# Versatile High-Throughput Platform for Focused Ultrasound In Vitro Application
## 4 Discussion  
During this study, an HTP platform for parallel cell sonication optimized for in vitro workflows based on multi-well plates was successfully developed. Traditional cell culture sonication setups are mostly based on single ultrasound transducers mechanically maneuvered below the well plate [16; 17; 18; 19; 20; 21; 22]. Our design enabled parallel sonication enhancing experimental workflow speed. However, handling data from many transducers in parallel presents challenges. For scenarios, like those using piston-like non-focusing transducers in the LIFU applicator model, we adopted parallel milling of 96 elements from a singular piezo composite plate. Although efficient, this fabrication method limited control over the homogeneity and yield of the applicator since defective elements were irreplaceable after characterization. In the LIFU model, this resulted in an element output standard deviation from 7 to 51% and yields from 70 to 100%. The inconsistent readings of homogeneity across LIFU models may arise from using bulk PZT or piezo composites and different milling parameters, warranting further investigation. In addition, the results showed that limiting the diameter of the ultrasonic transducer elements to the well diameter was ineffective for higher frequencies (>1 MHz). The natural focus with the maximum intensity was laterally located in the well diameter but was too far away in the direction of beam emission. In contrast, the HIFU model demonstrated improved performance with a consistent 94% yield and greater homogeneity with a 19% standard deviation of the acoustic power, which however comes at the price of a more complex assembly. When aiming for an  
Figure 16: Results of the maximum sound pressure with the 1 MHz HIFU platform along the z-axis in each well (96) from the bottom of the well (z = 1 mm) to the maximum filling height of 10 mm at a filling volume of 350 \(\upmu\)L. Thirty-two wells (4 rows of 8 wells each) were exposed to ultrasound. optimal homogeneity in the LIFU setup, a method similar to that of the HIFU applicator is recommended, with the elements mounted on a support with individual electrical contacts in an easily serviceable configuration. We aimed to develop versatile, easy-to-operate tools for sonication over a wide range of parameters, both in terms of frequencies and applied intensities. Depending on the diameter-to-thickness ratio and the corresponding material vibration modes, we selected either bulk PZT or piezo composite, allowing for efficient operation. With the dry-coupling approach, we observed that the heating of the material when operating the device at duty cycles greater than 5%. Accordingly, we built the HIFU version with cylindrically shaped transducers immersed in a stream of water, ensuring heat dissipation and allowing the transducers to operate at 100% of the DC supply. The difference in allowed duty cycles together with the achievable pressures, led to maximum \(\mathrm{I}_{\mathrm{SPPA}}\) values between 0.78 and 12.38 W/cm\({}^{2}\) for the LIFU version (1.5 MHz) and 72 W/cm\({}^{2}\) for the HIFU version, respectively. Importantly, our models achieved efficient confinement of acoustic energy to the wells. The lateral spread of the pressure distribution fields (\(-6\) dB width) was less than the 6.5 mm diameter of the well in the case of a 96-well plate. Achieving this presented a greater challenge in the HIFU model where the free field \(-6\) dB width was greater than 7 mm. We were able to reduce it to 5.6 mm by using separation elements between the individual transducers. The HIFU model allowed the sonication of 32 wells simultaneously, enabling the treatment of each well of a 96-well plate with only three repositioning steps of the plate. To ensure the most efficient application of the ultrasonic waves and minimize loss within the wells, a well plate with a minimal bottom thickness was utilized.