J. Soc.Cosmet.Chem., 24, 609-622 (September16, 1973)
InnovativeScanning Electron Microscopic Techniques for EvaluatingHair CareProducts SAL P. DxBIANCA, M.A.*
Synopsis-The utilization of microscopyin studying human hair is briefly reviewed. Reasons for selecting the SCANNING ELECTRON MICROSCOPE (SEM) over the transmissionelectron microscopeand the optical microscopeare discussed.The use of the SEM in evaluating HAIR CARE PRODUCTS is then described. A new technique employing a ROTATING HAIR STAGE, specially designed and fabricated for this study, is presented.The procedure devised allows one to view hair in the SEM while still attached to the panelist'shead. The technique is nondestructibleto the hair, permitting the study of sequentialtreatmentson the same hair. For example, the evaluation of a shampooon the hair after 0, 5, 10, and 20 treatmentsis now possible.The hair is removable from the SEM as many times as required for treatment without the necessity of cutting the hair from the scalp. In addition, the apparatusallows for complete axial rotation of the hair in the SEM.
The functionality of two hair care products(a shampooand a conditioner)is demonstratedusing this technique.MICROGRAPHS of hair damagesbefore and after treatment are categorizedand numericallyrated. The difference ratio was devised as an index to measurethe degreeof improvementof damagedsites. INTRODUCTION
In the past, evaluationof hair care productscenteredaroundsubjective beautysalonstudies.Recently,however,the value of the scanningelectron microscope(SEM) in revealingthe effectsof hair preparationshas cometo the forefront.This reportsummarizes part of our investigations into this area andrevealshow to employthe scanningelectronmicroscope asa tool to demonstratethe functionalityof a hair product.Beforegivingthe detailsof this *The Mennen Co., Morristown,N.J. 07960. 6O9
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investigation it is pertinentto brieflydescribethe capabilitiesand limitations of the threemicroscopic techniquesavailableand demonstrate why the SEM was chosen. MICROSCOPY OF HUMAN
HAIR
The scanningelectronmicroscope hasovercomemanylimitationsof optical microscopy and conventional transmission electronmicroscopy in elucidating the structure of human hair.
Usingthe opticalmicroscope, one viewsa pattern of light and dark areas producedby the reflectionor passagethrougha thin sliceof the specimen. Althoughhair can be viewed without interferences, while maintainingits natural colors,only thoseparts that lie in the sameplane can be reproduced sharply.Also,sincelight in the visiblerange of the spectrumis the energy source,the diameterof eachpart reproducedmustbe larger than the wavelengthof light. Only at low magnifications (below 200 diameters)is the light microscope usefulfor showingthe shapeanddepthof hair. In orderto avoidthis problem,earlystudiesof hair involvedthe viewingof very thin hair disks.Only limited informationcan be drawn from this technique,for onlyan extremelythin sectionof the specimencanbe viewed. The conventionaltransmission electronmicroscope(TEM) far exceedsthe magnificationof the optical microscope. This enormousmagnification(up to 200,000x) allowsthe study of detailswhich never appearedin the optical microscope. The TEM image is producedby monochromatic electronsthat have illuminated a specimenwhich transmitsor scattersthe electrons.Once pastthe specimen, the electronsare thenfocusedon a screenor sheetof film magnifyingthe image.What one seesis a two-dimensional pattern of light and dark areasproducedby passageof electronsthroughthe ultrathinspecimen.The useof ultrathinspecimens resultsin an extremelylow definitionof depth.Sincethe transmission electronmicroscope no longerworkswith light but electrons,the imagesproducedare not colored. Samplepreparationfor the TEM is complexand time consuming. Mostbiologicalsalnplesare replicated.Acetylcelluloseor a similarmaterialis placed overthe specimen whichis wettedwith methylacetate.After solventvolatilization the replicais carefullypeeledoff. This replica, or a secondreplica,is now shadowed.In this processa heavy metal suchas platinum or gold is evaporatedin vacuumon the samplesurface.What we seethen is not the samplebut a two-dimensional silhouette of the metaldeposits onthehair. Eliminatingthe opticalmicroscopy problemof narrowdepth of field and the transmissionelectron microscopelimitations of extensivespecimen preparations, the SEM has gainedpreferencein today'sresearchendeavors. It is an extremelyversatileinstrumentrevealingthe exacttopographical structure of the specimen.In normaloperation,the magnification rangeextends from 30x to 200,000x. The high depth of focus,a bonuscharacteristic of
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the instrument,revealsextreme architecturaldetail. Sample preparationis relatively simple.If the sampleis conductiveit need only to be fastenedto the samplemount.Our experiencehassho•vnthat micrographs of hair up to 1000x may be obtainedby this simplemountingprocedure.For greaterdefinitionof featuresandmagnifications to over 10,000x, the hair is coatedwith a thin layer of metal,usuallya gold palladiumalloy. The SEM is fundamentallydifferentfrom its TEM counterpartin that the electronsusedto producethe imagenormallyare not thosefrom the electron sourcebut lo•v-cncrgy(secondary)electronsreleasedfrom the surfaceof the sample.Althoughthe signalis typically producedby thesesecondaryelectrons,an imagecanbe producedby any signalresultingfrom the interaction of the high electronsourcewith the sample.SuchinteractionproducesX-rays, uv radiation,deflected(backscattered)electrons,ir radiation,etc., all of
•vhich•viththe properdetectionsystemcouldproducean appropriatesignal. The high-energy beam,usuallyoriginatingfrom a heatedtungstensource, is accelerated,alemagnified, and focusedto producea beam spotof approximately50 A. Deflectioncoilsplacedbetweenthe last lensprovidemeansfor X-¾ scanningof the specimenin a rectangularraster.When the electron sourcestrikesthe specimen, lo•v-cncrgy electronsare releasedfrom the surface. Thesesecondaryelectronsare dra•vnto a collectorand phototube.The instrumentelectronicsare such as to producea synchronism betweenthe electronbeam and a spot on a cathoderay tube, resultingin a one-to-one correspondence betweenthe positionof the electronbeam on the specimen andthat of the spoton the cathoderay tube.The resultis an imageproduced on a televisionscreenallowingthe viewerto infer a three-dimensional structure from a two-dimensional
screen.
The SEM was chosenfor this studybecausethe micrographs produced containmuchmoretopographical informationthan other microscopic techniques.The imagesproducedreveal the true surfacestructureover a •vide rangeof magnifications. It is obviousthat for evenrelativelylo•v magnifications,the SEM has distinctadvantagesover a standardoptical microscope; for example,only the SEM could reveal cuticleuplift or fiber flyaway as shownon Figs. 1, 2, and $. Historgt
Electronmicroscopy hasgreatlyextendedour insightsinto the structureof hair. The first applicationof the instrumentto this field •vas initiated by Zahnin Germanyin the early 1940's(1). This •vorkwascontinuedby othersin the United States,Netherlands,and Australia,andby 1948variousmethodsof replicatingthe surfaceof hair •vcrcdevised(2). Ho•vcvcr,becauseof instrument limitationsand the nonconductivcncss of hair, little work was performed
directlyon hair itself. Most of the •vorkinvolvedthe use of a metallicconductivecoating.
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Figure 1. Micrographo[ damagedhair fiber (250X) showingexposedcortex
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Figure2. Micrograph o[ hair fiberpulledapart(300X)
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Figure 3. Micrographof hair fiber pulled into a kn3t (800X) (note severecuticleuplift)
XVolframand Lindemannemployedthe SEM to reveal the morphological characteristics of hair (3). They pointedout that the cuticle,often neglected when consideringthe stress-strain propertiesof hair, may in reality have a substantialeffect.The conceptof cuticle-cortex ratio wasproposedto explain the supercontraction propertiesof hair. It was found that the level of supercontractiondecreases with increasingrelative cuticle (decreasingfiber diameter).
Swift investigatedthe architecturalchangesof hair surfacesresultingfrom simpletoiletrytreatments(4). Althoughthisstudygivessomeinsightsin how to choosethose productswith optimum characteristics(e.g., cleaningby shampooing),it still impliesthat manyhairsmustbe evaluated. Ayer and Thompson(5) utilized the SEM to stndy the coatabilityof hair sprayfihnson individual and small groupsof hairs.They investigatedthe,.use of severalsurfactantsto improvethe coatabilityand improvethe properties of hair spray formulationssuchas luster and flaking. The techniqueinvolved sprayinga hair swatch,drying,and gluingit to a mount.The samplewas then
shadowedwith a gold film, placedin the SEM, and viewed.
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EXPERIMENTAL
Rotating Hair Stage
All previous SEM studies of hair suffer from the primary experimental diffculty that hair exhibitsconsiderable heterogeneitynot onlyfrom personto personbut alsobetweenhairsfrom the samepersonand evendifferentsectionsalongthe samehair shaR.Consequently, when studyingthe effectof a productit is extremelycliffcult to concludethat a particularfeatureresulted from the treatmentand did not alreadyexistprior to the treatment. In orderto overcomethesedrawbacksthat have in the pastbeentakenfor grantedas being impossibleto overcome,a rotating hair stage (RHS)* was designedand fabricated (Figs. 4 and 5). The RHS providesfor mounting four differenthair shafts,each of which could be rotated aroundits axisby controlsexternalto the microscopevacuumsystem.After severalattempts,a satisfactory sea]was developedconsisting of a brassscrew-onnut, beveledon the inside,into which fit a carefully slit beveled rubber groinmet.When the hairswere placedin the slit, the pressureproducedby the beveledscrew-on nut wassufficientto producean excellenthigh vacuumseal. The rotatinghair stageallowedus to make a completelyvalid evalu.ation of hair before and after treatment.The RHS and the SEM parametersused resultedin the ability to performthe following: 1. The identical
area on the same hair was evaluated
in the before and
after micrographs.In order to ensurethis, completeaxial rotation in additionto the normal X-Y rotationof the SEM was performed,revealing the entire circumference of the hair. 2. The hair remainedon the panelist'shead when applyingthe product. The RHS allowed taking the samplein and out of ,the SEM without cuttingthe hair from the scalp. 3. The SEM instrumentparameterswere adiustedto obtain micrographs of hair without applyinga conductivemetallic c•ating. In otherwords, no specialtreatmentwas givento the varioushair samples.The charging problems,normally encountered,were minimized by operatingthe SEM a•treducedacceleratingvoltages(10 kv rather than 30 kv), low beam currents,and very specificsettingsfor the condenserlens and detectorpower supply. Test Protocol
Only girls with hair longer than 18 in. in length were selected;no other qualificationswere used.Eighteenincheswas our estimateof the minimum length requiredfor gettingthe hairs into and out of the microscope without breaking. *Designedjointly by the Mennen Co., Morristown,N.J., and StructureProbe Inc., West Chester,Pa.
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Figure 4. Rotating hair stage
A. Externalknobsfor rotatinghair shafts B. Face plate for bolting into microscope C. Vacuum seals for cables
D. Cable stabilizer
E. 4 mountingslotsfor mounting 4 hair shafts
Figure 5. Rotating hair stageshowingrubber grommetvacuum seal A. Slit rnbber grommet B. Beveled nut
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No effort was made to use panelists•vith or without any particularhair types (virgin, bleached,dyed, etc.), textures(fine, medium,coarse),amounts (thin, average,thick), and condition(oily, normal,dry). Table I showsthe percentages of differentcategories finally selected. Table
I
StatisticalBreakdownof Panelists'Hair Usedin Testinga Types
Amounts
Virgin
67
Thin
12
Bleached
25
Thick
41
Average
47
Dyed
8 Textures
Fine Medium Coarse
Condition
46 50 4
Oily Dry
47 2
Normal
51
"Figures given as percentages.
Each of four selectedhairs was color tagged near the scalp, then carefully fed throughthe rotatinghair stage.The hairs were not conditionedor treatedin any way. They xvereheld in placewith silver-platedwoodentoothpick ends.After providingsuffleientslackin the device,the rubber grommet xvasput in place,with the four hairsfitting xvithinthe slit. The apparatuswas thenboltedinto the opensidepostof the SEM. Usingthe televisionscanmode of the instrument,the damagedareasof each hair xverelocated. In some easesthe damagedareaswere located at considerable distancesalongthe hair shaft.To photographsuchdamages, montagesconsisting of as manyas 8-10 individualphotographs were made. Also,high magnification micrographs were madeof selecteddamagedareas. The panelist'shair wasremovedfrom the instrumentafter a suffleientnumber of "before"treatmentmicrographs xveretaken. The hairsxvereremovedfrom the rotatinghair stageand allowedto fall backinto place.The hairswere now randomlydistributedand for all practical purposes weresimilarto all otherson the head.That is, whenthe product was applied,thesehairs receivedno specialtreatment.For the shampoo product,the hair wasshampooed (txvolatherings)sixtimes.After eachshampooing,the hair wasthoroughlyrinsedand driedwith an electricdrier. The ha;rconditioner product,a leave-ontype,xvasappliedto slightlywet hair and
electricallydried.After treatmentthe panelist's taggedhairswerereturnedto the instrumentand the "after" micrographstaken.
By viexving the hairby meansof the TV modeandrotatingit, we wereable to find the exactarea of the hair shaftand photographthe repairedarea.In
additionto the photographs, videotapedatawerealsocollected. This ability to view the scanning of a hair shaftwhile rotatingit allowsoneto visually
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appreciate the improvement of eachhair shaft.The videotapedata contain, for severalreasons,a considerablygreateramountof informationthan could ever be recordedon Polaroidfilm. Of thesereasons,perhapsthe most importantis the fact that considerably higher magnificationinformationcan be recordedon videotape.This is possiblebecauseany image drift, which would ruin a 50-secphotographicexposure,doesnot have this catastrophic effectwhen videotaping.All videotapingis accompaniedby narrationwhich also documentsthe panelist'snumber, and time and place the data were recorded. DISCUSSION AND I•ESULTS
Healthy hair is composedof threeproteinaceous layers:the medulla,which is the central core-rarely found throughoutthe entire shaft; the cortex,extremelylongfibrilscomprisingmostof the hairsvolume;and the cuticle,the outer layer of overlappingplates.After enduringseveralyearsof exposure, abrasion,and styling,evenwell-treatedhair becomesdamaged. After viewing many hairs under the SEM, it was found th.at,with some
overlapping,the damageareascould be classifiedinto four generalcategories(i.e., flyawayfibers,exposed cortex,split ends,and generalshaftdamage).
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Figure 6. Damagecategory-flyawayfibers
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Examplesof eachcategoryare shownin Figs.6 through9. Also,a dramatic pair of beforeandaftermicrographs froman actualpanelist,showingthe improvementafter treatment,is shownin Figs. 10 and 11. To evaluatethe degreeof improvementresultingfrom the producttreatment a numericalsystemwas required.The differenceratio (DR), which is an indexto measurethe improvement of eachdamagedsite,wasdevisedand found to be a meaningfulsystemto communicatethe repair without being misleading. Each"before"and"after"pair of micrographs wasclassified into oneof the four mentioneddamagetypes,and eachdamagesiteassigned a danaage point from 0 to 4. (The higherthe nulnberthe more severethe damage.)The DR for ('achsitemay be calculatedby:
DR= damage points after treatment damagepointsbeforetreatment
A value of 1.0 indicatesno changewith treatment.DR valuesbelow 1.0 shoxvimprovementof a danaage site and thoseabove1.0 indicateadditional danaageresultingfrom the treatment.A summaryof the resultsis listed in Table II.
Figure 7. Damagecategory-exposed cortex
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Figure 8. Damagecategory--splitend
Figure 9. Damagecategory-generalshaftdamage
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Figure 10. Micrograph of general shaft damage before treatment with conditioning formulation
Table
II
Difference Ratio (DR) Values Conditioner•
Total number of panelists Total
number
of hairs evaluated
Total number of damage sites evaluated Average difference ratio calculated DR range per panelist
Shampoo•
50
15
192
59
442 0.26 0.03--0.66
127 0.47 0.28-0.71
"Shampooand leave-on conditionersuppliedby the Mennen Co., Morristown, N.J. Both productsare protein formulations.
SCANNING ELECTRON MICROSCOPIC TECHNIQUES
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