## 2 Materials and Methods
### Setup of an Ultrasound Application System for Ultrasound-Microbubble Cavitation  
To irradiate ultrasound and generate ultrasound-microbubble cavitation in the microfluidic cell culture flow chamber, an ultrasound application system was established. The ultrasound irradiation platform consisted of a focused immersion ultrasound transducer (A303S, Olympus NDT, Center Valley, PA, USA) with a central frequency of 1.0 MHz, an amplifier (1040L, Electronics & Innovation, Rochester, NY, USA) with a power gain of 53 dB, and a function generator (33220A, Agilent, Santa Clara, CA, USA) to drive the transducer. To estimate an experimental focal length of the focused ultrasound transducer, a profile of pressures generated by the transducer was measured by an acoustic intensity measurement system (AIMS III, Onda Corporation, Sunnyvale, CA, USA) and a hydrophone (HGL-400, Onda Corporation). As described in Figure 1b, The measured center focal distance was 17 mm from the surface of the transducer, and the generated peak negative pressure for the focal region was 0.6 MPa at 200 mV\({}_{\mathrm{P-P}}\) of an input voltage. Based on the profile data and an optimization of the input voltage that produces a level of ultrasound that induces cavitation and does not change the position of the microbubble itself, we applied 200 mV\({}_{\mathrm{P-P}}\), 10 cycles, and a single trigger as ultrasound irradiation conditions. To precisely match the focal region with the plane in which the cervical cancer cells and microbubbles were located, we employed multiaxis microscale stages that can accurately move the ultrasound transducer to the lateral (\(xy\)) and axial (\(z\)) axis. Also, we gave a constant angle to approach the transducer to the specimen for ensuring that the transducer did not overlap optical paths in an optical fluorescence imaging system. For this issue, additional rotation stage was applied to precise position adjustments of the ultrasound transducer. Multiaxis translational stages, the rotation stage, and mechanical components for accurate position adjustments of the ultrasound transducer were purchased from Science Town (Incheon, Republic of Korea) and Nam-il Optical Instruments Co. (Incheon, Republic of Korea). Figure 1: (**a**) Schematic of the optical imaging platform integrated with an ultrasound application system for obtaining fluorescence images of calcium flux changes and propidium iodide (PI) in a cervical cancer cell treated by ultrasound-microbubble cavitation. The meaning of each abbreviation in this figure is as follows. UST: an ultrasound transducer; S: a specimen that consists of a microfluidic cell culture flow chamber and a water tank; Obj. : an objective lens; DM: a dichroic mirror; Ex. : an excitation filter; Em. : an emission filter; LS: a light source for fluorescence excitation; EMCCD: an electron multiplying charge-coupled device camera. (**b**) Acoustic pressure fields (\(xy\) and \(yz\)-axis) at an input voltage of 50 mV\({}_{\mathrm{P-P}}\) and a graph of peak negative acoustic pressures via input voltages of a focused ultrasound transducer. To effectively deliver ultrasonic waves to the cervical cancer cells in the cell culture flow chamber with water medium, a small water tank was customized using transparent acrylics. The underside of the water tank was drilled to fit the size of the flow chamber. After that, the water tank and the flow chamber were attached using a waterproof tape (Ace Cross 071, [PERSON], Tokyo, Japan) so that the acrylic of the water tank did not cover the imaging area. So, a reduction of image quality from the thick acrylic that formed the water tank was prevented. To generate ultrasound-microbubble cavitation for calcium channel activation and drug release in the cervical cancer cell, DEFINITY(r) (Lantheus Medical Imaging, N. Billerica, MA, United States), an ultrasound image contrast agent, was applied with injection of PI solution after seeding of cells in the cell culture flow chamber and staining by Fura-2 AM.