## 3 Results and Discussion  
### Imaging of Calcium Flux Changes of a Cervical Cell by Ultrasound-Microbubble Cavitation  
As described in Figure 2, the cavitation between ultrasound and the microbubble in contact with the cell membrane causes cracks in the plasma membrane of the cell, resulting in changes in the permeability of the cell membrane [24,25]. This increase in cell plasma membrane permeability cause changes in calcium flux in the short term and allows the entry of chemicals that cannot enter living cancer cells generally. To measure transient variations of intracellular calcium flux, fluorescence images of Fura-2 AM excited by light with a wavelength of 340 and 380 nm were acquired using the optical imaging platform integrated with the ultrasound application system. Relative calcium flux changes in the cervical cancer cell, which was attached to the microbubble, were obtained by calculating images of the ratio (\(I_{\lambda\ =\ 340\ nm}/I_{\lambda\ =\ 380\ nm}\)) of two fluorescence images as described in Figure 3a. We confirmed that the investigated optical imaging platform provides time-varying calcium flux changes in a single cell. In images of calcium flux changes over time based on ultrasound irradiation, the ultrasound-induced microbubble cavitation offered a significant increase in the intracellular calcium concentration in the cervical cancer cell. This increase in calcium flux gradually decreased and lasted for about 50 to 60 s after the ultrasound-induced microbubble cavitation. After 120 s, it recovered to a similar level of intracellular calcium concentration distributions as the image before the ultrasound exposure (t = -1.8 s).  
We determined the ratio of fluorescence by excitation lights at 340 and 380 nm for an entire region of the cell as described in Figure 3b. Also, we  
Figure 3: (**a**) Time-elapsed images of a ratio (\(I_{\lambda\ =\ 340\ nm}/I_{\lambda\ =\ 380\ nm}\)) between fluorescence images of Fura-2 AM excited at a wavelength of 340 and 380 nm. Background signals were corrected in each excitation wavelength. A criterion of 0.0 s is when ultrasound was irradiated. An inlet yellow box indicates a region of interest to determine quantitative ratios that mean calcium flux of the cervical cancer cell with the ultrasound-microbubble cavitation. (**b**,**c**) Plots of quantitative ratios between fluorescence of Fura-2 AM in the cervical cancer cell with the ultrasound-microbubble cavitation and the cell without the cavitation. Figure 3b means the ratio for the entire region of cells, and Figure 3c indicates the ratio for a region of interest, which is represented as a yellow box, of the neighboring region in contact with the microbubble.  
(yellow box in Figure 3a) for a specific region of the cell adjacent to the microbubble, and the estimation of fluorescent signals for the region of interest (ROI) was performed. A quantitative ratio analysis (as described in Figure 3b,c) in the entire region of the cell and the selected region of interest within the cervical cancer cell also indicated that the intracellular calcium concentration was recovered to its previous level before ultrasound exposures with a decaying temporal constant of 72.6979 and 68.8918 s following an increase in calcium flux. There is little difference between the two temporal constants, which means that the calcium flux by the ultrasound-microbubble cavitation reacts and recovers over the entire cell domain.