# Bone Chemical Composition Assessment with Multi-Wavelength Photoacoustic Analysis
## 1 Introduction  
The number of patients with osteoporosis, a serious public health threat with significant physical, psychological, and economic impacts, is expected to grow, mainly due to the increase in the aging population worldwide. In osteoporosis, the bone mineral density (BMD) is reduced, bone microarchitecture (BMA) deteriorates, and the amount and variety of proteins in bone are altered. Currently, most clinically used non-invasive assessment methods are based on the use of X-ray or ultrasound [1; 2]. These methods, although often used to measure bone mineral density (BMD) and some mechanical properties, have limited sensitivity to monitor the chemical or molecular changes in the bone that result from disease or aging. In addition, X-ray-based techniques use ionizing radiation, which is not ideal for pediatric applications or long-term repetitive monitoring. Quantitative ultrasound (QUS) technologies provide practical and low-cost alternatives, and have already led to clinical instrumentations [3; 4]. The QUS bone assessment methods are usually based on the measurements of sound velocity (SOS) and broadband ultrasound attenuation (BUA) through agiven tissue. However, the specificity of QUS is limited when pathogenic bone diseases are determined by microstructure and chemical changes rather than the change in bone mass [5; 6; 7]. Bone quantity and quality are dependent on not only the mass and structure of the non-organic mineral matrix but also the organic matrix which is associated with the bone blood flow and cellular metabolism. The main components of the organic matrix in the bone are lipid, blood cells, collagen, and proteins. It is the mineral component of the non-organic matrix that makes bone hard and rigid, but the arrangement of the collagen fibers in the organic matrix makes bone strong. In addition, the other organic components, including bone marrow fat, marrow perfusion, and oxygen saturation, are related to the cellular metabolism of bone. Therefore, those features of chemical components may offer newly detectable information for the early diagnosis of osteoporosis [8; 9]. Recently, it has been reported that magnetic resonance imaging (MRI) can distinguish changes in bone marrow lipid content and bone microarchitecture between normal bone and osteogenic bone [10; 11; 12]. Due to its high cost and complexity, MRI examinations are not a feasible replacement for routine X-ray-based methods. In prior studies, optical spectroscopic techniques have been used to evaluate how alterations of bone composition contribute to bone quality changes related to aging, disease, or injury [13; 14; 15; 16]. Traditional optical techniques, however, suffer from low spatial resolution and limited detection specificity due to the overwhelming optical scattering in biological tissues. The emerging biomedical photoacoustic (PA) techniques have the unique capability to probe the highly sensitive optical absorption contrast in deep biological tissues [17; 18]. The PA signal generated by the bone contains both microstructural information and molecular and chemical information, both highly relevant to bone health. In previous research, [PERSON] et al. evaluated the cortical and trabecular bone structure and density variations in bone using a dual backscattered ultrasound and PA radar system [19; 20]. Our group has studied the feasibility of using the thermal photoacoustic (TPA) method and photoacoustic spectral analysis (PASA) method to assess the BMD and BMA of trabecular bones in rat models [21; 22; 23]. [PERSON] et al. introduces an optimized PA imaging technique to assess SO\({}_{2}\) within the femoral bone marrow cavity through disease progression in a murine model [24]. Recently, [PERSON] et al. used the dual-modality multispectral PA system to quantify the blood over fat ratio present in the marrow, which was correlated with the molecular changes in the long bone [25].