforce-distancemeasurements

for identifying stiffness values of PA nanofibers. Authors used

surfaceswithknownstiffnessvalues(siliconandPMMA)to

esti-mate and compare elasticmoduli of PA nanofiber films made

withcalciumionsorHCl.Elasticmoduliofbothnanofilmswere

0.1±0.05GPa,whileonemadewithcalciumhadslightlyhigher

stiffness(0.2±0.1GPa).Helenetal.(2011)alsoinvestigatedthe

effect of gelation conditions onadhesion and stiffness of

self-assembledorganicmaterialsandtheyusedforce-spectroscopyin

liquidtocorrelateadhesionandstiffnesspropertiestomacroscopic

mechanicalproperties.Smithetal.(2006)hasappliedforce

spec-troscopyonamyloidfibrilsself-assembledfrominsulin,byprobing

freestandingfibrilsongoldsurfacespatternedwithgrooves.Such

geometriesareparticularlysuitableforquantitativeanalysisdue

towelldefined boundaryconditions,andSmith etal.extracted

anelasticmodulusof3.3±0.4GPaandstrengthof0.6±0.4GPa.

Adhesionis also an importantparameter in understandingthe

mechanical andchemical properties ofpeptidic nanostructures.

inves-Fig.8.AFMimagingofPAnanofibersformedongold(A)andsilicon(B)surface. ReprintedwithpermissionfromJiangetal.(2007).Copyright©(2007)TheRoyalSocietyofChemistry.

tigateadsorptionmechanismofpeptidesonhydrophobicsurfaces,

tofindout that multiplemechanismsare responsiblein

deter-miningtheadsorptionstrength.Themechanicalmappingcanbe

performedbothinairandinliquid,enablingstudyofvarious

envi-ronmentsonmechanicalandadhesionproperties.Dongetal.has

studiedforce-extensiononself-assembledfibrils formedby

29-residueamphiphaticpeptidehormoneglucagoninbufferwithapH

of2.0(Mingdongetal.,2008).Kimetal.hasusedafunctionalized

tiptomeasurethebindingpropertiesoflipopolysaccharides(LPS)

onimmuneproteins(lipopolysaccharidebindingprotein[LBP]and

CD14).Intheirstudy,Kimetal.(2007)havedirectlyobservedthe

concentrationdependentinhibitoryeffectofantimicrobialpeptide,

polymyxinB(PMB)onbindingofLBP-LPSandCD14.In

function-alized tipstudies, typically a linker(or spacer)is used toaffix

biomoleculesonto thetip. Thisallows freemotionand

confor-mationofthemoleculeonthetip.Theeffectoflinkerproperties

onadhesionpropertieshavebeenstudiedpreviously(Craigetal.,

2008),anditwasfoundthatoptimalspacerlengthsyieldedhighest

adhesion,underlyingthecomplex natureof suchdirect

quanti-tativemeasurements.Innanomechanicalmappingandadhesion

studies,typicallyalargenumberofmeasurementsareperformed

anddataisanalyzedusinghistogramstoobtainstatistically

sig-nificantinformation about the sample. During nanomechanical

measurements,forforce-extensionexperimentstypicallysoft

can-tileverswithspringconstantsontheorderof 0.01–0.1N/mare

used,whileforelasticmodulusmeasurementsstiffercantilevers

(1–10N/m)arepreferred.

AFMisahighlysophisticatedtechnique,whichcanbringout

highresolutionimagesnotonlyaboutthree-dimensional

topogra-phyofsurface,butalsomechanicalpropertiesofmoleculesonthe

surface.Besidesthis,laborioussamplepreparationtechniquesare

notrequiredinthismethod.AllthesemakeAFMveryattractivefor

researchersworkingwithpeptidenanomaterials.

5. Opticalmicroscopy-basedtechniques

Despitehavinglowerresolutionandmagnification,several

opti-cal microscopy techniquessuchas polarizing,fluorescence and

confocalmicroscopy provideinvaluableinformation for

charac-terizationofpeptidenanostructures.Immunostainingofsamples

provideexcellentcontrastforfinestructuresandallowsobtaining

imageswhichcannotbeobtainedwithTEM,SEMorAFM.

Inpolarizingmicroscopy,thesampleisilluminatedby

polar-izedlight,whichinteractswithanisotropicdomainsandgenerates

contrastbetweenanisotropicdomainsand otherpartsof

mate-rial.Self-assemblyofpeptidemoleculesproducedifferentliquid

crystallinedomainsin gels. Polarizinglight microscopyallowed

identificationofauniqueopticalproperty–birefringency–ofthese

anisotropicdomains.Hartgerinketal.(2002)showedorientation

ofliquidcrystallinephaseofpeptideamphiphilegelsintherange

oftensofmicrons(Fig.9A).Concentrationdependentvariationof

birefringencepropertyofpeptideamphiphilegelshasalsobeen

demonstrated(HungandStupp,2007).Hexagonalliquidcrystalline

phasechangestonematicphasewithweakerbirefringentproperty

asPAconcentrationdecreases.Uniformandlarge(tensof

millime-ters)birefringentdomainswereobservedinpolarizingmicroscopy

imagesofalignedmonodomainPAgelfilms,whereuniformityof

birefringencyindicatedalignmentofnanofiberstoconstitutegel

film(Fig.9B–D) (Zhanget al.,2010).Stainingalsostands tobe

apowerfulmethodtodetectpeptidenanostructuresinsolution.

Congoreddye,whichbindsto␤-sheet-richregions,iswidelyused

todetectamyloid-likestructures.Congored-stainedamyloid-like

peptidecoatedsurfacesshowbirefringentyellow-greendomains

(GileadandGazit,2004).

Orderedassembly of nanostructurescan giveriseto

higher-orderstructures,whichcanbeobservedevenbylightmicroscopy.

Forexample,peptidenanotubes(basedondiphenylalanine

self-assembly)filmsformedondifferentsurfacessuchasgold,SiO2and

InPwereimagedwithopticalmicroscopyalongwithotherimaging

techniques(Hendler et al.,2007).Peptide nanotube

crystalliza-tiongaverisetowell-organizedspherulites,whichwereclearly

observedatthismagnification.

Immunostainingofnanostructuresisanothermethodfor

visu-alization under fluorescence microscope. Limiting step here is

thelackofspecificantibodiesagainstpeptidemoleculesforming

the nanostructures. Amyloid nanofibrils are appropriate

exam-plesforthiscasesincetheyareformedbyaggregationofnatural

polypeptides.Inonestudy,fibrilsformedindrosophilabrainby

␤-amyloidpolypeptide,whichplaysacriticalroleinthepathology

ofneurodegenerativediseasessuchasAlzheimer’sdisease,were

fluores-Fig.9. Polarizinglightmicroscopyimagesofpeptideamphiphilegels.(A)WellknownPAgel,formedbyshortanisotropicdomains.(B)PAgelfilm,formedbynoodle-likegels, showinglargeandsimilaranisotropicdomains.(C)Onenoodle-likegelstringshowsalignedmonodomainextendingovercentimeters.(D)Lightextinctionatcross-points oftwogelstringsshowsuniformalignmentineachstring.

FigureAwasreprintedwithpermissionfromHartgerinketal.(2002).Copyright©(2002)NationalAcademyofSciences,U.S.A.FigureB-Dwerereprintedwithpermission fromZhangetal.(2010).Copyright©(2010)MacmillanPublishersLtd.

centmicroscopewhereself-assembledfibrillarstructuresstained

with␤-amyloidantibody couldbe observed(Scherzer-Attali et

al.,2010).Filamentoustemperature-sensitiveproteinZ(FtsZ)isa

prokaryoticmonomericprotein(homologoustoeukaryoticprotein

tubulin)whichself-assemblesintofilamentousstructures

form-ing contractile ring or Z-ring. Formation of contractile ring is

criticalforprokaryoticcelldivision.Inspiredbyself-assemblyof

FtsZmolecules,OstrovandGazit(2010)synthesizednanowires,

withthesemolecules.Forthispurpose,FtsZproteinsexpressedin

bacteriaweretaggedwithgold-binding,silver-reducingor

biotiny-lation motives at gene level. Biotinylated FtsZ polymers were

stainedwithfluorescentavidinandimagedbyconfocalmicroscopy,

whichallowedobservingfluorescentlystainedfilamentous

struc-tures.

Peptide-basednanostructurescan alsobe labeledcovalently

withfluorescentdyesforreal-timeimagingofdynamicprocesses.

Kametaet al. labeled bolaamphiphileswith Alexadye through

aminegroupsandobservedfluorescentnanotubesformedbythese

molecules.ToinvestigateencapsulationandtransportationofGFP

inAlexananotubes,time-lapsefluorescencemicroscopywasused

(Fig.10A).Excitationand absorptionfilters used inmirror unit

ofmicroscopealloweddetectionofFRET(fluorescenceresonance

energytransfer)fromGFPtoAlexa,which occursinnanotubes,

whilecuttingbulkGFPfluorescenceinsolutionand direct

exci-tationofAlexa(Kametaetal.,2008).

Staining ␤-sheet-rich nanostructures with fluorescent dyes

suchasThioflavinT(ThT)isanotherpossibilityforfluorescence

imagingofpeptidenanostructures.ThTbindsto␤-sheetsin

amy-loidfibrilsandgivesacharacteristicshiftinitsemissionspectrum.

Tamamisetal.(2009)stainedtriphenylalanineassembliesbyusing

ThTandanalyzedthesesampleswithconfocalmicroscopywhich

showedelongatedfibrillarstructures(Fig.10B).

Fluorescent imaging alsoallows following each molecule in

bulk gel, when latter one is formed by mixing two or more

moleculessuchasheparin and heparin-bindingPA. Fluorescent

labelingofheparinallowedobservingheparininPAgelbyusing

confocalmicroscopy(Fig.10C)(Rajangametal.,2006).Sometimes,

chemicalstructuresofsomepeptidemoleculesallowdisplaying

photoluminescencepropertywithoutfurtherstainingprocedure.

Diphenylalanine(FF)peptidenanotubesshowphotoluminescence

in blue and UV regions of excitation origin caused by

Fig.10.Fluorescentstainingofpeptidenanostructures.(A)Time-lapsedimagingofencapsulationandtransportationofGFPinnanotubeslabeledwithAlexa.2brightlines, appearingattwoendsofnanotubeandextendingtocentralpartlaterareduetoFRETfromGFPtoAlexa.(B)Fluorescenceimageofself-assembledtriphenylalaninenanofibrils stainedwithThTdye,staining␤-sheetstructures.(C)ConfocalmicroscopyimageofgelformedbymixingPAandfluoresceinheparin.(D)Fluorescentmicroscopyimageof surfacepatternedwithdiphenylalaninepeptidenanotubes.Bluesquaresarephotoluminescencefromnanotubes,whilepurplecircleatthecenteriscausedbyreflectionsof excitationbeamfromsurface.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

FigureAwasreprintedwithpermissionfromKametaetal.(2008).Copyright©(2008)JohnWileyandSons.FigureBwasreprintedwithpermissionfromTamamisetal. (2009).Copyright©(2009)Elsevier.FigureCwasreprintedwithpermissionfromRajangametal.(2006).Copyright©(2006)AmericanChemicalSociety.FigureDwas reprintedwithpermissionfromAmdurskyetal.(2009).Copyright©(2009)AmericanChemicalSociety.

et al., 2009). Patterned surface withFFnanotubes showed

flu-orescence (excitation at 340–380nm) from expected regions

(Fig.10D).

FRETcanbeusedtodetectspecificinteractionsin

biomolec-ularanddynamicsystemssuchaspeptideamphiphilegels.Ina

co-assembly systemcomprised of fluorophore attachedPA and

non-fluorescentsecondaryPA,FRETwasobservedbetweendonor

– fluorescent PA and fluorophore carrying heparin – acceptor,

which binds tonon-fluorescent PA (Behanna et al., 2006).The

energytransferwasverifiedbyacceptorphotobleaching

experi-ment,whichrecoveredemissionfromthedonor.

Ramanspectroscopyisanotherpowerfultechniqueto

inves-tigate the presence of various groups and their binding

prop-erties. Raman microscopy provides diffraction limited imaging

of biomolecular structures and conventionally cannot provide

single molecule resolution. Typically, the imaging volume is

about a micrometer cube. Even though individual fibers

can-not be resolved with confocal Raman microscopy, Matsui and

Douberly (2001) demonstrated that Raman signatures can be

usedtoidentifybundlesandindividualnanotubesself-assembled

out of the bolaamphiphilic peptide monomer

bis(N-R-amido-glycylglycine)-1,7-heptanedicarboxylate.Theyhaveshown that

Ramansignatures can be usedto discriminate peptidic

nanos-tructures bound to the bundles and the peptide nanotubes

disassembledfromthebundle.

TipandsurfaceenhancedRamanspectroscopy(TERSandSERS)

havebeenextensivelyusedtocharacterizebiomoleculesaswell.

TheversatilityoftheTERSmethodisdemonstratedbyNeugebauer

etal.(2006)whocollectedlocationdependentRamandataon

bac-terialsurfaces(S.epidermidiscells),showingpeptidicRamanbands.

Yeoetal.(2008)performedTERSonCytochromec(Cc),and

demon-stratedthesuperiorperformanceofTERSmethodonresolvingboth

thehemeandaminoacidvibrationalbands.Deckert-Gaudigand

Deckert(2010) demonstrated extremely highspatial resolution

(nanometer)ofTERSoninsulinfibrils.TERStechniqueistypically

appliedwithacombinedRamanandAFM/STMsystem,whereas

SERScanbeperformedusingasimplerRamanspectrometer.Aliaga

etal.(2011)havedemonstratedthatalargenumberofbandscan

beobservedandidentifiedusingSERSwithsilvernanoparticles,on

syntheticcarboxyterminalpeptideofhumanchorionic

gonadat-ropinb-subunit.ReproducibleSERSpectrawereobtainedbyadding

thecolloidalAgNPsolutionontothedriedanalytesample.

Simplic-ityoftheSERStechniqueallowswideapplicabilitycomparedto

TERS,inapplicationsnotrequiringspatialresolution.

Optical microscopy techniques take a snapshot of peptide

nanostructuresfromadifferentaspect.Thesetechniquesshould

beusedtoidentifyspecificdomainsorstructuresbylabelingfor

fluorescencemicroscopyandFRETtechniqueorbyusing

special-izedtechniquessuchaspolarizingmicroscopyandRaman-based

6. Conclusion

Peptidenanomaterialsarepromisingcandidatestosolvemany

issues regarding health, energy and information technology.

Progressin this field benefitsimmensely from characterization

studiesperformedbyhighlyadvanced microscopes.Capabilities

ofeachmicroscopytechniqueallowinvestigationof

nanomateri-alsfromdifferentaspects.TEMimaging,whichreliesonelectrons

travellinginsidethesample,allowsidentifyingultrafinepatterns

onnanostructures.Ontheotherhand,SEMimaginggets

informa-tionfromelectronsscatteredonthesurfaceofthesample,soitis

advantageousforimagingthickerand bulkiersamples.AFMcan

providetopographicalandmechanicalviewofthesurface,with

aneasiersamplepreparationprotocol.Opticalmicroscopy

tech-niques,inspiteoflowerresolution,provideveryusefulinformation

suchasidentificationofanisotropicdomainsinpeptidegelwith

polarizinglight microscopy.It isimportantfor researcherswho

workinthisareatounderstandlimitationsandadvantagesofeach

microscopytechniqueandchoosetherightoneforcharacterization

studies.

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