## 3 Results and Discussion
### Discussion  
In this work, we established the optical imaging platform integrated with the ultrasound application system for in vitro assays of ultrasound-mediated drug delivery. Effective observations of changes in intracellular calcium concentration and fluorescent signal changes of intracellular PI over time indicate that the platform can be used efficiently for validation of ultrasound-mediated drug delivery techniques. Using the optical imaging platform integrated with the ultrasound application system, we derived the ultrasound-based cavitation of a microbubble attached to a membrane of a cervical cancer cell, and obtained longitudinal fluorescence images of calcium flux and intracellular PI uptake to explore the feasibility of effective and noninvasive drug or gene delivery in cervical cancer by microbubble-mediated sonoporation. Also, it is confirmed that chemicals such as anti-cancer drugs can be introduced into the cervical cancer cell by the ultrasound-microbubble cavitation by observing the gradual increase of fluorescence signals from intracellular PI after the induction of the ultrasound-microbubble cavitation. As the result indicates, we confirmed that there is a significant difference between the decaying temporal constant of calcium flux (=67.8918 s.) and the growing temporal constant of fluorescence from intracellular PI (=191.188 s.), which implies a temporal mismatch between a membrane recovery for calcium ion channels and a closure of pores for drug delivery. (In this study, the time mismatch was about 2 min (=123.296 s.) for the region of interest corresponding to the neighboring region in contact with the microbubble.) It is correlated to the result of temporal mismatches in prior studies [14] related to drug delivery using ultrasonic cavitation, and to derive meaningful biophysical information, it is important to obtain standardized temporal constants in a specific cell line through measurements on a large number of cells. In addition, the temporal constants of intracellular PI differed scientifically in the entire region of the cell and the region of interest corresponding to the membrane region approached by the microbubble. Further studies in membrane recovery dynamics based on the temporal mismatch are expected to provide important information on drug delivery and cell therapy by ultrasound-microbubble cavitation. In particular, comparative studiesare needed of important parameters from each cancer cell to apply ultrasound-microbubble cavitation to other carcinomas, since the cellular dynamics of carcinoma in various regions are different, and the established platform is a useful tool in the comparative studies. Improvements that can provide higher performance in the optical imaging platform with the ultrasound application system are as follows. Currently, the optical imaging platform obtains fluorescence images to measure intracellular calcium flux and to confirm intracellular PI uptake. The system can adopt an additional fluorescent indicator to measure other cellular properties such as cell viability [28; 29]. In particular, we believe that it is necessary to observe cell growth by adding additional fluorescent channels to this platform for verification of changes in long-term cell growth during and after ultrasound-microbubble cavitation. Although the temporal resolution of measuring each image sequence was slightly reduced, we are convinced that the additional implementation provides valuable information to understand cellular properties and dynamics of drug delivery. Currently, one image sequence is measured in approximately 1.8 s since two filter sets to measure three fluorescence images are operated using electrically operated turrets, so there is a time loss caused by switching. When only calcium imaging or PI imaging is performed individually, the overall measurement time can be further improved.