# Ultrasound-Guided Detection and Segmentation of Photoacoustic Signals from Bone Tissue In Vivo
## 3 Results and Discussion  
The results of the US and PA wave propagation simulations are presented in Figures 3 and 4, respectively. Figure 3a-c provide overviews of the wave propagated in the bone in the US mode at 1 us, 10 us, and 20 us, respectively. In the US mode, as shown in Figure 3, the acoustic source was from the US transducer at the left side of the bone, which generated US wave propagation through the bone with attenuation and reflection. The reflection US wave was received by the transducer placed at the left side of bone, which can be used to measure the distance between the bone and transducer. The attenuated US wave that penetrated the whole bone was received by another transducer placed contralaterally at another side of the bone tissue. For the PA mode, the simulation results of the light distribution are shown in Figure 4a. The light distribution employed in this study was then normalized and converted into the PA initial pressure using Equations (3) and (4). Figure 4b,c provide overviews of the waves propagated in the bone in the PA mode at 1 us and 10 us, respectively. The PA signal from the trabecular bone (TB) and the soft tissue (ST) are marked in red and cyan, respectively. The following can be observed from the PA simulation results presented in Figures 3 and 4: (1) for the high optical attenuation in the bone, the overlying soft tissue and the cortical bone on the right side (near the transducer) had a very low PA signal; (2) the TB signal arrived at the transducer first because it was the closest to the transducer; and (3) the overlying soft tissue and the cortical bone signals near the laser side were propagated along two paths. One group of signals, the Lamb waves, propagated along the cortical bone surface. The 2D numerical simulation results are provided in the Supplementary Materials. The other signal had free-space propagation, which accounted for a large proportion of the signals. The Lamb waves constituted a small proportion; however, they propagated along the cortical bone, where the SOS was approximately 4000 m/s, which was higher  
Figure 3: Ultrasound simulation results. (**a**) Simulation results at t = 1 us in the US mode. (**b**) Simulation results at t = 10 us in the US mode. (**c**) Simulation results at t = 20 us in the US mode. TT, transmitting transducer; RT, receiving transducer. Figure 2: Experimental setup. (**a**) US and (**b**) PA measurement system for calcaneus bone in vivo. than that of the trabecular portion. From the simulation, we confirmed that the Lamb wave generated by the cortical bone arrived after the compression wave generated by the TB. This was due to the propagation path of the Lamb waves; their propagation along the bone surface is longer than that of the compression waves, which propagate inside the bone. The whole PA signal could be segmented into two parts from the time domain. The first comprises the TB, which is the range of interesting (ROI), whereas the other part comprises the mixed PA signal from the overlying soft tissue, cortical bone, and their echoes. However, the distinctions of the PA signals from the TB and other parts are not ascertained owing to the complicated propagation modes of the laser-induced US signal in the bone. This causes an overlap of the PA signals from different parts in the time domain. The US transmission mode can help distinguish the PA signal of the trabecular portions from the others. The setups and typical US signal profiles are shown in Figure 5, where the left column shows the setups for the US reflection mode, US transmission mode, and PA transmission mode, respectively. The three setups in Figure 5a,c,e are similar, whereas the differences are the excitation source and the receiver. The corresponding signals are shown in Figure 5b,d,f, respectively. In Figure 5b, the dashed line indicates the time of the first received reflection signal from the bone surface; \(d\) is the thickness of the overlying soft tissue.