Bone is a material that is essential for the study of humans in long-ago times. More precisely, the collagen, or fibrous tissue, which provides the groundwork for these bones, is required for radiocarbon dating of the fossil, as well as studies on ancient diet and the protein composition of older humans.
Many methods have been used to assess the collagen content of a bone specimen, which determines its usefulness for this type of study. However, they tend to destroy the sample used. Now a new study shows the value of near-infrared (NIR) spectroscopy as a portable, non-destructive and rapid tool for this application.
Archaeological excavations research on human burial - Image Credit: Masarik / Shutterstock
Many crucial events of the past have been dated by using radiocarbon and other dating methods on human and other bones. Determination of the molecular fingerprint, or the types and relative concentrations of different isotopes in bone collagen, have helped to determine the diet of those times, including the emergence of various food crops and the place of animal food consumption. The protein profile determined from these bones also shows the difference between ancient and modern humans. Thus scientists have been able to find out much of importance about the past from bone collagen.
Like all ‘soft tissues’, collagen deteriorates over time. Moreover, the degree to which the collagen in ancient bones is preserved varies a lot from site to site, and even at different locations and soil depths within the same site. Thus archaeologists may spend a lot of their time and energy, not to mention precious funds, on preparing a sample for analysis, only to find out that it doesn’t contain sufficient collagen. In defence, scientists have devised ways to first test the bone for the degree of preservation, by examining its percentage of nitrogen, microporosity or by its FTIR spectroscopic findings. However, these cannot be done locally in many cases, and may result in the destruction of the bone sample.
In contrast, NIR spectroscopy yields results within seconds, can penetrate the bone to a greater depth of millimeters compared to microns in other methods, can be scaled down to achieve portability, and preserves bone in intact condition. The depth of effective penetration is especially important in that a lot of bone surfaces are typically encrusted after deposition.
To test the efficacy of this bone screening technique, the scientists used it on 50 specimens of ground bone, still within their protective glass vials. They were sorted by the percentage of collagen and assigned to two sets, calibration and validation respectively, by their odd and even numbering. Using a model based on the 25 samples in the calibration set, the percentage of collagen in the validation set samples was estimated.
The technique was repeated on 49 whole bone samples. These were then coupled with ground bone scans to produce two combined sets of both ground and whole bones. This was used to produce a model that would hopefully cover the whole range of possible bone samples from whole to completely degraded. All the samples were supposed to be up to 45,000 years old. They calibrated the model using a 49 sample set, and tested it on 48 samples in the validation set.
The data was used to classify the specimens as Sample (above 3% or 1% collagen, respectively) and Do Not Sample groups (below 3% or 1% collagen, respectively). Collagen was then extracted from the samples using conventional methods.
The model based on a calibration set of 25 samples performed well on a 21 sample validation set, with 100% accuracy for above and below 3% collagen. In the second round, it was able to correctly classify 30/32 specimens with over 3% collagen, and 88% of 16 specimens that had less than 3% collagen.
The most important finding that 100% of specimens which were predicted to have over 3% collagen had at least 1% collagen, which is enough for radiocarbon and dietary analyses of ancient bones. This may not be good enough to determine whether or not a sample is really well-preserved or not. But it should be useful in answering the question as to whether any given specimen should be sampled at all – is there a fair chance of success to compensate for the risk?
Advantages of NIR spectroscopy
The NIR spectroscopic tool can be used to look at the preservation status of collagen in archaeological specimens all over the world to a putative age of up to 45 000 years. Other preliminary screening tools, such as percent nitrogen, FTIR and Raman spectroscopy are also able to achieve over 70% success in separating bones into those which have more or less than 1% collagen. However, NIR spectroscopy will be most useful where specimen preservation is required, where a large number of specimens are to be processed, and where specimen surfaces are heavily contaminated, requiring deeper penetration depths. The portability is another important facet of its usefulness – the entire apparatus fits into a briefcase-sized bag with a handheld probe.
At a single dig site there may be thousands of bone fragments possibly useful for analysis. Even though as a whole the bones contain less than 1% of well-preserved collagen, some specimens with much better collagen ratios will be found, and these relatively rare specimens can be identified by scanning hundreds or thousands of samples over a couple of days.
The technique also allows certain regions of the specimen to be highlighted for further analysis. This is vital in sites where all the finds are poorly preserved. This could also help compare variations in specimen quality between sites with respect to their preservation and the presence of post-depositional artefacts.
Finally, NIR spectroscopy can help reveal which contaminants are present in the specimen before it is subjected to further analysis, thus preventing much tedious work on identifying and nullifying the effect of artefactual data. This technique may also be eventually adapted to screen for DNA in the bones, an even more precious find.
The technique has gone through a pilot run already, at a Czech site with among the oldest remnants of art known to exist. The researchers had only 6 vials of samples taken from human burial sites, but when these were analyzed by NIR spectroscopy, they could define how much they needed to subject to destructive analytical methods, rather than run the risk of losing them all. The results were published earlier this month in the Journal of Archaeological Science: Reports.
Saving Old Bones: a non-destructive method for bone collagen prescreening. Matt Sponheimer, Christina M. Ryder, Helen Fewlass, Erin K. Smith, William J. Pestle & Sahra Talamo. Scientific Reports, volume 9, Article number: 13928 (2019). https://doi.org/10.1038/s41598-019-50443-2. https://www.nature.com/articles/s41598-019-50443-2#Abs1