J. Soc.Cosmet.Chem., 24, 609-622 (September 1973)
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
transmission electron 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, per-
mitting 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 demon-
stratedusing this technique.MICROGRAPHS of hair damages before and after treatment
are categorizedand numericallyrated. The difference ratio was devised as an index to
measurethe degreeof improvementof damagedsites.
In the past, evaluationof hair care productscenteredaroundsubjective
beautysalonstudies. Recently,however,the value of the scanning electron
(SEM) in revealingthe effectsof hair preparations cometo
This reportsummarizes of our investigations this area
the forefront. part into
andreveals how to employthe scanning electron as
microscope a tool to dem-
onstrate functionality a hair product.Beforegivingthe detailsof this
*The Mennen Co., Morristown,N.J. 07960.
610 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
investigation is pertinentto brieflydescribe capabilitiesand limitations
of the threemicroscopictechniques why the SEM
MICROSCOPY OF HUMAN HAIR
The scanningelectron has
microscope overcome of
microscopy conventional electron
the structure of human hair.
Usingthe opticalmicroscope, viewsa pattern of light and dark areas
produced the reflection passage througha thin sliceof the specimen. Al-
thoughhair can be viewed without interferences, its
while maintaining natu-
ral colors,only thoseparts that lie in the sameplane can be reproduced
sharply.Also,sincelight in the visiblerange of the spectrum the energyis
source, diameterof eachpart reproduced mustbe larger than the wave-
lengthof light. Only at low magnifications (below 200 diameters)is the light
usefulfor showing shape anddepthof hair.
In orderto avoidthis problem, of
earlystudies hair involvedthe viewingof
very thin hair disks.Only limited information can be drawn from this tech-
nique,for onlyan extremely of can
thin section the specimen be viewed.
The conventional transmission electron microscope (TEM) far exceeds the
magnification the optical microscope. This enormous magnification (up to
200,000x) allowsthe study of detailswhich never appearedin the optical
microscope. TEM image is produced monochromatic by electrons that
have illuminated a specimen which transmitsor scattersthe electrons. Once
the are on
pastthe specimen, electrons thenfocused a screen sheet film or of
magnifying image.What one seesis a two-dimensional pattern of light
and dark areasproduced passage electrons of throughthe ultrathinspeci-
men.The useof ultrathinspecimens in
results an extremely low definition of
depth.Sincethe transmission electron no
microscope longerworkswith light
but electrons, images are
produced not colored.
Sample for and
preparation the TEM is complex time consuming. Mostbio-
logicalsalnples replicated. or
Acetylcellulose a similarmaterialis placed
overthe specimen whichis wettedwith methylacetate. After solventvolatili-
zation the replicais carefullypeeledoff. This replica, or a second replica,is
now shadowed. this process heavy metal suchas platinum or gold is
evaporated vacuumon the samplesurface. What we seethen is not the
sample a two-dimensional of on
silhouette the metaldeposits thehair.
Eliminatingthe opticalmicroscopy problemof narrowdepth of field and
the transmission electron microscope limitations of extensivespecimen
preparations, SEM has gainedpreference today's in research endeavors.
It is an extremely versatile instrument the
revealing exacttopographical struc-
ture of the specimen. normaloperation, magnification rangeextends
from 30x to 200,000x. The high depth of focus,a bonuscharacteristic of
SCANNING ELECTRON MICROSCOPIC TECHNIQUES 611
the instrument,revealsextreme architecturaldetail. Sample preparationis
relatively simple.If the sampleis conductive need only to be fastenedto
the sample has
mount.Our experience sho•vn of
that micrographs hair up to
1000x may be obtained this simplemounting procedure. For greaterdefi-
nitionof features to
andmagnifications over 10,000x, the hair is coatedwith a
thin layer of metal,usuallya gold palladiumalloy.
The SEM is fundamentally in
differentfrom its TEM counterpart that the
electrons usedto producethe imagenormallyare not thosefrom the electron
source lo•v-cncrgy (secondary) electrons released of
from the surface the
sample.Althoughthe signalis typically producedby thesesecondary elec-
trons,an imagecanbe produced any signalresulting from the interaction
of the high electronsource with the sample.Suchinteraction produces X-rays,
uv radiation,deflected(backscattered) ir
electrons, radiation,etc., all of
•vhich•viththe properdetection an
couldproduce appropriate signal.
The high-energy from a heatedtungsten
alemagnified, focused producea beam spotof approxi-
is accelerated, and to
mately50 A. Deflectioncoilsplacedbetweenthe last lensprovidemeansfor
X-¾ scanning the specimen a rectangular raster.When the electron
strikes specimen, electrons released
lo•v-cncrgy are from the sur-
face. Thesesecondary are
electrons dra•vnto a collector and phototube.The
electronics 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 cathode tube.The resultis an image produced
on a televisionscreen struc-
allowingthe viewerto infer a three-dimensional
ture from a two-dimensional screen.
The SEM was chosen this studybecause micrographs produced
containmuchmoretopographical information than other microscopictech-
niques.The imagesproducedreveal the true surfacestructureover a •vide
rangeof magnifications. is obviousthat for evenrelativelylo•v magnifica-
tions,the SEM has distinctadvantagesover a standardoptical microscope;
for example,only the SEM could reveal cuticleuplift or fiber flyaway as
shownon Figs. 1, 2, and $.
Electron has our into
microscopy greatlyextended insights the structure of
hair. The first application the instrument this field •vas initiated by
in (1). by
Zahnin Germany the early 1940's This •vorkwascontinued othersin
the United States, and
Netherlands, Australia,andby 1948various methods of
replicating surface hair •vcrcdevised because instru-
(2). Ho•vcvcr, of
ment limitationsand the nonconductivcncss hair, little work was performed
directlyon hair itself. Most of the •vorkinvolvedthe use of a metalliccon-
619. JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Figure 1. Micrographo[ damagedhair fiber (250X) showingexposedcortex
.,,'.:%.. •.'•.•,,;..:•.•.:":::....•.. .-.-c',,....'
,."'."'•';..;...::;: ,-i- '.:. .......... .?--:...•..•-•:•....::¾:•-; '--
• -..:-•,•'.. .• ......
..., ß .• .:;':, .... .•
'* ....,,. .........
'-"• •,•.,,.,..'•'•." • ' ....
.... .; '*' ..... ....
"".;::'""':'.Z•"'•...... .." '-"•'•'.
• --'-•'•-'••' .
a... ,. % " •. % .*,,. .
. ',-,,,, . .½•. .::..:-..-.-. ...
. .•,f%.•.•--:c•..;-'.'-.-.•-•-,, .-
• , ,•."" • .... .,.. :.?,-.,?:•:..•.-•,-•......•,.............:-.
•' • ,.'"• "'•. '"'../'-'-
'-i.. "....'"'"'??S?'"--,.•?."•'•:/:" .....
Figure2. Micrograph hair fiberpulledapart(300X)
SCANNING ELECTRON MICROSCOPIC TECHNIQUES 613
Figure 3. Micrographof hair fiber pulled into a kn3t (800X) (note severecuticleuplift)
XVolframand Lindemannemployedthe SEM to reveal the morphological
characteristics hair (3). They pointedout that the cuticle,often neglected
when considering stress-strain of
properties hair, may in reality have a
effect.The concept cuticle-cortex
substantial of to
ratio wasproposed explain
properties hair. It was found that the level of super-
the supercontraction of
contractiondecreaseswith increasingrelative cuticle (decreasingfiber diame-
Swift investigated architectural of
changes hair surfaces resultingfrom
simpletoiletrytreatments (4). Althoughthisstudygivessome in
to choosethose productswith optimum characteristics (e.g., cleaningby
shampooing), still impliesthat manyhairsmustbe evaluated.
Ayer and Thompson(5) utilized the SEM to stndy the coatabilityof hair
sprayfihnson individual and small groupsof hairs.They investigated use
of severalsurfactants 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
with a gold film, placedin the SEM, and viewed.
614 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Rotating Hair Stage
All previous SEM studies of hair suffer from the primary experimental
diffculty that hair exhibitsconsiderable not
heterogeneity onlyfrom person to
person but alsobetweenhairsfrom the samepersonand evendifferentsec-
tionsalongthe samehair shaR.Consequently, the
when studying effectof a
productit is extremelycliffcult to conclude that a particularfeatureresulted
from the treatmentand did not alreadyexistprior to the treatment.
In orderto overcome thesedrawbacks that have in the pastbeentakenfor
grantedas being impossible overcome, 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
controls externalto the microscope vacuumsystem. After severalattempts,a
satisfactory was developed of
consisting a brassscrew-on nut, beveledon
the inside,into which fit a carefully slit beveled rubber groinmet.When the
hairswere placedin the slit, the pressure by
produced the beveledscrew-on
nut wassufficient producean excellent high vacuumseal.
The rotatinghair stageallowedus to make a completely valid evalu.ation
of hair before and after treatment.The RHS and the SEM parameters used
resulted the ability to performthe following:
1. The identical area on the same hair was evaluated in the before and
after micrographs. order to ensurethis, completeaxial rotation in
additionto the normal X-Y rotationof the SEM was performed,reveal-
ing the entire circumference the hair.
2. The hair remainedon the panelist's head when applyingthe product.
The RHS allowed taking the samplein and out of ,the SEM without
cuttingthe hair from the scalp.
3. The SEM instrumentparameters were adiustedto obtain micrographs
of hair without applyinga conductive metallic c•ating. In otherwords,
no specialtreatmentwas givento the varioushair samples. The charging
problems,normally encountered, were minimized by operatingthe
SEM a•treducedaccelerating voltages(10 kv rather than 30 kv), low
beam currents,and very specificsettingsfor the condenser lens and
Only girls with hair longer than 18 in. in length were selected; other
were used.Eighteenincheswas our estimateof the minimum
length requiredfor gettingthe hairs into and out of the microscopewithout
*Designedjointly by the Mennen Co., Morristown,N.J., and StructureProbe Inc., West
SCANNING ELECTRON MICROSCOPIC TECHNIQUES 615
..... I Ilfilfillllll .........
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
616 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
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 differentcategories finally selected.
Statistical of Hair Usedin Testinga
Virgin 67 Thin 12
Bleached 25 Thick 41
Dyed 8 Average 47
Fine 46 Oily 47
Medium 50 Dry 2
Coarse 4 Normal 51
"Figures given as percentages.
Each of four selectedhairs was color tagged near the scalp, then care-
fully fed throughthe rotatinghair stage.The hairs were not conditioned or
treatedin any way. They xvere held in placewith silver-plated woodentooth-
pick ends.After providingsuffleient slackin the device,the rubber grommet
xvas in place,with the four hairsfitting xvithin slit. The apparatus was
thenboltedinto the opensidepostof the SEM.
Usingthe television the
scanmode of the instrument, damagedareasof
each hair xverelocated. In some easesthe damagedareaswere located at
considerable distancesalongthe hair shaft.To photograph suchdamages,
consisting as manyas 8-10 individualphotographs were made.
Also,high magnification micrographs were madeof selected damagedareas.
The panelist's hair wasremoved from the instrument number
after a suffleient
of "before" treatmentmicrographs xvere taken.
The hairsxvereremovedfrom the rotatinghair stageand allowedto fall
backinto place.The hairswere now randomly and
distributed for all practi-
cal purposes on
weresimilarto all others the head.That is, whenthe product
was applied,thesehairs receivedno specialtreatment.For the shampoo
product, hair wasshampooed six
(txvolatherings) times. After eachsham-
pooing, hair wasthoroughly rinsedand driedwith an electricdrier. The
product, leave-on type,xvas appliedto slightlywet hair and
dried.After treatment panelist's
electrically the tagged to
the instrumentand the "after" micrographstaken.
the of it,
By viexving hairby means the TV modeandrotating we wereable
to find the exactarea of the hair shaftand photograph repairedarea.In
addition the photographs,videotapedatawerealsocollected.This ability
to view the scanning a hair shaftwhile rotating allowsoneto visually
SCANNING ELECTRON MICROSCOPICTECHNIQUES 617
the of data contain,
appreciate improvement eachhair shaft.The videotape
for severalreasons, considerably greateramountof informationthan could
ever be recordedon Polaroidfilm. Of thesereasons, perhapsthe most im-
portantis the fact that considerablyhigher magnificationinformation can
be recorded videotape.This is possible becauseany image drift, which
would ruin a 50-secphotographic exposure, doesnot have this catastrophic
All is by
effectwhen videotaping. videotaping accompanied narrationwhich
also documents the panelist'snumber, and time and place the data were
DISCUSSION AND I•ESULTS
Healthy hair is composed threeproteinaceous layers:the medulla,which
is the central core-rarely found throughoutthe entire shaft; the cortex,ex-
tremelylongfibrilscomprising mostof the hairsvolume;and the cuticle,the
outer layer of overlapping plates.After enduringseveralyearsof exposure,
abrasion, styling,evenwell-treatedhair becomes damaged.
After viewing many hairs under the SEM, it was found th.at,with some
overlapping, damageareascould be classified into four generalcate-
(i.e., flyawayfibers, split ends,and general
Figure 6. Damagecategory-flyaway
618 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
of are in
Examples eachcategory shown Figs.6 through9. Also,a dramatic
from showing im-
pair of beforeandaftermicrographs an actualpanelist, the
provement is in
after treatment, shown Figs. 10 and 11.
To evaluatethe degreeof improvement resultingfrom the producttreat-
ment a numerical system was required.The difference ratio (DR), which is
an indexto measure improvement eachdamaged site,wasdevisedand
found to be a meaningful to the
system communicate repair without being
Each"before" "after"pair of micrographs classified oneof the into
four mentioned damage types,and eachdamage a
siteassigned danaage point
from 0 to 4. (The higherthe nulnberthe more severe damage.)The DR
for ('achsitemay be calculated by:
= points treatment
DR damage after
A value of 1.0 indicates changewith treatment.DR valuesbelow 1.0
shoxv of site
improvement a danaage and thoseabove1.0 indicateadditional
danaageresultingfrom the treatment.A summaryof the resultsis listed in
Figure 7. Damagecategory-exposed
SCANNING ELECTRON MICROSCOPIC TECHNIQUES 619
Figure 8. Damagecategory--split
Figure 9. Damagecategory-general
620 JOURNAL OF THE SOCIETY OF COSMETIC CHEMIST$
Figure 10. Micrograph of general shaft damage before treatment with conditioning
Difference Ratio (DR) Values
Total number of panelists 50 15
Total number of hairs evaluated 192 59
Total number of damage sites evaluated 442 127
Average difference ratio calculated 0.26 0.47
DR range per panelist 0.03--0.66 0.28-0.71
"Shampoo and leave-on conditionersuppliedby the Mennen Co., Morristown, N.J. Both
productsare protein formulations.
SCANNING ELECTRON MICROSCOPIC TECHNIQUES 6•1
:}'.•... •,,--:•: :...
-. \ ":'? •
..... <• •.-•'
. •.. .... •..
• ß • '"'
of repair of shaftdamage
Figure 11. Micrograph samearea as in Fig. 10 showing with
one applicationof conditioner
A closer lookat the damage categories that split endsaccount
35%of all hair damage studies III
(Tables and IV). Flyawayfibersand gen-
eral shaftdamage werefoundin 32 and25%of the damaged respective-
Results of Shampoo Treatment
Category Per Cent
DamageCategory Per Cent Improved
Flyaway fibers 35 93
Exposed cortex 7 88
Split ends 35 60
Shaft damage 23 93
622 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Results of Conditioner Treatment
Category Per Cent
DamageCategory PerCent Improved
Flyaway fibersfrom cuticle 28 94
Exposedcortex 8 93
Split ends 35 95
General shaft damage 26 92
Miscellaneous 3 80
improvement all damage typesaftertreatment.
For example, on
based a minimum of
reduction onedamage point in our rat-
ing system, site
over90%of the damaged improved after treatment with the
hair conditioning In actuallydosed60%of
formulation. fact, the conditioner
the split ends(improved zero column). The shampoo formulation tested
improved typesof hair damage(Table III). This studynot .onlydemon-
stratesimprovementof damage sites but revealsthe products'ability to
repair different types of damages.
approach evaluatehair careproducts
A systematic to usingthe SEM has
beendeveloped. employing rot.
The technioue, a ating hair stage(RHS) al-
lows for maximumrotational freedom of the hair in the SEM. The hair, while
still attachedto the scalp,may be removed into the SEM
as often as necessary.
The difference ratio (DR), a numericalsystemshowingthe degreeof
improvement damagedsitesafter treatment
with a hair conditioner and shampoo.
The author is grateful to Dr. C. Garber, Presidentof StructureProbeInc.,
for his continuing All
effortsand suggestions. micrographs shownwere taken
at Structure Probe Inc.
( Received 29,
(1) Fraser, R. D. B., and Rogers,G. E., Aust. ]. Biol. Sci., 8, 129 (1955).
(2) Barnes,R. B., Burton, C. J., and Scott, R. G., Electron microscopical replica technique
for the studyof organicsurfaces, Appl. Phys.,16, 730 (1945).
(3) Wolfram, J. L., and Lindemann, M. K. O., Some observationson the hair cuticle,
]. Soc.Cosmet.Chem., 22, 839 (1971).
(4) Swift, J. A., New developments electronmicroscopy, Ibid., 22, 477 (1971).
(5) Ayer, P. A., and Thompson, J. A., Scanning electron microscopyand other new
approaches hair spray evaluation,Ibid., 23, 617 (1972).