Abstract
This study conducted a comprehensive analysis of the pale pink human bones unearthed in Baoji, Shaanxi, China. A range of scientific techniques were employed in the analysis, including optical microscopy, scanning electron microscopy coupled with an energy-dispersive spectroscopy, Fourier transform infrared spectroscopy, micro-Raman spectroscopy, stimulated Raman scattering microscopy, and X-ray photoelectron spectroscopy. The objective of this study is to explore the reasons for the observed colouration of the bones. The pink bones are poorly preserved and contain high levels of Fe³⁺, most likely causing their colour and poor condition. This is linked to the region’s semi-leached soil. Furthermore, this study SRS to bioarchaeology, providing a new technical approach for studying the preservation status of human bones unearthed in archaeological excavations. This development holds significant positive implications for the scientific analysis of human bones in archaeology.
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Introduction
Research into the degradation and contamination mechanisms of human bones unearthed during archaeological excavations is essential for assessing their state of preservation, evaluating their historical and scientific significance, and carrying out conservation work1,2,3. Bones are complex biological composites, consisting of 70% inorganic minerals (such as hydroxyapatite) and 30% organic matter (e.g., collagen)4. Over long periods of burial, diagenesis replaces collagen with inorganic salts while external pollutants infiltrate porous structures, altering the bones’ chemical and biological properties5,6,7,8. This process damages the bone’s microstructure and reflects its deteriorating preservation status via macroscopic features such as colour changes and increased porosity6,7,8,9. This can affect the analysis of results from studies on human bone isotopes and ancient DNA, since these studies require the identification of bone degradation processes and contamination states.
The conventional view primarily attributes abnormal colours in archaeological bones to the use of minerals such as hematite or cinnabar in ancient funerary rituals10,11. However, metal artefacts in the burial environment can also affect bone colour through ion replacement and mineral co-precipitation12,13,14. Furthermore, metal ions can catalyse collagen oxidation and degradation, thereby damaging the bone’s structure and reducing its mechanical properties15,16,17. Nevertheless, the chemical mechanisms of bone colour changes and their relationship to preservation remain unclear15,16,17,18. Current techniques for analysing bone colour changes and deterioration mostly rely on macroscopic observations and elemental quantification 19,20,21,22,23, which cannot reveal the distribution of phases at the microscale or the dynamics of chemical bonds15,16,17,18. For example, traditional Raman spectroscopy can detect the hydroxyapatite peak (~960 cm−1)24, but it struggles with strong fluorescence interference in biological samples such as bones. This interference masks the collagen C-H bond signals (2800–3050 cm−1)22,25,26,27 and complicates dynamic tracking of organic degradation28,29,30. Recent studies have shown that stimulated Raman scattering (SRS) technology offers a new approach due to its high sensitivity and label-free imaging capabilities22. Freudiger30 used SRS to achieve high-resolution, 3D imaging of lipids and proteins in biological tissues. This paves the way for applying SRS to study the organic degradation of archaeologically excavated human bones31.
This study examines the pale pink human bones excavated in Baoji, Shaanxi, in order to gain a better understanding of the impact of environmental factors on bone preservation and colour changes. Advanced techniques such as Super-Depth Microscopy, SEM-EDS, FT-IR, XPS and SRS were employed to analyse the bones’ condition and colouring mechanisms. SRS is a non-invasive and highly specific method that allows for the detailed analysis of collagen distribution and degradation in bones. This offers an innovative approach to the study of protein-based artefacts. This could lead to technological breakthroughs in the under-researched field of bone degradation. Understanding the relationship between bone colour changes and degradation/pollution mechanisms is crucial for evaluating the integrity of bone information and for developing targeted conservation strategies.
Methods
Samples
The human bone samples used in the study were excavated from Baoji, Shaanxi Province. This region is located in the Guanzhong area of Shaanxi, which was one of the major political centres in ancient China32,33. Two types of samples were analysed: normal-coloured bone samples and pink-coloured bone samples. Both types were excavated from two archaeological sites (The archaeological excavation data from Chejiasi Cemetrey and the Fengxiang Highway Emergency Maintenance Centre are currently being organised. The relevant data from the excavations will be released to the public after the preliminary report is published): Chejiasi Cemetrey and the Fengxiang Highway Emergency Maintenance Centre site. The seven tombs from which the pink human bones were unearthed are all of low status, and no iron artifacts were found. Among the pink-coloured bone samples, five were recovered from Chejiasi Cemetrey, and two were from the Fengxiang Highway Emergency Maintenance Centre site. Normal-coloured bone samples are used as a control to evaluate colour differences in colourimetric analysis, elemental differences relative to pink samples in energy-dispersive X-ray spectroscopy (EDS), and spectral data comparisons between normal-coloured bone (M58 control group) and pink samples in FTIR and Raman analyses. The Fig. 1 colour comparison of complete femurs from normal and pink bone samples shows the colour comparison of complete femurs from normal coloured and pink samples collected from the two sites.
colour comparison of complete femurs from normal and pink bone samples.
Table 1 Samples information summarises the detailed information of the samples used in the study. To objectively quantify the colour characteristics of the pink bone samples, seven pink bone samples and two normal bone samples were ground to powder and spread evenly on glass slides. The colourimetric data of these samples were obtained using a colourimetre. Figure 2 visually shows the apparent colourimetric differences between the normal and pink bone samples after spreading. The specific colourimetric data are given in Table 2. The colourimetric data reported in Table 2 also in Fig. 2 for an immediate visualisation of the difference in colour.
Apparent colour difference between pink human bone (left) and ordinary human bone (right).
The results of the colourimetric analysis show significant colour differences between the pink and normal human bones. The a values (red-green axis) of the pink bone samples are all greater than 3, and the b values (yellow-blue axis) are all less than 11, whereas the a values of the normal bone samples are less than or equal to 0.40, and the b values are greater than 11. These data indicate that the colour of the pink bone samples is significantly redder than that of the normal bones, which are relatively more yellow. The colourimetric results objectively demonstrate the clear colour differences between the study samples and normal human bones (Fig. 3).
L*a*b* Visualisation results.
Colourimetric analysis
The experiment was carried out at the School of Cultural Heritage, Northwest University, Xi’an. The L*a*b* colour values of the sample were measured using an X-Rite VS450 colourimetre. Samples were ground into a powder and placed on a standard white board for measurement. The test aperture is 13 mm, the light source is the D65 standard illuminant, and the observer field of view is 10°.
OM analysis
The experiment was carried out at the School of Cultural Heritage, Northwest University, Xi’an. Super-depth-of-field microscopy was carried out using the Smartzoom 5 intelligent super-depth-of-field 3D digital microscope manufactured by Carl Zeiss, a German company. The surface erosion marks of the samples were observed at 200x magnifications.
SEM-EDS
The experiment was carried out at the School of Cultural Heritage, Northwest University, Xi’an. The VEGA3XM tungsten filament scanning electron microscope from TESCAN, Czech Republic, combined with the X-ACT X-ray energy dispersive spectrometre from Oxford Instruments, UK, was used to observe the microstructure of the samples and to analyse their elemental composition. The surface of the sample was sprayed with gold and this element was removed from the element data. The samples are mounted using conductive adhesive, with an operating voltage of 20 kV. Statistical errors were estimated based on X-ray counting statistics, with typical values of 0.5–2% for major elements and 2–5% for minor elements, consistent with EDS performance at 20 kV. The quantitative results for all elements are based on K-shell characteristic X-ray emissions.
Fourier transform infrared spectroscopy
The experiment was carried out at the School of Cultural Heritage, Northwest University, Xi’an. A Bruker TENSOR 27 Fourier Transform Infrared spectrometre was used. Potassium bromide (KBr) pellets were employed as background standards. To prepare the samples, small amounts of pink bone residue were mixed with dry KBr, and the mixture was ground into a fine powder and pressed into transparent pellets for FTIR analysis. The scan was conducted over a wavenumber range of 4000 cm−1 to 400 cm−1, with a resolution of 4 cm−1 and 128 scans. The Omnic 9 software was used to process and analyse the spectral data, with a particular focus on changes in the organic and inorganic components of the samples.
Micro-Raman spectroscopy
The experiment was carried out at the School of Cultural Heritage, Northwest University, Xi’an. Raman spectroscopy analysis was performed using an InVia Qontor micro-confocal Raman spectrometre manufactured by Renishaw, a UK company. The instrument is equipped with lasers at 532 nm, 633 nm, and 785 nm, with a 20 μm slit. The spectral detection range is from 50 to 3500 cm−1, with 10x magnification in the eyepiece and 50x magnification in the objective. The study used a 633 nm laser for the test, with exposure time 5 s, 3 accumulations, laser power 50%.
Stimulated Raman scattering
The experiment was carried out in Xi’an. Stimulated Raman scattering microscopy was performed using a Vibroni X Superview microscope equipped with a Spectra-Physics laser. The laser at 801 nm has a power of 30 W, and the laser at 1045 nm has a power of 50 W. This instrument was used for in situ microstructural phase observation of the surface of pink bone samples, with a spectral capture range of 2800–3050 cm−1.
X-ray photoelectron spectroscopy
The experiment was carried out at the college of Chemistry & Materials Science, Northwest University, Xi’an. A model PHI 5000 VersaProbe III X-ray photoelectron spectrometre (XPS) manufactured by ULVAC-PHI is used with an Al Kα excitation source. The X-ray source power is 150 W, pumped to 6.65 × 10−7 Pa under vacuum environment, and the sample binding energy (EB) is charged with contaminated carbon C1s(284.8 eV). This instrument was used to detect the valence state behaviour of Fe elements in the pink cortical bone of the samples, thereby investigating the cause of the pale pink colouration.
Ethics approval and consent to participate
Ethic approval and consent to participate This article does not contain any studies with living human participants performed by any of the authors. All experimental samples were obtained from archaeological excavations. Ethics Committee approval was obtained from the Institutional Ethics Committee of School of Culture Heritage, Northwest University to the commencement of the study. Moreover, we followed guidance for the ethical treatment of human remains following “Ethics of DNA research on human remains: five globally applicable guidelines” (https://www.nature.com/articles/s41586-021-04008-x).
Results
Micromorphology
The surface of the samples was examined by super-depth-of-field microscopy, and the results are shown in Fig. 4.
OM analysis results.
OM analysis shows that the pink human bone samples have varying degrees of pink colouration. Sample 1-1 from M51 at the Chejiasi cemetery shows a pale pink colour with a yellowish tinge, and its surface is relatively uniform but rough, with clear signs of erosion. Similarly, sample 1-2 from M127 shows a pale pink colour with a very rough surface and significant erosion. Sample 1-3 from M157 shows a pale pink colour and a rough surface with obvious signs of erosion. Sample 1-4 from M187 shows a pale pink colour with a yellowish tinge and erosion marks are clearly visible on the relatively rough surface. Sample 1-5 from M204 shows a pale pink colour and a rough surface with quite visible erosion. From the Fengxiang site, Sample 2-1 from M79 shows a pale pink colour, although lighter in shade, with a rough surface and quite visible erosion marks. Sample 2-2 from M27 shows a deep pink colour with extremely dark red patches in the field of view and very obvious erosion marks. Overall, all samples show signs of mineralisation, with sample M27 showing more pronounced surface erosion and a deeper pink colour. The consistency of the pink colouration and surface erosion across the samples suggests a possible relationship between the colour changes and the observed degree of mineralisation and erosion.
Quantitative analysis results
To further determine the elemental composition of the samples, energy dispersive X-ray spectroscopy (EDS) analysis was performed on the surface of the samples using a scanning electron microscope. The quantitative elemental analysis results obtained from EDS are presented in Table 3. To investigate whether pink human bones differ from normal human bones in terms of elemental composition, energy dispersive spectroscopy analysis was also performed on 20 normal-coloured skeletal samples excavated from the Chejiasi Cemetrey and the Fengxiang Emergency Conservation Centre, with 10 samples from each site. The results of the analysis are also detailed in Table 3. It is noteworthy that these skeletal fragments showed a significant difference in hardness compared to the pink human bones during the compositional analysis experiments; these samples were much harder, while the pink bone samples were more fragile.
The analytical data show that the sample contains Ca in the range of 38.67% to 40.83%, O in the range of 32.31% to 40.31% and P in the range of 15.48% to 17.81%. The Ca/P ratio ranges from 2.05 to 2.51, indicating hydroxyapatite contamination34. All samples contain Fe with concentrations ranging from 0.17% to 0.52% and its distribution is positively correlated with Si. The Fe content in the deep pink sample 2-2 is particularly significant. Elemental analysis suggests that Fe may be the primary colouring element responsible for the pink colouration of the bones. In the study of red human bones from the Campu Stefanu site, which is located in the village of Sollacaro in southern Corsica, France, elemental analysis revealed that iron is the key factor causing the red colouring, with concentrations ranging from 2% to 6%. This phenomenon is attributed to the use of ochre in burial practices11,35. In contrast, the iron concentration in the bone samples of this study is significantly different, suggesting that the pink colour may not be due to red pigments used in burial practices. In the detection area of sample 1-2 with higher Fe content, tunnel-like erosion traces were observed under the scanning electron microscope at 800x magnification. Through SEM measurements, the tunnel-like erosion in the pink human bone sample shows pores with diametres ranging from 20 to 40 μm. Specifically, the pore diametre at the location shown in the figure for sample “1-2” is 36.8 μm (Fig. 5).
SEM observation results of samples 1-2 at 800x magnification.
At 800x magnification, similar observations were also made on the surface of Ordinary bone samples, some of which are shown in Fig. 6.
SEM observation results of ordinary human bone at 800x magnification.
Normal human bone and pink human bone have significant differences in their microstructure. In particular, the surface of ordinarily coloured human bones samples from the same archaeological site appears uniform and dense, with fewer and smaller pores. Elemental analysis of normal bone samples shows that the calcium content (Chejiasi Cemetrey: 39.03–67.58%, Fengxiang: 37.25–43.11%), oxygen content (Chejiasi Cemetrey: 37.25–43.11%, Fengxiang: 29. 94–41.32%) and phosphorus (Chejiasi Cemetrey: 2.12–19.34%, Fengxiang: 16.76–19.82%) are generally within normal ranges, with a few exceptions, indicating a low degree of mineralisation36,37,38. Although iron is present in all samples, it is extremely low and insufficient to cause colour changes11,35,39,40. Silicon is also low in the white samples. The chemical composition of white bone samples is similar to that of normal bone, and variations in trace element content may be related to an individual’s lifetime intake. However, these changes are not significant enough to cause colour changes.
Correlation statistics of elements and soil conditions
The elemental correlation statistics of the pink bone samples are shown in Table 4.
Considering the soil conditions in which the buried bones were found, it is likely that some of the Mg and Fe elements in the samples were introduced from the burial soil. The soil samples from both archaeological sites are classified as semi-leached41. In a semi-leached soil environment, the abundant calcium and magnesium in the soil, together with the low precipitation of iron oxide and manganese, provide favourable conditions for the co-migration of Fe, Mg and Al elements. The semi-leached nature of the soil leads to the leaching and accumulation of free calcium carbonate in the profile, accompanied by the formation of humus layers which provide a medium for the migration of Fe, Mg and Al. Correlation analysis shows that the correlation coefficients between Fe and Mg and Al are both 0.96, indicating a significant positive correlation between the three, suggesting that they are likely to have a common source and similar migration pathways39,42,43,44. In addition, the large amount of humus released during body decomposition may further enhance the migration capacity of these elements45. This co-migration of elements may affect bone preservation and colour, especially as the correlation coefficients between Fe and Ca and P are both -0.36, indicating a similar negative correlation. This suggests that Fe competes with key elements in the hydroxyapatite (HAP) structure during migration, leading to destruction of the HAP structure44,46. In addition, the chemical reactivity of Fe may promote complexation with phosphate, affecting the deposition and crystallisation of calcium phosphate46, thereby influencing the degree of mineralisation and mechanical strength of bone.
Therefore, the characteristics of the semi-leached soil in which the human bone samples were buried may not only affect the migration and deposition of elements, but may also significantly affect the structure and stability of the bones through the action of Fe. In addition, the redox activity of Fe may accelerate the decomposition of organic matter in the bones, affecting their preservation and making them more fragile. The study will combine phase analysis and stimulated Raman scattering results to further investigate how these elements affect the preservation and structural stability of the bones.
Fourier transform infrared spectroscopy
To investigate the composition of the pink bone samples, the study ground a small amount of fallen pink bone residue into powder and pressed it into pellets to obtain more accurate analysis results from infrared spectroscopy (Fig. 7) and pink-coloured human bone (two samples, 1-1 and 2-1), excavated from the same site.
FT-IR results of samples.
The pink bone samples (1-1, 1-2, 2-1) and the white bone sample (M58) both show typical characteristics of organic and inorganic components in bone tissue47. The pink bone samples show an O-H stretching vibration peak at 3500 cm−1; the amide I band at 1500 cm−1 indicates the presence of collagen; and absorption peaks at 1436.18 cm−1, 1029.87 cm−1, 604.54 cm−1 and 564.15 cm−1 are also associated with carbonate vibrations. These signals are common in the infrared results of ancient bone samples, suggesting the possible presence of secondary mineral phases22. The spectral characteristics of the white bone sample are similar to those of the pink bone, also showing O-H stretching vibrations and the Amide I band, as well as absorption peaks associated with carbonates and phosphates48,49,50. These spectral features indicate that both bone samples contain collagen and minerals. In addition, the results of the infrared spectroscopy analysis show similar organic components in both, suggesting that differences in organic content do not account for the colour differences in the bones.
Micro-Raman spectroscopy
From the microscopic observation results, it can be seen that the pink bone samples are significantly mineralised. To further determine the phase structure of the samples’ mineralisation, Raman microscopic observations of the cortical bone surface were performed on seven samples using a 633 nm laser. RRUFF database was used for the careful comparison of the spectra. Raman analysis provided consistent phase peak data, with a distinct signal peak at around 963 cm−1, which is the characteristic peak of [Ca₅(PO₄)₃OH]. The sampling locations and analysis results of the micro-Raman experiments for samples 1-3, 1-5, and 2-2, which showed stronger signals, are now presented (Fig. 8).
Micro-Raman sampling points and results of samples.
A mineral phase that appears pale pink under the microscope was selected as the target for Raman spectroscopy analysis, as shown in the left image. Careful comparison of the spectral data obtained confirmed that the Raman spectral signal of this phase matched the characteristics of hydroxyapatite [Ca₅(PO₄)₃OH], a common inorganic component of human bone. However, as bone samples are biological tissues, their inherent fluorescence properties produce a strong fluorescence signal during Raman spectroscopy analysis51,52. The intensity of this signal far exceeds that of the Raman signal from the bone surface minerals themselves. This strong fluorescent background severely interferes with the detection of Raman spectra, making it difficult to accurately identify Raman characteristic peaks other than those of hydroxyapatite. This is clearly reflected in the test data provided, especially in the spectral regions from 250 to 750 cm−1 and above 1000 cm−1, where the envelope effect of the fluorescence signal is particularly significant, with intensities even exceeding those of the characteristic peaks of hydroxyapatite. To overcome the interference of the fluorescent background in biological tissues, it is therefore essential to use appropriate spectral acquisition and data processing techniques.
In current Raman spectroscopy research, the interference of fluorescence signals is a pressing issue, particularly in the fields of biology, medicine and food science. To overcome this challenge, researchers have mainly adopted two strategies: first, using mathematical methods and computational modelling techniques to effectively suppress and eliminate the fluorescence signals generated during experiments, thereby extracting pure Raman signals52; second, enhancing the intensity of the Raman scattering signals to improve the signal-to-noise ratio and thus better reveal the molecular structural information of the samples52,53.
In order to further investigate the cause of the pale pink appearance of the samples and to obtain richer surface phase signals from the samples, stimulated Raman scattering analysis was carried out on the samples used in the experiment. Through this analysis, micro-regional imaging results of the samples were successfully obtained, providing important experimental data for further understanding the chemical composition and structural characteristics of the samples.
Stimulated Raman scattering
Using SRS, the C-H bond vibrations were observed in the wavenumber range of 2800 cm−1 to 3050 cm−1, which is particularly important for the detection of organic phases such as lipids and proteins54. This region includes the characteristic peaks of C-H bond vibrations in lipids and proteins, such as symmetric CH₂ vibrations, CH₃ vibrations and high frequency C-H vibrations, which can effectively distinguish the distribution characteristics of organic phases55,56. Figure 9 SRS results of samples.shows the results of Scanning Raman Scattering (SRS) microscopic observations of the samples. The left image shows the results of the light pink bone sample 1-2 at 2889.98 cm−1, while the right image shows the observation results of the dark pink bone sample 2-2 at 2854.98 cm−1.
SRS results of samples.
As SRS is not affected by non-resonant terms in the third-order non-linear polarizability, it is free from spectral distortion caused by non-resonant background interference. This ensures that the spectral peak shapes obtained are identical to those of spontaneous Raman scattering, with no spectral distortion51. The ROI Manager is used to perform Multiple Measure on all 51 stimulated Raman scattering results obtained for Sample 1-2 in this wavenumber range, resulting in the spectral data shown in Fig. 10 1-1 Multiple measure results. In these results, ‘bright spots’ refer to distinct bright spots of significantly higher intensity than other signal-rich areas, while ‘lamellar regions’ refer to areas of uniform signal intensity distribution.
1-1 Multiple measure results.
The intrinsic Raman signals at 2977.65 cm−1 (C-H high-frequency vibrations or collagen fibril lamellar structure) and 2820.78 cm−1 (CH₂ or CH₃ vibration noise region)56,57,58 are relatively weak but appear as bright yellow spots in the stimulated Raman scattering (SRS) microscopic observations. This is attributed to fluorescence background signals generated by Fe under SRS excitation. Specifically, when Fe-O or Fe-P bonds are doped or when there is crystal lattice distortion, their Raman-active peaks shift to higher wavenumbers59, overlapping with other high-frequency vibration signals (broad peaks from C-H bonds)60. This causes the overall signal intensity of the bright spots in the Fe distribution to be significantly enhanced by fluorescence. The non-specific nature of the fluorescence signal causes it to appear as bright regions in SRS images, in contrast to the surrounding organic phases. This phenomenon is consistent with the fluorescence behaviour of Fe-doped hydroxyapatite, where fluorescence signals interfere with the imaging contrast of organic phases51,61.
Analysis of sample 1-2 (pale pink) showed that the internal organic phase arrangement was relatively regular, a feature which suggests that the protein distribution in sample 1-2 may have a positive effect on bone strength. However, although sample 2-2 shows a higher signal intensity in the experimental results, the irregularity and dispersion of the organic phase distribution suggests a limited contribution to bone support. In addition, the number of metal impurity spots is significantly higher in sample 2-2 than in sample 1-2, a phenomenon which suggests that the pink colouration may be closely related to the iron content. These observations are consistent with the actual preservation conditions of the bones of samples 1-2 and 2-2.
Based on the combined results of SRS and elemental analysis, the study suggests that the colouration of pink bones is most likely due to the presence of internal Fe elements. To further investigate the colouring mechanism of Fe elements in pink bones, the study used X-ray photoelectron spectroscopy (XPS) to analyse the valence state behaviour of Fe elements.
X-ray photoelectron spectroscopy
The valence state of elements is crucial in explaining the colouring mechanism; therefore, X-ray photoelectron spectroscopy (XPS) analysis was performed on pink bone sample 2-2 and the Fe 2p spectral lines were subjected to peak fitting62, with the results shown in Fig. 11.
XPS of Sample 2-2.
The ratio of Fe²⁺ to Fe³⁺ is 33.7% to 66.3%, indicating that iron in pink human bones is predominantly in the form of Fe³⁺. This finding suggests that Fe³⁺ is the main factor causing the pink colouration of bones. Fe³⁺ is more stable than Fe²⁺ and, due to its red nature, an increase in its concentration can cause the bone colour to become redder, thus affecting the macroscopic colour appearance of the bones63.
Based on elemental correlation analysis and stimulated Raman scattering imaging results, it is likely that in the burial environment, Fe elements can replace calcium ions in the form of ions and incorporate into the apatite structure, competing with calcium ions. The ionic radius of Fe²⁺ is similar to that of calcium ions, while Fe³⁺ is slightly smaller, making it theoretically more inclined to occupy the Ca (II) sites in apatite51,59,60,64. The additional positive charge of Fe³⁺ leads to an electrical imbalance in the chemical structure after doping, which can be compensated by structural adjustments or co-doping with other ions. This imbalance can also promote the formation of complexes between iron ions and both organic and inorganic components in bone, exacerbating the destruction of the supporting structure of the organic phases shown by SRS imaging. This results in poorer sample preservation and affects the apparent colour of the bones36,37.
Discussion
Based on the results of microscopic observation, elemental analysis, phase analysis and valence state analysis, the following conclusions were drawn:
-
1.
The pink colour of the human bones unearthed at Baoji, Shaanxi, China is related to the accumulation of Fe³⁺ in the semi-leached soil within the bone structure. Fe³⁺ not only affects the appearance of the bones, making them appear pink due to its stability and colouring properties, but also competes with calcium ions in the apatite structure. It can replace calcium ions and incorporate into the apatite structure, reacting with organic phases to cause their dispersion, thereby disrupting the support that organic phases provide to the bone structure and significantly affecting the mechanical strength of the bones.
-
2.
The results of the phase analysis show no significant differences between bones of normal colour and pink bones, suggesting that the discolouration of bones may not be caused by factors specific to the bones themselves, but is primarily influenced by the burial environment. In light of this, it is crucial to promptly identify and remove human bones from specific burial environments and to strictly control preservation conditions. Failure to take these measures may result in the destructive effects of Fe³⁺ on other samples in the same burial environment, thereby affecting the structural stability of the bones. In the long term, this destructive effect could lead to the deterioration or even disappearance of archaeological materials, resulting in the loss of valuable information for the study of ancient human activity and environmental change.
-
3.
This study applies SRS technology to the field of archaeology, revealing the distribution of organic phases in human bones at high resolution, providing a new perspective on the analysis of archaeological materials. It provides a non-destructive, highly specific analytical method for the in-depth study of the material composition, manufacturing techniques and historical information of cultural artefacts. In collagen identification, SRS technology allows detailed observation of collagen fibre microstructures and chemical changes. By detecting C-H bond vibration characteristics, it reveals collagen degradation and distribution in burial environments. This helps to understand how soil components, such as iron, accelerate collagen degradation through redox reactions. This non-destructive in situ analysis method has a very low detection limit. In addition to bones, it can be applied to the scientific analysis of other protein-based cultural heritage samples, such as organic residue studies. This technique is likely to provide a breakthrough in technical approaches to the study of bone degradation mechanisms and other underdeveloped areas of study.
Future research will use SRS technology to investigate how iron ions chemically affect the crystallinity and stability of hydroxyapatite and how they accelerate collagen degradation. This will clarify the role of iron ions in bone degradation and support the development of targeted preservation strategies.
Data availability
Data is provided within the manuscript or supplementary information files.
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Acknowledgements
The bones samples used in this study were provided by Baoji Archaeological Research Institute, Shaanxi province.
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J.L.: Writing original draft, Visualisation. XL: Supervision, Methodology, Investigation, Conceptualisation. C.Z., Q.W., W.H., and H.W.: Excavation, Providers of archaeological materials.
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Li, J., Zhang, C., Wang, Q. et al. Preliminary analysis on the cause of the pale pink colouration of human bones from Baoji archaeological sites by stimulated Raman scattering imaging. npj Herit. Sci. 13, 294 (2025). https://doi.org/10.1038/s40494-025-01832-6
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DOI: https://doi.org/10.1038/s40494-025-01832-6
