j. Soc.Cosmet. Chem.,48, 199-208 (July/August1997)

Use of atomicforce microscopyfor high-resolution non-invasive structural studies of human hair JAMES R. SMITH, Scanning ProbeMicroscopy Laboratory, School of Pharmacy, Biomedical and PhysicalSciences, University of Portsmouth, St. Michael'sBuilding,WhiteSwanRoad, Portsmouth PO1 2DT, UnitedKingdom.

Accepted for publication September 3O, 1997.

Synopsis

The morphologyof the fine cuticularstructureof human hair hastraditionallybeeninvestigatedusing scanningelectronmicroscopyand transmission electronmicroscopy.Although thesetechniquesare very useful,they requirespecimens to be coatedwith metallicfilms or to be suitablystained.In addition,high vacuumconditionsare requiredthat may damageor alter the appearance of delicatecuticular structures. Atomicforcemicroscopy is a relativelynewscanning probetechnique,capableof imagingsurfaces at high resolutionunderambientconditions. In this communication, the potentialapplications of atomicforce microscopy for structuralinvestigations of humanhair surfaces are discussed. Fine surfacestructures,such as the exocuticle,the endocuticle,and the marginalband (A- or oMayer),could be easilyidentified.The technique hasalsobeendemonstrated to imagehair surfaces in liquid environments, openingthe wayto in situstudiesof the effectsof hair-careproductsand treatments.

INTRODUCTION

The humanhair is protectedby an almostformidablebarriercomprisedof manythousandsof scale-likeplates,calledcuticles.Thesearearrangedin approximately six to ten layers(1), eachof a thicknessof 0.3-0.5 pm (2,3). The cuticularcell consistsof essentially two layers,the endocuticleand exocuticle(1), the formerbeingthe inner onethird of the cellandcontainingcellulardebristhat caneasilyundergohydrolysis by proteases (4,5). A significantdegreeof keratinizationoccursnearthe surfaceof the cuticle,and trichohyalingranules(6) are depositednearthe lateralcell wall; it is this layer,termed the exocuticle,that is responsible for the hair'sresistance to detergents,organicsolvents, and many other harsh environments(7).

Structuralinformationconcerning hair cuticleshaslargelybeenobtainedfrom scanning electronmicroscopy (SEM) and transmission electronmicroscopy (TEM) studiesof human hair fibres(3,4,7-9). While theseare valuabletechniques,their applicationin structuralhair analysisis limited by the requirementfor samplesto be coatedwith a thin metallic film, or, in the caseof TEM, the needto suitablystain specimens prior to investigation.In addition, both these microscopictechniquesrequire high vacuum 199

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conditionsfor successful imaging, which could damageor alter the appearance of fine cuticular

structures.

Atomic forcemicroscopy(AFM) is a relativelynew technique(10) and is a memberof a generic classof related scanningprobe microscopy(SPM) techniques(11,12). The principleof AFM relieson the useof a sharp,pyramidaltip mountedon a cantileverthat is brought into closeproximity to the sampleof interestwhere intermolecularforces acting betweenthe tip and the surfacecausethe cantileverto bend. Cantileverdeflectionsare detectedby a laserbeamfocusedon the top of the cantileverand areconverted to a digital voltage signal that is then applied acrossa piezoelectricceramicelement to which the sampleis mounted.This hasthe effectof maintaininga constantforce,in the order of a few nano-Newtons, between the scanningtip and the sample. A threedimensionalmap of the specimen'stopographycan thereforebe obtainedby plotting this feedbackvoltage signal againstX and Y coordinates.

Confocalmicroscopy hasrecentlybeendemonstrated to be a goodtechniquefor imaging hair surfacesin their natural environment (13). However, the resolution limit for this

method is of the orderof 0.25 pm, an improvementon conventionallight microscopy (0.6 pm), but much lessthan that of electronmicroscopy. SinceAFM doesnot require the focusingof light or other radiationfor its operation,a very high resolutionis achievable,of the orderof 1-2 nm for biologicalmaterials.This high resolvingpower, togetherwith the ability to imagesurfaces in aqueousenvironments, makesAFM a very attractivetechniquefor imaging hair surfaces. Only a few reportson the use of AFM in this applicationarea have appearedin the literature(14-17). Here, the potentialapplications of AFM for structuralinvestigations of human

MATERIALS

hair surfaces are discussed.

AND

METHODS

Human scalphairswere obtainedfrom biopsiesfrom Europeanbrown-hairedsubjects who had washedtheir hair approximately5 h prior to the investigations.A sodium lauryl sulphate-based shampooand conditionercontaining dimethiconesor 1% zinc pyrithionewas used,and hair wasallowedto dry naturallyat room temperature.'Unwashed'samplesrefer to hair that had beenwashedthe previousday and had not been groomed.Hair sectionsof 1 cm in length, no further than 5 cm from the scalp,unless otherwisestated,were cut and fixed on a samplemounting assemblyusing carbonloaded double-sidedadhesivetape. AFM studieswere performedusing a Discoverer TMX2000 scanningprobe microscope(SPM) (TopoMetrix Corporation,SaffronWalden, Essex,UK) under atmosphericconditions,although in someexperiments,a liquid cell was used to obtain imagesin an aqueousenvironment.A relative humidity of 60-65% was notedduring all the experiments,althoughthis wasnot accuratelycontrolled; room temperaturewasrecordedto be 22øC. Standardprofile pyramidalsilicon

nitridetipsmounted oncantilevers offorceconstants in therange0.036to0.072N m-• wereused.The samplewasheld in positionon a piezoelectrictripod scannercapableof a maximum XYZ translationof 75 x 75 x 12 pm. Imageswere left-shadedto enhance topographical featuresand displayedon a monitorwith a resolutionof 500 lines x 500 pixels. Quantitativedata, such as height measurements and surfaceroughness,were obtainedusingTopoMetriximageanalysissoftware(18). The arithmeticsurfacerough-

AFM

FOR

STUDIES

OF HUMAN

HAIR

201

nessaverage,Ra, is the arithmeticaverageof the absolutevaluesof the measuredprofile height deviations,given by: n

•lZ i- Zl i=l

Ra =

(1)

n

where,n -- numberof heightpositionsalongline profile,Z i -- heightat positioni (nm), and Z -- averageheight (nm). For this study,a line profile of 4 pm wasused. --

RESULTS

AND

DISCUSSION

Figure 1 showsa three-dimensional AFM micrographof the surfaceof a typical human scalphair taken from a Europeanbrown-hairedsubject.Cuticleswerefoundto be very well defined and lying flat on the hair shaft. The arrangementof cuticular cells is multilayered,due to cuticlesoverlappingand not because of multiple cell layering.An interestingscratchmark featurecan be observedtraversingfrom the top left to the bottom right in Figure 1 in the generaldirection of the fiber axis;suchfeatureshave been observedelsewhere(19). On closerexaminationof the image, three types of cuticularcell can be identified: the exocuticle,the endocuticle,and the marginal band (A- or u-layer). Thesestructuresare indicatedin Figure 2. The marginalband (20,21), denoted"A" covereda largepart of the exocuticleand wasvery smoothin appearance. The thicknessof this layerwastypically in the rangeof 30-70 nm, in agreementwith the 45-50 nm rangeof valuesreportedelsewhere(22). The exocuticles,denoted"B" in

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0 nm

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Figure 1. Three-dimensional representation of an AFM micrographof humanEuropeanbrownscalphair.

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ß

20prn.... -90.0 ø

B B 90.0 ø ,' (

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Figure 2. An AFM micrographof humanEuropeanbrownscalphair. A = marginalband(A- or c•-layer), B = exocuticle, C = endocuticle.

Figure 2, were much more rough, showinga woodgrainappearance in the directionof the fiber axis. Exocuticleswere very jaggedat their brokendistal ends,in agreement with SEM studies.In somecases,wherethe breakagewasfragmented,a rougherdeposit of endocuticularmaterial, "C," could be seenon the top surfaceof the underlying exocuticle.Similar breakagepatterns, together with postulatedmechanismsof their formation, have been discussedelsewhere(3).

The AFM is able to provide quantitative data as well as high-resolutionvisual information. In Figure 3, two cross-sectional profilesshowthe surfacerelief of an exocuticle (upper)and an endocuticle(lower).As expected,the exocuticlewasmuchsmootherthan the exposedunderlyingendocuticle(Ra:9 nm cf. 30 nm). The useof surfaceprofilometry to recordsurfaceroughness alongthe fiber axisof humanhairshasbeenreportedin the literature (23). While there are obviouslysomesimilaritiesin surfaceprofilometryto AFM, the macroscopic techniqueis muchlesssensitive.When operatingon a submicron scale,height informationcanbe grosslyaffectedby the very largeradiusof curvatureof the tip (2.5 Fm). Suchartefactsarenot observedin profile measurements obtainedusing AFM, becausethe radius of curvature is much smaller (