## 3 Results and Discussion
### Fluorescence Imaging of Propidium Iodide  
Figure 4a is a result of time-elapsed fluorescence images, which were acquired by the optical imaging platform with the ultrasound application system, for the detection of the increase in intracellular PI to confirm a possibility of drug delivery in the cervical cancer cell. Background fluorescence signals were corrected in each transient fluorescence image, and the detailed steps are as follows. A region without cells was set and an average of background intensities was calculated for the determined background area. After that, background correction was performed by subtracting the average value from the intensities of the acquired fluorescence images. Unlike the rapid increase in calcium flux immediately after the ultrasound exposure, the gradual increase in the fluorescence from intracellular PI was confirmed by fluorescence images of each lapsed time.  
Figure 4: (**a**) Time-varying fluorescence images of intracellular PI in a cervical cancer cell with an ultrasound-microbubble cavitation. Background fluorescence signals were corrected. An inlet yellow box indicates a region of interest to determine intracellular PI fluorescence of the cervical cancer cell with the ultrasound-microbubble cavitation. (**b,c**) Plots of quantitative intensities of fluorescence from intracellular PI in the cervical cancer cell with the ultrasound-microbubble cavitation and the cell without the cavitation. Figure 3b indicates the quantitative intensities of intracellular PI for the entire region of cells, and Figure 3c means the intracellular PI intensities for a region of interest, which is represented as a yellow box, of the neighboring region in contact with the microbubble.  
To determine the quantitative characteristics of the increasing tendency of intracellular PI, fluorescence intensities in time were estimated from a selected region of interest (yellow box in Figure 4a) within the cervical cancer cell. The result is described in Figure 4b,c. For the result to be qualitatively determined in the fluorescence images, fluorescence signals from intracellular PI were gradually increased compared to the cervical cancer cell with no cavitation. We applied an exponential growth fitting to fit the increase of fluorescence signals from intracellular PI, and a growing temporal constant was 327.2194 s for the entire region of the cell and 191.188 s for the region of interest corresponding to the membrane in contact with the microbubble. The fitting curve of the fluorescence intensity of intracellular PI for the entire cell region did not have a meaningful R-square value because the average of the overall fluorescence intensity was too small. The result indicates that PI, which cannot be normally permeated into the cancer cell, entered the cell by the ultrasound-induced microbubble cavitation. PI reacts interchangeably between bases with no sequence preference and binds to DNA (deoxyxribonucleic acid) in cells. Due to its inability to pass through the membrane of living cells, PI is commonly employed as a fluorescent marker for dead cells [14,26,27]. When detecting dead cells using PI, the fluorescence intensity of PI in the dead cell is \(10^{3}\) to \(10^{4}\) times higher than the background due to binding to DNA. In this assay, although the fluorescence of intracellular PI was increased after the ultrasound-microbubble cavitation as described in Figure 4b,c, the increase of fluorescence intensity was lower than that of PI in dead cells because the viability of the cervical cancer cell was not varied by the cavitation. The results from calcium flux and intracellular PI fluorescence images mean that the sonoporation did not cause irreversible damage to the cervical cancer cells, but only provided a temporary opening that can be recovered after a short period of time. It can serve the purpose of minimally invasive drug delivery, which does not cause permanent changes to cells and biological tissue while delivering chemicals.