Technical note: A new three-dimensional technique for high resolution quantitative recording of perikymata

Technical Note: A New Three-Dimensional Technique forHigh Resolution Quantitative Recording of Perikymata

E. Bocaege,1,2* L.T. Humphrey,2 and S. Hillson1

1Institute of Archaeology, University College London, London WC1H 0PY, UK2Department of Palaeontology, Natural History Museum, London SW7 5BD, UK

KEY WORDS enamel; incremental markings; perikymata; three-dimensional analysis

ABSTRACT The number and spacing of incrementalmarkings at the enamel surface, known as perikymata,are considered important indicators of dental growth pat-terns, as they provide information on crown formationtimes and the underlying developmental processes. Thisstudy explores the potential of a new three-dimensionaltechnique for the reconstruction of dental growth profiles,using teeth from a medieval child from Abingdon, Oxford-shire. The crowns of three anterior teeth were imaged and

analyzed using the Alicona 3D InfiniteFocus imagingmicroscope. Individual perikyma grooves can be unambig-uously identified on a profile of the reconstructed enamelsurface and direct distances between successive pairs ofperikyma grooves can be calculated from coordinate data.This quantitative approach constitutes a more objectiveway to record perikymata spacing than current methods.Am J Phys Anthropol 141:498–503, 2010. VVC 2009 Wiley-Liss,

Inc.

The process of enamel formation leaves its mark as aseries of microscopic grooves running around the toothcrown surface known as perikymata. These are the sur-face expression of a regular layered structure seen insections of enamel (Fitzgerald, 1998; Reid and Dean,2000; Dean et al., 2001; Smith, 2008). For the first yearor so of crown formation, the layers pile up to form thecuspal (appositional) enamel and it is only after thispoint that perikymata are formed down the crown side(imbricational enamel). To estimate crown formationtimes, assumptions need to be made about age of crownformation initiation, duration of appositional enamel for-mation, and the number of days between long periodlines (Retzius lines beneath the enamel surface and peri-kymata at the enamel surface). Nevertheless perikymatacounts have proved useful for estimating crown forma-tion times in fossil taxa and for estimating age at deathin cases where tooth crown formation was incomplete atthe time of death (Bromage and Dean, 1985; Dean et al.,1986; Dean, 1987; Smith et al., 2007b). Perikymata pack-ing or spacing patterns differ between species and maybe of value as taxonomic indicators (Dean, 1987; Deanand Reid, 2001; Ramirez-Rozzi and Bermudez de Castro,2004; Guatelli-Steinberg et al., 2005, 2007). Detailedanalysis of perikymata spacing can be used to identifyenamel hypoplasias (Hillson and Bond, 1997; King et al.,2002, 2005; Guatelli-Steinberg et al., 2004).Perikymata have conventionally been imaged on the

crown surface with reflected light microscopy or usingscanning electron microscopy (Boyde, 1971; Newmanand Poole, 1974; Hillson and Bond, 1997; Dean andReid, 2001; Ramirez-Rozzi and Bermudez de Castro,2004; King et al., 2002, 2005; Guatelli-Steinberg et al.,2005, 2007). Conventional reflected light microscopy canonly be used to image a small area of the crown at onetime, because the strongly curved surface means thatother parts of the enamel surface are out of focus. Mod-erately high magnifications are needed for the sharpestdefinition of perikyma grooves and for the most accuratemeasurements of spacing, but higher power objective

lenses are associated with smaller depth of focus, so it isnecessary to find a compromise.Two measurement methods can be used to derive peri-

kymata spacing patterns. One is to fit a graticule in theeyepiece, calibrate it and then count the number of peri-kyma grooves within a given distance over the crown(Reid and Dean, 2000; Dean and Reid, 2001; RamirezRozzi and Bermudez de Castro, 2004; Guatelli-Steinberget al., 2005, 2007).This yields a density of perikymata(count per decile of crown height for example), whichcan be used to determine average perikymata spacingwithin that region of the crown surface. It is not practi-cal to use a graticule to measure the spacing individu-ally. As the crown surface is curved, its changing anglerelative to the optical axis of the microscope must intro-duce measurement errors.An alternative is to use an engineer’s measuring

microscope to record coordinates for each perikymagroove along a transect down the crown side and toderive spacing mathematically from these coordinates(Hillson and Jones, 1989; King et al., 2002, 2005). Thisrelies on precise focusing on each groove to give thevertical coordinates. In the authors’ experience, theobjective lenses required for practical imaging have adepth of focus of around 7 lm (i.e., the zone of heightvariation within which any features appear to be infocus). In effect, that dictates the precision of the meas-urements. Perikymata in the central part of the crownare spaced at around 60 lm so a potential error of 7 lmrepresents 12% of the distance between neighboring

*Correspondence to: Emmy Bocaege, Institute of Archaeology,University College London, London WC1H 0PY, UK.E-mail: [email protected]

Received 11 May 2009; accepted 14 October 2009

DOI 10.1002/ajpa.21233Published online 1 December 2009 in Wiley InterScience

(www.interscience.wiley.com).

VVC 2009 WILEY-LISS, INC.

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 141:498–503 (2010)

grooves. In practice, the accumulated error over a seriesof perikymata tends to be rather less but it still makesdetailed comparisons difficult.In this article we evaluate the potential of a new tech-

nique for high resolution quantitative recording of peri-kymata. The Alicona InfiniteFocus is an optical systemthat constructs a three-dimensional replica of a surfacefrom a stack of images and incorporates softwarefor high resolution three-dimensional analysis of thereconstructed surface. Major applications include themeasurement of surface textures on bearing surfaces inengineering, for which high resolution is at a premium(Wanske et al., 2008). One of the first applications to bio-logical materials involved analysis of bone surface modi-fications (Bello and Soligo, 2008).

MATERIALS AND METHODS

Images of developing permanent incisors from a singlejuvenile (individual 2212) from a Medieval cemeterysample from Abingdon, Oxfordshire were produced andanalyzed. The three teeth utilized in this study were alower central incisor, an upper central incisor, and anupper lateral incisor. These incisors had incompletelyformed roots and were relatively unworn, with mame-lons remaining on the occlusal edges and Tomes’ processpits visible on many perikyma ridges. Prior to imaging,teeth were cleaned with industrial methylated spirit.Teeth were placed on a black plastazote surface andscanned without a nonreflective coating.

Imaging

The Alicona InfiniteFocus scans a series of imageplanes over a defined focus range, set to include the fullrange of features in the specimen under study. Theinstrument captures the spectral variation between over-illuminated and under-illuminated surfaces. A detailedthree-dimensional model of the surface is constructedfrom the stack of images, calculating x, y, and z coordi-nates for any point within the resolution of the scan.A resolution of up to 0.4 lm horizontally and 0.01 lm

vertically can be obtained using the highest magnifica-tion objective lens and slowest scan speed. Scanning atthis resolution is time consuming, but it is possible toreach an effective compromise (below). The very highvertical resolution is particularly useful for perikymataas their grooves are extremely shallow features.Overlapping images of the crown surfaces were cap-

tured along a band of enamel at the centre of the labialsurface of each incisor running from the cusp tip to thecemento-enamel junction. Each image field measuredaround 1.45 mm in length and around 700 lm in width(see Fig. 1). Two additional strips were imaged on themesial and distal extremities of the labial surface of thelower central incisor.The mamelons of the unworn incisors were scanned

with the teeth tilted 408, at a vertical resolution of 1.4 lm,a horizontal resolution of 0.875 lm, and at a working dis-tance of around 13 mm. Images of the occlusal and mid-crown sections of the teeth were taken with the teethpositioned orthogonally to the beam splitter, at a verticalresolution of 1.4 lm, a horizontal resolution of 0.875 lm,and at a working distance of around 13 mm. The cervicalpart of the teeth was captured with the teeth tilted 208, ata vertical resolution of 0.109 lm, a horizontal resolutionof 0.875 lm, and at a working distance of around 10 mm.Images of the occlusal and mid-crown zones of the crownwere captured using a 203 lens. Images of cervical part ofthe crown, which has the closest spaced perikymata werecaptured using a 503 lens.

Profile analysis

The three-dimensional model is rendered as a rotata-ble, shaded image by the Alicona InfiniteFocus 2.0, IFM2.1 software (see Fig. 1). Perikyma grooves are revealedas shadows. Prior to profile analysis, the plane to whichall the measured points are referenced must be adjustedto correct for bias introduced by the stereoscopic images.This is accomplished using numerical algorithmsbased upon pixel-to-pixel image intensity and depth.Subsequently, a profile path orientated perpendicular to

Fig. 1. Three-dimensional image of mid-crown perikymata on the upper lateral incisor.

499QUANTITATIVE RECORDING OF PERIKYMATA

American Journal of Physical Anthropology

the line of the perikyma grooves was defined on eachimage field. After filtering the high-frequency irregular-ities on the profile, which separates the roughness fromthe waviness and the overall geometrical form of the sur-face, the coordinates of the profile path were used to con-struct a quantitative profile diagram (Russ, 2007). Thisdiagram represents a selected part of the profile paththat is delimited according to internationally standar-dized surface roughness parameters (ISO 4288). The pro-file diagram reveals many structural irregularities onthe enamel surface but there are clearly and sharplydefined minima which, in every case, can be visuallymatched to the line of a perikyma groove (see Fig. 2).

Perikymata counts and spacing

On each incisor, perikymata counts were made on astrip running along the midline of the tooth from the oc-clusal extremity of the crown (the tip of the centralmamelon) to the cemento-enamel junction at the cervicalbase of the crown. The lower central incisor was selectedfor detailed investigation of variation between differentparts of the labial crown surface. Counts were madealong two additional strips, running from the tip of themesial and distal mamelons to the cemento-enamel junc-tion and parallel to the midline strip.Since the minima of the filtered profiles were unam-

biguously identified as perikyma grooves, the distancebetween minima represents the distance between thecorresponding grooves on the crown surface. The directdistance between perikyma grooves was automaticallycalculated by the Alicona software by drawing a straight

line between the points identified by the user as relevantminima on the profile diagram (see Fig. 2).

RESULTS

Perikymata counts

Total counts of perikyma grooves along the midline ofthe labial surface of the Abingdon teeth were 168 for thelower central incisor, 159 for the upper central incisor,and 135 or the upper lateral incisor. Perikymata countsare within the range of values recorded by previousstudies (Table 1). For the lower central incisor, lower

Fig. 2. Image field and filtered profile diagram of cervical region of a lower incisor crown.

TABLE 1. Comparison of perikyma counts in crownformation studies

Tooth typeNumberof teeth

Totalperikymagrooves

Standarddeviation Reference

Lower centralincisor

2 170–200 Stringeret al., 1990

1 197 Dean andBeynon, 1991

15 133 11 Dean et al., 20011 168 This study

Upper centralincisor

2 185 Stringeret al., 1990

15 165 21 Dean et al., 20011 177 King et al., 20021 159 This study

Upper lateralincisor

16 134 16 Dean et al., 2001

1 135 This study

500 E. BOCAEGE ET AL.


numbers of perikyma grooves were counted on the distal(148) and mesial (153) strips of the labial surface thanalong the midline.The varying prominence of perikymata was used to

match the sequences of the perikymata in the threestrips on the lower central incisor so that the spacingcould be compared directly between exactly equivalentpairs of perikyma grooves (see Fig. 3). The striking mor-phological similarity of the 100th perikyma groove onthe middle and mesial strips and the 95th perikymagroove on the distal strip allowed the three strips to bealigned with confidence. Differences in overall number ofperikyma grooves were primarily associated with differ-ences in the cervical extension of the crown. The cervicalextremity of the middle strip had 16 more perikymagrooves than the mesial strip and 15 more perikymagrooves than the distal strip. The mesial and middlestrips each had five more perikyma grooves on the occlu-sal extremity of the crown than distal strip, implyingthat the lower number of perikyma grooves on the distalstrip is partially accounted for by the reduced height ofthe mamelon on this part of the tooth.

Perikymata spacing within lower central incisor

The spacing between adjacent pairs of perikymagrooves ranges from 100 to 200 lm occlusally to lessthan 50 lm cervically along all three strips of the lowercentral incisor. The mean spacing between pairs of peri-kyma grooves in the occlusal part was 93 lm in the mid-dle strip, 96 lm in the mesial strip, and 99 lm in thedistal strip. At mid-crown height the equivalent meanspacing was 45 lm for the mid-sagittal strip, 44 and 47lm for the mesial and distal strip, respectively. In thecervical part of the crown, the mean spacing was 28 lmin the middle and mesial strips and 25 lm in the distal

strip (see Fig. 4). These differences were not significant(Anova, P 5 0.999).Spacing between equivalent pairs of perikyma grooves

can vary substantially across the labial surface. Thespacing between the 103rd and 104th perikyma groovesvaries in a particularly striking way between mesial,middle, and distal strips. The grooves are only 5 lmapart in the middle strip, which is the smallest perikymagroove spacing observed anywhere on the tooth. By con-trast, in the mesial and distal strips these grooves are46 lm and 41 lm apart, respectively, which is within therange of variation shown in the neighboring parts of thecrown surface.The rate of increase in crown surface height can be

illustrated by plotting the cumulative spacing betweenperikyma grooves against perikyma groove count (seeFig. 5). The initiation of imbricational enamel occurslater on the mesial and distal strips than on the middlestrip, but these areas of the tooth crown exhibit a higherrate of increase in crown surface height at the occlusalend. This is achieved by a wider spacing between succes-sive perikyma grooves and leads to a convergence incumulative tooth crown height attainment. Comparisonof cumulative plots for perikyma groove spacing for thethree strips demonstrates that the narrow spacingbetween the 103rd and 104th perikyma grooves on the

Fig. 3. Montage of matched perikymata on the mesial, mid-dle, and distal strips of the lower central incisor.

Fig. 4. Mean spacing for the occlusal, mid and cervical partsof the distal (black), middle (striped), and mesial strips (darkgrey) crown of the lower central incisor.

Fig. 5. Cumulative graph of perikymata spacing from theocclusal to the cervical extremity of the matched distal (darkgrey), middle (light grey), and mesial (black) strips of the lowercentral incisor.



middle strip has little or no effect on the overall trend(see Fig. 5).

Perikymata spacing differences between teeth

Comparison of cumulative plots for perikyma groovespacing along the middle strip of each incisor reveals areduced rate of increase in crown surface height overtime, indicating a more tightly packed perikymata spac-ing pattern at the cervical end relative to the occlusalpart of the crown in all three teeth (see Fig. 6). Thelower lateral incisor has 168 perikyma grooves comparedto only 135 grooves in the upper lateral incisor. Despitethis the upper lateral incisor achieves a similar crownheight measured along the surface curvature becausethe perikyma grooves are less closely spaced.

DISCUSSION

This first exploration of the potential of the Alicona3D Infinite focus imaging microscope has illustrated theadvantages of this new recording technique. The highresolution images yield a detailed reconstruction of thecrown surface with each point represented by three-dimensional coordinates. Profile analysis enhances theobjectivity of perikymata identifications as the minimaon the filtered profile diagrams correspond unambigu-ously to perikyma grooves.A further advantage of the Infinite Focus software is

the option which automatically calculates the referenceplane, allowing adjustment for the curvature of the toothcrown surface. Curvature of the crown surface can influ-ence the accuracy of distance measurements and previ-ous analyses have had to incorporate a mathematicaladjustment for this bias after measurements wererecorded (Dean and Reid, 2001; Guatelli-Steinberg et al.,2007; Smith et al., 2007a).Actual measures of perikymata spacing rather than

average perikymata spacing enable the objective identifi-cation of irregularities within a particular region of thecrown surface. By comparing the actual perikyma groovespacing to the underlying trend along the crown surface,irregularities in perikymata spacing can be detected.These irregularities could be matched across a dentitionto develop a chronological alignment (registration)between successively forming teeth in a developmental

sequence. This approach has previously been used toestimate age at death of fossil juveniles (Smith et al.,2007b). Another application is the accurate identificationof enamel hypoplasias. Disruptions in the contour of thecrown surface resulting from a greater perikymata spac-ing than expected for that region of the crown can beused to identify enamel defects if they can be crossmatched between teeth with overlapping developmentalschedules (Hillson and Bond, 1997; King et al., 2002,2005). It is important to distinguish these systemicgrowth disruptions from local variations in perikymagroove morphology.Lengthy scanning times required for the capture of

high resolution images are a limitation of the technique.Nevertheless the quantification of perikymata spacing isless time-consuming than previous 3D techniques(Hillson and Bond, 1997; King et al., 2002, 2005), as thedistances between perikyma grooves are calculated auto-matically from points identified on the profile diagrams.As with other techniques for the identification and anal-ysis of perikymata, abrasion of the enamel and the pres-ence of dirt and calculus could influence the accuracy ofthe observations and measurements.For the Medieval individual from Abingdon, periky-

mata counts are within the documented range of humanvariation. Three separate image strips taken from thedistal, middle, and mesial mamelons of the lower centralincisor could be matched by a following a single peri-kyma groove across the labial surface of the tooth. Quan-titative analysis showed that differences in perikymatacounts on these strips occurred primarily as a result ofdifferences in the cervical extension rather than differen-ces in mamelon height. The cervical extremity of themiddle strip had more perikyma grooves than the mesialand distal strips.There is an overall decrease in perikyma groove spac-

ing from the occlusal to the cervical end of the crown ofall three incisors. The upper lateral incisor achieves asimilar crown height to the other incisors within ashorter amount of time and this can be largely attrib-uted to wider spacing in the occlusal and mid-crownenamel, since all three incisors exhibit similar spacingpatterns at the cervical end. This accords with previousstudies of perikyma grooves spacing on the anteriorteeth of modern humans based on the number of peri-kyma grooves per decile of tooth height (Reid and Dean,2000). The strikingly small perikyma groove spacingbetween the 103rd and 104th perikyma grooves on themiddle strip of the lower central incisor, relative to thewider spacing between the corresponding perikymagrooves on the distal and mesial strips, is interpreted asa local variation in perikyma groove morphology.Future research will compare the number and spacing

of striae of Retzius recorded in histological sections withthe information about perikymata numbers and spacinggenerated by the Alicona 3D profile analysis to test theaccuracy of the method outlined in this study. It will alsobe useful to explore to what extent perikyma grooves onworn teeth can still be identified using this instrument.A further evaluation of the technique will involve com-parison of measurements taken on actual teeth to thosetaken on high quality replicas.

ACKNOWLEDGMENTS

The authors are grateful to Jonathan Krieger andSilvia Bello for technical advice.

Fig. 6. Cumulative graph of perikymata spacing from theocclusal to the cervical extremity of the middle strips of thelower central incisor (dark grey), upper central incisor (lightgrey), and upper lateral incisor (black).

502 E. BOCAEGE ET AL.


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Technical note: A new three-dimensional technique for high resolution quantitative recording of perikymata