# Bone Chemical Composition Assessment with Multi-Wavelength Photoacoustic Analysis
## 3 Results and Discussion
The EDTA treatment also reduced the contents of both oxygenated hemoglobin and deoxygenated hemoglobin, because soaking the bone in the EDTA solution led to the removal of blood from the bone. The bar chart in Figure 4b summarizes the changes in mineral (hydroxyapatite) content, blood content (including the two forms of hemoglobin), and the overall content of other materials including lipid, collagen, and water. To examine whether these changes in chemical components in the bone caused by the EDTA treatment are significant, un-paired \(t\)-tests were performed to compare the results in Figure 4b. After a 13-hour EDTA treatment and 26-hour EDTA treatment, the mineral content, the blood content, and the overall content of other materials (including lipid, collagen, and water) all show statistically significant changes (\(p\)-value \(\approx\) 0.01, 0.02,0.01, respectively). The changes in chemical contents between the two treatment groups (i.e., 13-hour EDTA treatment and 26-hour EDTA treatment) are not significant. Figure 3: (**a**) The averaged PA spectra measured from the three groups of porcine ribs, including fresh bones (Group 1), bones treated with EDTA for 13 h (Group 2), and bones treated with EDTA for 26 h (Group 3). (**b**) Optical absorption spectra of major chemical components in the bone, including hydroxyapatite, collagen, oxyhemoglobin (HbO2), deoxygenated hemoglobin (Hb), lipids, and water. (**c**) The averaged PA spectrum from Group 2 (the bones treated with EDTA for 13 h) in comparison with the fitted spectrum by using the least-squares method. Spectral unmixing also enables us to evaluate the contribution from each chemical component to the PA signal from the bone at each specific optical wavelength. The contribution \(c_{i}(\lambda)\) for each chemical component was calculated by using  
\[c_{i}(\lambda)=\frac{\left[\mu_{a}(\lambda)\right]_{i}\cdot\left[C_{r}\right]_{i }}{\left[F(\lambda)\right]_{i}} \tag{5}\]  
where \(\left[\mu_{a}(\lambda)\right]_{i}\) is the optical absorption spectrum of different chemical components at a wavelength of 680-950 nm, \(\left[C_{r}\right]_{i}\) is the relative content of different chemical components in the bone, as shown in Figure 3b, and \(\left[F(\lambda)\right]_{i}\) is the fitting line, as shown in Figure 3c. Based on Equation (5), the quantified contribution \(c_{i}(\lambda)\) of different chemical components in the bone can be obtained. Figure 5 shows the quantified contribution \(c_{i}(\lambda)\) from each of the six chemical components (i.e., hydroxyapatite, deoxygenated hemoglobin, oxygenated hemoglobin, collagen, lipid, and water) to the bone signal as a function of optical wavelength from 680 nm to 950 nm. For fresh bones, the major contributors to the PA signals over the studied spectrum were hydroxyapatite, deoxygenated hemoglobin (680-780 nm), and oxygenated hemoglobin (800-950 nm). After EDTA treatment, the contribution from hydroxyapatite was weaker over the entire spectrum; the contribution from oxygenated hemoglobin was almost gone, as not only the blood was removed but it also became older and deoxygenized as a result of the treatment. In addition, because of the loss of both hydroxyapatite and blood after treatment, the relative contributions from lipid, collagen, and water became larger, especially in the spectral range of 900-950 nm. This further explains the peak around 920-930 nm that was observed in the PA spectral curve from the bones treated with EDTA for 26 h, as shown in Figure 3. To indicate the reproducibility, the contribution of each chemical component to the PA signal from the bone as the function of optical wavelength was conducted for each bone sample in those three groups.