# Bone Chemical Composition Assessment with Multi-Wavelength Photoacoustic Analysis
## 3 Results and Discussion  
Fresh human bone contains non-organic mineral (hydroxyapatite) and water, as well as organic compounds, including collagen, lipid, and hemoglobin [34; 35; 36]. Each non-organic and organic material in the bone has its own unique optical absorption spectrum, and may contribute to the PA signal from the bone. In the spectral range of 680-950 nm, the main optically absorbing components in the bone are hydroxyapatite, oxygenated hemoglobin, deoxygenated hemoglobin, and lipids, while the light absorption of other materials is relatively low [13; 37; 38; 39; 40; 31]. Figure 3 shows the MWPA results for the three groups of bones. The three solid lines in Figure 3a show the averaged PA spectra measured from the three groups of bones, while the standard deviation and error bars are shown by the shaded area next to each curve. An obvious difference can be seen in the PA spectra from the three groups. In the PA spectrum from the fresh bones (Group 1), we can see relatively strong absorption around 700 nm, 760 nm, and 860-950 nm. The overall shape of the PA spectrum matches well with results in the literature [13; 41]. The optical absorption spectra of the main chemical components in the bone are shown in Figure 3b. We can see that the main characteristics of these absorption spectra, especially for hydroxyapatite, hemoglobin, and lipid, are reflected in the measured PA spectrum from the fresh bone in Figure 3a [21]. We expect that both deoxygenated hemoglobin and hydroxyapatite contribute to the strong absorption in the range of 680-700 nm [42; 43]. The peak around 760 nm in the PA spectrum may come from the absorption of both deoxygenated hemoglobin and lipid [44]. The strong absorption around 900-950 nm may be associated with lipid, oxygenated hemoglobin, and water. According to the literature, lipid has a strong absorption peak at 930 nm, while collagen has an absorption peak near 920 nm [44; 45]. However, these peaks at approximately 920-930 nm can only be noticed in the PA spectrum from the bones treated by EDTA for 26 h (Group 3). For the other two groups (i.e., Group 1 and Group 2), these peaks cannot be seen, probably because the 900-950 nm spectral range is still dominated by oxygenated hemoglobin which was not fully removed from the bones in these two groups. With the optical absorption spectra of all the major chemical components in the trabecular bone known [21; 31], the relative contribution of each chemical component to the PA spectrum can be derived by performing a spectral unmixing. A least-squares method was used, which tries to fit the PA spectrum via a linear combination of the optical spectra of all the chemical components leading to the minimum sum of the squared residuals of measurement points from the fitting line. As an example, Figure 3c shows the averaged PA spectrum from Group 2 in comparison with the fitted spectrum based on the least-squares method. A very high R\({}^{2}\) of 0.97 was achieved, demonstrating that a successful fitting with a high accuracy was achieved in spectral unmixing. For Group 1 and Group 3, the same procedure also led to a satisfactory R\({}^{2}\) of 0.96 and 0.94, respectively. After the spectral unmixing, the relative contents of the six chemical components in the bone, including hydroxyapatite, deoxygenated hemoglobin, oxygenated hemoglobin, lipid, collagen, and water, were derived, as the results show in Figure 4 for the three groups of bone samples. The spectral unmixing here did not lead to the measurements of molar concentrations of the chemical components because the data in Figure 3b utilized in spectral unmixing were not the molar extinction coefficients. The relative content derived from the spectral unmixing reflects the weight of each optically absorbing chemical component in the measured PA spectral. As expected, when the EDTA treatment time increased, the content of hydroxyapatite decreased [46], while the contents of lipid, collagen, and water increased accordingly.