# Bone Chemical Composition Assessment with Multi-Wavelength Photoacoustic Analysis
## 4 Conclusions  
Via the experiments on clinically relevant porcine rib bones, we studied the feasibility of assessing bone chemical composition by using MWPA method. The quantified relative contents of the chemical components in the three groups of bones, including untreated, treated with EDTA for 13 h, and treated with EDTA for 26 h, were compared, and statistically significant chemical changes due to the treatments were demonstrated. Besides detecting the changes in mineral content in the bone, MWPA could provide additional information of other optically absorbing chemical materials, such as lipid, collagen, oxygenated hemoglobin, deoxygenated hemoglobin, and water. Evaluation of the contents and changes of these organic and non-organic materials in the bone may provide highly valuable information on bone health. In the future, the MWPA could be developed into a low-cost, non-invasive, and patient friendly tool for early diagnosis and longitudinal monitoring of a variety of bone conditions resulting from diseases or aging. The present study also has some limitations. First, the EDTA bone models do not create changes in bone chemical composition exactly the same as those in osteoporosis or osteopenia bone. Therefore, to pave the way toward clinical applications, extensive studies on clinically relevant animal models of bone diseases followed by clinical trials on human subjects would be necessary to fully understand the performance and limitations of the MWPA method described in this work. Second, the effect of light attenuation was not considered in this study. For in vivo studies, human bones are large, which leads to high light attenuation in them. Therefore, compensation for light attenuation should be considered in future work. Third, for the large ultrasound attenuation in bone for in vivo studies, especially for the high frequency component, therefore, the transducer used for in vivo studies should have a relatively low center frequency. Hence, parts of the future work would be studying the relationship between the PA frequency and the amount of the different chemical components in the bone, as well as the PA spectral curves as functions of the center frequency of the transducer. Fourth, for practical use, the bone assessment technique based on the PA detecting method shown in our manuscript may not available and should be improved with a better design. In particular,  
Figure 5: The contributions of each optically absorbing chemical component to the PA absorption curves, including (**a**) hydroxyapatite, (**b**) deoxygenated hemoglobin (Hb), (**c**) oxygenated hemoglobin (HbO\({}_{2}\)), (**d**) whole blood (Hb + HbO\({}_{2}\)), (**e**) lipid, (**f**) collagen, (**g**) water, (**h**) and the total of lipid, collagen, and water, to the bone PA signals at different optical wavelengths from 680 nm to 950 nm. the mode of detection should be improved based on the target bone in vivo. For example, we used the transmission PA mode for multi-wavelength PA measurement of calcaneus bone in vivo in our recently published study [29]. Fifth, the optical penetration depth at different wavelengths and the ultrasound penetration depth at difference frequencies should be further studied. Based on them, the optimized range of wavelengths and center frequency of the transducer should be used. Despite these limitations, this study successfully proved the feasibility of using the emerging PA techniques to assess the chemical information in bone. In comparison with established gold standard dual-energy X-ray absorptiometry (DEXA) imaging modalities, the presented PA bone assessment method has many advantages, such as being target specific, non-ionizing, low cost, and patient friendly. Furthermore, the development of PA techniques as a way to accurately quantify organ-level chemical and molecular changes, such as lipid content, blood content (perfusion), and hydroxyapatite content, may allow for early identification of changes in bone metabolism and quality. Conceptualization, T.F., J.Y., and Q.C. ; methodology, T.F. and Y.Z.