A303s su

To image calcium flux, the spheroids were loaded with freshly dissolved, cell-permeable 1 µM Fluo 4-AM (F14201, Thermo Fisher Scientific), a green fluorescent, intracellular Ca²⁺ indicator, for 30 min at 37 °C. Then, the spheroids were moved to a glass bottom dish (P50G-0-30-F, MatTek, Ashland, MA, USA) containing media with 1 × 10⁶ microbubbles. A coverslip was placed above the spheroids. The presence of microbubbles on the spheroids was microscopically confirmed. Using an in-house designed cone housing and a water immersion transducer (A303S-SU, 0.5-inch diameter, Olympus NDT), US was delivered (frequency 1 MHz; PNP 250 kPa; pulse length 10 µs; pulse interval 10 ms; treatment duration 10 s), and green fluorescence was measured for up to 1 min (Supplementary Videos S3, 4). The involvement of mechanosensitive channels in UTMC-induced Ca²⁺ influx was confirmed by pretreating spheroids with 1 µM GsMTx4 (STG-100, Alomone labs, Jerusalem, Israel) for 30 min. The occurrence of sonoporation was confirmed by loading spheroids with 120 µM PI (P3566, Thermo Fisher Scientific) in the same setup (Supplementary Video S2). The videos were quantified using Fiji.

The hydrogel (containing the channel) was placed in a tank of de-gassed and de-ionised water under a 40 × water immersion objective lens (model: LUMPLFLN, numerical aperture: 0.8, working distance: 3.3 mm; Olympus, Tokyo, Japan). A focused transducer (model: A303 S-SU, diameter: 13 mm, focal distance: 15.2 mm, centre frequency: 1 MHz; Olympus, Essex, UK) was placed to target the centre of the focal plane of the lens. This alignment was performed using a needle hydrophone (diameter: 0.2 mm, Precision Acoustics, UK). The hydrophone was also used to calibrate the peak-rarefactional pressure in situ. An LED light source (KL 2500, Schott, Dorset, UK) provided illumination from below (Fig. 1a).
Each sample was sonicated with a single pulse (pulse length: 10 ms, centre frequency: 1 MHz, peak-rarefactional pressure: 600 kPa). The transducer was driven by sinusoidal pulses generated by a function generator (33500 B Series, Agilent Technologies, Santa Clara, CA, USA) and passed through a 50-dB amplifier (E&I, Rochester, NY, USA). A frequency of 1 MHz was chosen as it is commonly used in clinical systems and many proposed applications. Lower-frequency pulses can also penetrate deeper into the body. For the stiffest gels (E = 8.7 kPa), a peak-rarefactional pressure of 1 MPa was used instead, as no deformation could be seen at lower pressures.