## 2 Materials and Methods
### Instrumentation of an Optical Imaging Platform and Imaging Procedures  
An optical imaging platform (as illustrated in Figure 1a) integrated with an ultrasound application system was investigated based on an inverted optical microscope (Ti-E, Nikon Corporation, Tokyo, Japan) to acquire both fluorescence images of Fura-2 AM and PI. The detailed description of each optical component according to the optical path and sequence is as follows. The excitation light of a light source (Lambda DG-4, Sutter Instrument, Inc., CA, United States) that consisted of a high-power xenon arc lamp and high-speed wavelength switching module was irradiated to the specimen through excitation filters, optical components, and an objective lens (S Plan Flour ELWD 20x/0.45, Nikon Corporation). To obtain relative Ca\({}^{2+}\) concentrations from Fura-2 AM, there were two excitation filters applied, which allowed the excitation light to be adjusted with two central wavelengths of 340 and 380 nm (ET340x and ET380x, Chroma Technology, Bellows Falls, VT, USA). Also, a filter (69000x, Chroma Technology) corresponding to a fluorescence excitation band of PI to detect fluorescence signals from PI penetrated into the cervical cancer cell. Fluorescence with a central wavelength of 510 nm, emitted from Fura-2 AM, was measured by a high-sensitivity electron multiplying charge-coupled device (EMCCD) camera (iXon 885, Andor Technology PLC, Belfast, Northern Ireland). To eliminate noise components with different wavelengths, an emission filter (ET510/80m, Chroma Technology) with a central wavelength of around 510 nm was employed. Likewise, another emission filter (69000m, Chroma Technology) was employed to measure the fluorescence emitted from PI permeated into the cervical cancer cell. Also, a multiband dichroic mirror (T400 lp and 69000 bs, Chroma Technology) was applied as the optical component separating the path of the light source and the detector. The platform was equipped with a multiaxis sample scanning stage (BioPrecision2, Ludl Electronic Products, Ltd., Hawthorne, NY, United States), and ultrasound irradiations and fluorescence imaging were set to be focused in the same area. Thus, it allowed the platform to move samples and perform ultrasound-microbubble cavitation and fluorescence imaging on cells in the same chamber or other chambers. A theoretical lateral resolution for fluorescence imaging of Fura-2 AM is 692 nm and the resolution for fluorescence imaging of PI is 814 nm.  
A single set including fluorescence images of Fura-2 AM for excitations at 340/380 nm and a fluorescence image of PI was acquired every 1.8 s. An overall measurement time was 945 s (\(-\)52.2 to 892.8 s) when a trigger time of ultrasound irradiation was set to 0.0 s. To determine the formation of microbubbles, brightfield images were captured prior to fluorescence image acquisition. Also, brightfield images were obtained instead of fluorescence images in a time domain of -3.6 to 1.8 s for the confirmation of whether microbubbles attached to the membrane of the cervical cancer cell cavitated after ultrasound irradiation. A white light illuminator (pE-100, CoolLED Ltd., Andover, UK) mounted above the specimen was applied as a light source to acquire brightfield images.  
A semi-automated operation of the optical imaging platform, brightfield/fluorescence image sequence acquisition, preprocessing, and postprocessing of acquired images were progressed by MetaMorph(r) (Molecular Devices, Inc., Downingtown, PA, United States). Also, we applied GNU Octave to additional postprocessing of fluorescence images and quantitative data analysis.