## 2 Materials and Methods  
### Design of the Integrated Probe  
The integrated probe prototype is depicted in Figure 1a, which displays a single-element imaging probe positioned within the central hole of a spherical-focused therapeutic probe. The therapeutic probe operates at a center frequency of 4.5 MHz, while the imaging probe operates at 12 MHz. Two probes were fixed to the mechanical structure by connectors. Compared to the therapy system guided by a linear array B-mode imaging probe, the volume is significantly reduced, which is depicted in Figure 1b. The imaging probe utilized in this study was a 12 MHz single-element probe, which was located horizontally in the central hole of the therapeutic probe for imaging the treatment focus. The echo amplitude and spectrum of the imaging probe can be observed in Figure 2. The use of this frequency probe allowed for effective imaging of shallow and complex tortuous venous morphology, while also allowing for the detection of potential deep venous thrombosis. This is advantageous for the development of a comprehensive treatment plan due to the probe's excellent imaging depth and resolution. The use of HIFU treatment requires the selection of an appropriate frequency for the therapeutic probe. Generally, the therapeutic probe frequency is set between 1 and 3 MHz [22]. However, some studies have suggested that higher frequency focused ultrasound probes can achieve better treatment outcomes for vascular treatments [23]. Therefore, this system developed three higher-frequency focused ultrasound probes with the same physical parameters, including diameter and curvature, except for the frequency, at 3 MHz, 4.5 MHz, and 7.5 MHz. The increase in the excitation frequency of the transducer in most human tissues leads to a power-law increase in absorption and attenuation. An increase in absorption coefficient results in a larger sound absorption and heating rate, while an  
Figure 1: Prototype probe for integrated ultrasound imaging and therapy: (**a**) system guided by a single-element imaging probe; (**b**) dimensional comparison, the right is a system guided by a B-mode imaging probe. Figure 2: Echo test results of the 12 MHz imaging probe. increase in attenuation coefficient decreases the penetration depth. Therefore, in this experiment, both the heating rate and the penetration depth of blood vessels under ultrasound exposure need to be considered simultaneously. In previous studies, the primary reason for the contraction and closure of blood vessels under ultrasound exposure was determined to be the high-temperature denaturation reaction of collagen in the blood vessels [24; 25]. To compare the effect of these three treatment probes on protein denaturation, a one-minute treatment comparison experiment was conducted using 15 W of sound power to excite these probes with a sinusoidal wave on a PAA phantom. The PAA phantom was made of polyacrylamide gel with 30% added serum, whose acoustic parameters were similar to those of ex vivo liver and muscle tissue [26]. When the temperature of the PAA phantom reaches 60 \({}^{\circ}\)C or above, it changes from transparent to opaque white. Additionally, 60 \({}^{\circ}\)C is the critical temperature for irreversible coagulation necrosis of cells. Therefore, a PAA phantom is often used in HIFU ablation experiments, and the ablation range can be determined by observing the white area inside the PAA phantom. Each frequency of the focused ultrasound transducer underwent three experiments, and the average width, height, and area of the resulting lesions were calculated. The results are shown in Figure 3.  
To test the protein heating rate generated by the three different frequency probes during treatment, a thermocouple was embedded in the PAA phantom at the focus of the probe, and the temperature rise was recorded and plotted using a communication converter. Subsequently, the same method as the aforementioned experiment was used for one minute of treatment.