## 2 Materials and Methods
### Ex Vivo Experiment with PAI System  
Before the experiments, bovine hemoglobin (Hb) (H3760, Sigma-Aldrich, MO, USA), and gelatin (G-2500, Sigma-Aldrich, MO, USA) were used to mimic breast tumors [38]. First, 10 mL saline was blended with 1 g of Hb powder and stirred at 60 \({}^{\circ}\)C for 15 min. Then, 2% gelatin was poured into the completely dissolved Hb powder and stirred again in 10 min to achieve a uniform Hb distribution. Next, to remove the accumulated bubbles produced in the mixing process, the prepared solution was placed into a vacuum machine (PVH-PO-27, Labpartners, Cincinnati, OH, USA). Additionally, chicken breast tissues with the same dimensions as described in the previous section were also prepared. Then, a cylindrical part in the middle of tissues with an 8-mm circular diameter was removed and placed on a petri dish. To create the tumor phantom, the Hb solutions were injected into the detached cylindrical tissues until the sample color turned black. Then, the petri dish was stored in a refrigerator at 0 \({}^{\circ}\)C for 5 min to freeze the sample. Afterward, the developed artificial tumor was inserted back to the hole left after rejecting the tissue earlier. Finally, the whole sample was stored again in the refrigerator for 10 min at 0 \({}^{\circ}\)C to prepare it for the experiment. With this procedure, the tumor and the normal tissue region are completely separated so that they are visually distinguished by the PAI system and it is much easier while conducting ex vivo experiments. For tissue treatment and monitoring, a customized PAI module was constructed and integrated with the HIFU system. The entire configuration of the ex-vivo experiment, including HIFU and PAI system, is described in Figure 5a. The laser source used a pumping Q-switched diode (SPOT-10-100-532, Elforlight, Daventry, UK) operating at 5 kHz repetition rate. The 532-nm laser output wavelength was generated to scan the artificial tumor including Hb particles [39]. The laser beam was coupled to a 2-m single-mode fiber (P3-460B-FC-2, Elforlight, Daventry, UK) through a fiber coupler and a half-wave plate. The output laser of the fiber with the intensity energy under 13.3 mJ/cm\({}^{2}\) was led in a PAI probe before irradiating the tissue sample as demonstrated in Figure 5c. The self-made acrylic PAI probe  
Figure 4: Proposed Fuzzy Logic Control System. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**No.** & **Fuzzy Set** & **Fuzzy State** & **Fuzzy Rule** \\ \hline
1 & 57–58 \({}^{\circ}\)C & Very cold & IF (temperature is “very cold”) THEN (“heat fast” sample by \(\pm\)18 V) \\
2 & 58–59 \({}^{\circ}\)C & Cold & IF (temperature is “cold”) THEN (“heat” sample by \(\pm\)15 V) \\
3 & 59–61 \({}^{\circ}\)C & Warm & IF (temperature is “warm”) THEN (“remain” sample by \(\pm\)12 V) \\
4 & 61–62 \({}^{\circ}\)C & Hot & IF (temperature is “hot”) THEN (“cool” sample by \(\pm\)9 V) \\
5 & 62–63 \({}^{\circ}\)C & Very hot & IF (temperature is “very hot”) THEN (“cool fast” sample by \(\pm\)6 V) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Fuzzy set, fuzzy state and fuzzy rule of the proposed control system. was designed and delicately fabricated by the milling machine. Figure 5b illustrates the structure of the PAI probe consisting of a focusing lens, an aluminum layer which reflects the laser beam to the sample. PA signals induced under the laser irradiation were detected using a flat single element 75-MHz ultrasound transducer (V2025, Olympus, Norfolk, VA, USA). A linear positioning in the \(z\)-axis direction was justified to calibrate the focal beam of the transducer to attain the maximum PA signal amplitude. A 2D linear stage (x-, y-axis) with 10 \(\upmu\)m spatial resolution synchronized with the laser trigger was also used to scan the sample for constructing a \(14\times 14\) mm\({}^{2}\) 2D image.