Age

at death evaluaFon by tooth cementum annulaFon (TCA)

a soLware for an automated line counFng (Auto-TCA)
Andrea Czermak1, Adrian Czermak2, Gisela Grupe3, Hartmut Ernst2
1 Ins<tut fr Archologische WissenschaMen, Frhgeschichtliche Archologie und Archologie des MiPelalters, Albert-Ludwigs-Universitt Freiburg, Germany; 2 LMU Biozentrum, Ludwig Maximilians Universitt Mnchen, Germany;
3 Faculty of Computer Sciences, University of Applied Science Rosenheim, Germany;

Principle of tooth cementum annulaFon (TCA)

A valid age at death estimation is required in historical and

forensic anthropology. Tooth cementum annulation (TCA)
provides a technique for age-at-death estimation of adult
individuals. The approach uses light microscopic images
acquired from tooth root cross sections. Age is estimated by
counting the number of visible tooth cementum incremental
lines and adding the result to the assumed age of tooth
eruption. Manual line counting, however, is time consuming,
potentially subjective and the number of individual counts is
insufficient for quantitative evaluations.
Here a custom-made software (AutoTCA) is presented that
allows automated evaluation of TCA images. It involves
Fourier filtering, line-by-line scanning and the counting of
grey scale intensity peaks within a selected region of interest
(ROI). Each scanning process of a particular ROI yields up to
400 counts, thus minimizing the potential error induced by
manual line counting. This simple and time saving program
can substitute manual counting and provides reproducibly
consistent and user independent unbiased results.
Reliability of the results, however, still depends largely on the
state of preservation of the analysed material, the
preparation, the choice of the thin section and image quality,
underlining the need to standardize these factors.

Figure 2. CorrelaFon of bands, ber orientaFon,

crystal structure and season. Dark and bright
lines, visible in transmiPed light microscope are
supposable correla<ng with variable orienta<on
and dierent mineraliza<on of the collagen
bers. Bright bands seem to be developed in
winter, dark lines in the summer season
(Liebermann 1994; Stutz 2002). The changeover
of the bands happens in March/April and
September/October (Wedel 2007).
(A) The varying orienta<on of collagen bers
(Liebermann 1993, 1994; assumed orienta<on of
the sharpey bers in the course of one year aMer
Wedel 2007) and (B) the (presumably)
consequen<al orienta<on and/or size of the
crystals (Cool 2002) seem to create the
phenotype of the rings. (Figures: Czermak).

Figure 8. Region of interest

(ROI). Several points have to
be marked to span a
polygon around the region
to be evaluated. The
boundary should follow the
bright erup<on line on
one side and the dark
border to the embedding
resin on the other side.

Figure 10. ROI aLer

rotaFon. Incremental
lines are now accurately
orientated in verFcal
direcFon. The lines are
oMen disrupted by
refrac<on or dirac<on
ar<facts (Fig. 6), by
par<al decomposi<on of
the tooth or by linear
kerf marks caused by
saw blade (arrows).
These kerf marks are
similar to incremental
lines and in case of a
parallel course they can
inuence the coun<ng
result

Figure 11. Power spectrum of

the rotated ROI. (A) Ver<cally
orientated incremental lines
are grouped near the central
horizontal line of the power
spectrum. In contrast, non
linear structures or linear
structures orientated in
dierent angles to the
incremental lines in the
original image (e.g. saw blade
marks) are represented by
pixels that are more distant
from the horizontal line.
(B) To completely eliminate
interfering ar<facts a point
symmetric masked was
applied an angular lter (20)
was used. (C) Masked power
spectrum of rotated ROI.

Frhadult

width of ROI

References

[1] Czermak A, Czermak AM, Ernst H, Grupe G (2006): A New Method for the Automated Age-at-Death Evalua<on by Tooth-Cementum
Annula<on (TCA). Anthopologischer Anzeiger 64 (1): 25-40.
[2] Czermak A (2012): Social Stra<ca<on in the Early Middle Ages - Evidence by Demography, Physical Stress and Nutri<on. (Soziale
Stra<zierung im frhen MiPelalter Aussage und Nachweismglichkeiten anhand von biologischen Indikatoren). Disserta<on, Mnchen.
[3] Cool SM, Forwood MR, Campbell P, Bennet MB (2002): Comparison between bone and cementum composi<ons and the possible basis for
their layered appearances. Bone 30 (2): 386-392.

Figure 7. DiracFon arFfacts. Ar<fact

lines (arrows) are oMen visible on the
interface of prepara<on and
embedding material (magnica<on
20x). They could be mistaken for
incremental lines, mainly on images
with lower magnica<on than 40x
diameters (Czermak 2006, 2012).

25

TCA:
aged 27

20
15
10
5
0
2

Applied data

1 tooth

8000 each tooth

mean value of all images

5 images

1600 each image

mean value of all ROIs

4 ROI each image

400 each ROI

mean value of the mode

of the single counts of a ROI

middle adult (aged 25-31)

(1) Using the Auto-TCA-

soMware is much more
<me saving than manual
coun<ng.
(2) The soMware provides
user independent,
consistent and r

20

TCA:
aged 41

15
10
5

8 10 12 14 16 18 20 22 24 26 28 30
slice3/count2
Schnitt3/Zhlung2
Schnitt4/Zhlung2
slice4/count2
Schnitt5/Zhlung2
slice5/count2

25

TCA (1):
aged 37

15

5
0
10

15

20

25

30

35

40

45

50

Incremental lines
slice3/count1
Schnitt3/Zhlung1
slice4/count1
Schnitt4/Zhlung1
slice7/count1
Schnitt7/Zhlung1
slice8/count1
Schnitt8/Zhlung1

slice3/count2
Schnitt3/Zhlung2
slice4/count2
Schnitt4/Zhlung2
slice7/count2
Schnitt7/Zhlung2
Schnitt8/Zhlung2
slice8/count2

Schnitt3/Zhlung1
slice3/count1
Schnitt5/Zhlung1
slice5/count1
Schnitt6/Zhlung1
slice6/count1

TCA (2):
aged 40

10

15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

slice3/count3
Schnitt3/Zhlung3
slice4/count3
Schnitt4/Zhlung3
Schnitt5/Zhlung3
slice5/count3

Sptadult
late adult
(aged 32-38)

Mode of each ROI

Table 1. Number of single-counts each counFng level and applied data for
further examinaFon . Each coun<ng process generates depending on ROI size
300-500 single counts. The Auto-TCA soMware shows the results in of one
coun<ng-process in a window, sorted by the most counted line-number, in
downward order. The most counted value, the mode, of a ROI was taken for
further examina<on.

slice3/count1
Schnitt3/Zhlung1
Schnitt4/Zhlung1
slice4/count1
Schnitt5/Zhlung1
slice5/count1

Hufigkeit [%]
Occurrence

Single counts

Incremental lines

EvaluaFon of the counFng results

TCA-method

Mitteladult

Conclusions

25

20

400 Single-counts each ROI

Figure 4. (A) LocalizaFon of

slices. Tooth-scheme (lateral
view). 8-10 sec<on were
taken (B) Thin secFons on a
slide.

early adult (aged 20-24)

VerFcally orientaFon of the naturally grown incremental lines

Figure 12. (A) ROI aLer image
analysis. AMer Fourier back-
transforma<on of the masked
spectrum all interfering structures
are eliminated from the image.
(B) Example for a line scan. AMer
image processing the applied
algorithm creates a line-by-line scan
of pixel-by-pixel gray scale values of
the processes ROI. Local maxima
and local minima and local minima.
Local maxima correspond to the
number of incremental lines in this
row. They are detected and counted
by a programmed peak-nder
algorithm.

Figure 6. Slice-secFon in a bright

eld microscope. Cementum-layer
with incremental lines (right side),
den<ne-layer (leM ) (magnica<on of
20x diameters). Picture taken with
the incremental lines in ver<cal
direc<on for a bePer program-run.

Figure 5. SecFon cut and microscopic image. Sec<on cuts taken

in an 90-angle to the root orienta<on show coincide ring
structures. In the bright eld microscope image there is a higher
contrast between bright and dark lines (medium box). Sec<on
cuts along the axis of the tooth root are not ver<cal to the
growing line of the cementum and they show rings which are
shiMed and not completely overlapping (lower box) (Maat
2006). Sec<ons in the upper and medium part of the root show
the most dis<nctly and visibly lines. (Figure modied aMer Maat
2006)

EliminaFon of interfering structures

grayscale level

Figure 3. Tooth embedded in

a epoxide resin bloc (Biodur
E12) and xed in a
microtome saw (Leica 1600).
The tooth crown of the
sample was cut o.

QuanFtaFve evaluaFon using Auto-TCA-soLware

Digital image processing

VerFcally orientaFon of the naturally grown incremental lines
Figure 9. ScaWerplot aLer
11x11 Gauss low-pass lter
and DFFT. The ROI was
segmented into equal
rectangles (128 pixels x
length of ROI). Each segment
was rotated to a horizontal
posi<on, using the outer
dark line as a reference.
Applica<on of Gauss low-
pass lter and Fourier
transforma<on (DFFT:
discrete fast Fourier
transforma<on). (A) The
scaPer plot shows a
preferred direc<on, which is
extracted by dng a straight
line. (B) This line determines
the ideal rota<on angle

(1) CreaFon of an unbiased method

to subsFtute manual line
counFng
(2) OpFmize sample preparaFon
and imaging
(3) QuanFtaFve evaluaFon using
Auto-TCA-soLware

Hufigkeit [%]
Occurrence

DetecFon of the ROI

Figure 1. Tooth scheme (longitudinal cut). The

root area is surrounded by the tooth-
cementum. The cementum is added in layers on
the bone-side of the tooth, comparable with
tree-rings. The cementum is nerved by
collagen bers (sharpey bers), which are
xing the tooth in the alveolar bone.
(Figure modied aMer Schroeder 2001).

Hufigkeit [%]
Occurrence

Program run

Sample preparaFon, microscopy and imaging

Aims of this project

Hufigkeit [%]
Occurrence

Abstract

slice3/count3
Schnitt3/Zhlung3
slice4/count3
Schnitt4/Zhlung3
slice7/count3
Schnitt7/Zhlung3
slice8/count3
Schnitt8/Zhlung3

Incremental lines
slice3/count2
Schnitt3/Zhlung2
slice5/count2
Schnitt5/Zhlung2
Schnitt6/Zhlung2
slice6/count2

slice3/count3
Schnitt3/Zhlung3
slice5/count3
Schnitt5/Zhlung3
Schnitt6/Zhlung3
slice6/count3

Mittelmatur

middle mature (aged 46-52)

25
20

TCA (1):
aged 45

15

TCA (2):
aged 52

10
5
0
15

20

25

30

35

40

45

50

55

60

Incremental lines
slice2/count1
Schnitt2/Zhlung1
slice3/count1
Schnitt3/Zhlung1
Schnitt8/Zhlung1
slice8/count1

slice2/count2
Schnitt2/Zhlung2
slice3/count2
Schnitt3/Zhlung2
Schnitt8/Zhlung2
slice8/count2

slice2/count3
Schnitt2/Zhlung3
slice3/count3
Schnitt3/Zhlung3
Schnitt8/Zhlung3
slice8/count3

Figure 13. Example for Auto-TCA counFng results of the incremental lines of individuals in dierent age groups
(morphologically determined). The soMware was tested on samples of individuals with unknown age-at-death from
historical excava<ons. The coun<ng results of one slice show a Gaussian distribu<on curve and several coun<ng on
dierent spots at one slice largely coincide. The younger an individual, the slender the Gaussian curve and the denite
the result (A). Proceeding (morphologically determined) age leads to a at curve and more scaPering results (B). In
higher age groups dierences between slices near the tooth crown and slices from the middle of the root become
clearly visible (C). The older one individual, the longer can the distance from the curves of dierent slices become (D)
(Czermak 2006, 2012).

[4] Liebermann D E (1993): Life History Variables Preserved in Dental Cementum Microstructure. Science 261: 1162-1164.
[5] Liebermann DE (1994): The Biological Basis for Seasonal Increments in Dental Cementum and their Applica<on to Archaeological
Research. Journal of Archaeological Science 21: 525-539.
[6] Maat G, Gerretsen R, et al. (2006). Improving the visibility of tooth cementum annula<ons by adjustment of the cudng angle of
microscopic sec<ons. Forensic Science Interna<onal 159(S): 95-S99.

[7] Schroeder H (2001). Orale Strukturbiologie. StuPgart, Thieme.

[8] Stutz A (2002). Polarizing Microscopy Iden<ca<on of Chemical Diagenesis in Archaeological Cementum. Journal of Archaeological Science
29: 1327-1347.
[9] Wedel VL (2007). Determina<on of Season at Death Using Dental Cementum Increment Analysis. Journal of Forensic Science 52 (6):
1334-1337.

Auto-TCA-soLware
(1) Using the Auto-TCA-
soMware is much more
<me saving than manual
coun<ng.
(2) The soMware provides
user independent,
consistent and
reproducible results.
(3) Sta<s<cal error is
minimized by larger
number of single counts
compared to manual
coun<ng.

Image quality is
crucial for valid line
counFng.
The soLware
provides reliable
counFng results, but
does not validate the
TCA-method.

Acknowledgement
This research work was supported by the
Hanns-Seidl-S<Mung, Munich, Germany
(PhD grant to Andrea Czermak)

Corresponding address
czermak_andrea@web.de