Characterization of Second Membrane Perforation by Ultrasound-driven Single Microbubble

The cells will undergo repeated sonoporation by flowing microbubbles in the blood vessel. Based on a platform for performing acoustic cavitation and observing cellular responses at the single-cell level, we observed 38 sonoporated cells, all of which underwent the primary and second reversible sonoporation. Our results show that: 1) The reversibly sonoporated cells were categorized into mild and moderate sonoporation. 2) In 34 out of 38 primarily sonoporated cells, the resealing time of 18 mildly sonoporated cells was less than 16 s, which was faster than that of 16 moderately sonoporated cells. 3) In 34 out of 38 sonoporated cells, the time for membrane resealing was shorter in cells that experienced the second sonoporation compared with cells in primary sonoporation. These results provide fundamental sights into the biophysical process of sonoporation.