5.3. Digital Image Processing (DIP)

An innovative tool used in this research is Digital Image Processing (DIP) (for our previous applications in human paleobiology and paleoanthropology, see Macchiarelli et al., 1990, 1995, 1996a, b, 1997a, b; Bondioli et al., 1993; Macchiarelli and Bondioli, 1994, 1995; Geusa et al., 1995, 1996a, b, 1997a, b; Galichon et al., 1996; Rossi et al., 1996, 1997a, b, c; Salomone et al., 1997; Savorè et al., 1997; see also Liebermann et al., 1990; Pesce Delfino and Lettini, 1990; Potente et al., 1991, 1992).





This approach permits a significant enhancement of the quality and quantity of (histo)morphological, (histo)morphometric, and densitometric information directly or indirectly obtainable from osteodental remains. By means of DIP procedures, it is possible to produce a detailed record of (micro)structures that would otherwise be difficult to obtain and, above all, would produce highly subjective results (Rosenfeld and Avinash, 1982; Watkins et al., 1993).



Each section was first observed at 100x in order to evaluate the presence and the extension of Wilson bands and their position on both buccal and lingual aspects of the tooth. Prism bending, as well as the neonatal line, were observed at a higher magnification (400x).

In order to enhance contrasts, each magnified thin section picture was transferred into numerical format by means of a digital image analysis system.

The slices were examined with an optical microscope (optical transmitted light microscope Laborlux S, Leica AG) under polarised light and using the Leica l filter. The microscope is equipped with a high resolution CCD video camera (TK 1281 Colour Video Camera, JVC Ltd.) connected to an analogue-to-digital (A/D) converter board (Image Grabber IG/PC, Neotech Ltd.) plugged into a Pentium PC. The PC is linked with a mixed network of computer workstations (PC Pentium, Apple Power Macintosh, Indigo Silicon Graphics). This computer network has been designed in order to maximise performance in terms of digital image processing power and versatility. The network design mixes different software/hardware workstations that are able to run the majority of modern digital image analysis software. This tool set, combined with a large on-line storage facility (greater than 20 Gb), guarantees productivity and efficient use of time during all phases of data collection.

The portion of the thin section covered by the microscope field at 40x magnification (4.8 mm²) was shot by means of the camera connected to the microscope, and the resulting analogue image was converted into numerical format through the image grabber board.

The software (Image Grabber PC 1.01, Neotech Ltd.) that drives the board returns the final digital image as an average of 16 - or more, according to user choice - subsequent shots of the same field. This iterative procedure was employed in order to filter and reduce the noise coming from the CCD device and from signal deterioration. The spatial resolution was 6.62 mm for each image pixel, and each final image was stored as an 8 bits, greyscale, GIF file.

To estimate neonatal line thickness (after Eli et al., 1989), three images were taken at 400x in three different positions along the buccal aspect: 1) close to the dentino-enamel junction; 2) in the middle of the dental crown; and 3) close to the apex. At 400x magnification the spatial calibration coefficient was 0.66 mm for each image pixel.

The spatial calibration coefficients were derived from a series of images - shot at various combinations of magnification and image size - of a micrometry slide (Leica AG) bearing an engraved metric scale

(40x magnification factor)  

(400x magnification factor)

Each image covered only a single portion of the tooth crown section, thus the final image of the whole crown is the result of the digital photomosaic of 7 to 15 partial images, depending on individual tooth size. In order to standardise the images and to keep the instrumental parameters constant, the program setting of tone and brightness corrections and microscope light were left unmodified throughout the same thin section. The photomosaics were created using the computer program Adobe Photoshop 3.2 (Adobe System Inc.).

In order to estimate the age of formation of the microscopic defects, different techniques of digital image analysis were used.

The software used for processing is a compound of three different DIP packages: NIH Image v. 1.61 (National Institute of Health, USA); Optilab Pro 2.5 (Graftek srl); and UTHSCA Image Tool 1.28 (Univ. of Texas, San Antonio Health Science Center, USA).

Contrast enhancement convolution filters (3x3 and 5x5 kernels) were run to get sharper details, while a change in the look-up table function was performed to increase site-specific contrasts of intensity profiles.

Further image enhancements of the enamel banding was obtained by the application of directional embossing filters (usually NE and NW 3x3 and 5x5 kernels) capable of enhancing the transition between the dark and clear regions of a band.

Special care was taken to avoid the introduction of artefacts, especially during the convolution and embossing procedures. After each electronic manipulation, the digital results were carefully compared to the original images, and each new highlighted feature was considered reliable only when confirmed by using different analytical tools. All measurements were taken on images that were elaborated as little as possible.

Measurements on the photomosaics were performed using the National Institute of Health NIH Image v.1.61 program running on an Apple Power Macintosh. The path of the measurements was traced by hand with a mouse on a PC high resolution 21" screen using the straight polyline measurement tool provided by the program. In order to closely follow curved paths, the number of the polyline vertex was proportionally increased where the tightness of the curve increased.



As shown by repeated tests for intra- and interobserver concordance, the systematic use of DIP techniques as described above ensure a high degree of repeatability and replicability of the estimates derived from the histomorphometric record.

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