Transient sonoporation can essentially be epitomized by two fundamental processes: acoustically induced membrane perforation and its subsequent resealing. To provide insight into these processes, this article presents a new series of direct evidence on the membrane-level dynamics during and after an episode of sonoporation. Our direct observations were obtained from anchored fetal fibroblasts whose membrane topography was imaged in situ using real-time confocal microscopy. To facilitate controlled sonoporation at the single-cell level, microbubbles that can passively adhere to the cell membrane were first introduced at a 1:1 cell-to-bubble ratio. Single-pulse ultrasound exposure (1-MHz frequency, 10-cycle pulse duration, 0.85-MPa peak negative pressure in situ) was then applied to trigger microbubble pulsation/collapse, which, in turn, instigated membrane perforation. With this protocol, five membrane-level phenomena were observed: (i) localized perforation of the cell membrane was synchronized with the instant of ultrasound pulsing; (ii) perforation sites with temporal peak area <30 μm(2) were resealed successfully; (iii) during recovery, a thickened pore rim emerged, and its temporal progression corresponded with the pore closure action; (iv) membrane resealing, if successful, would generally be completed within 1 min of the onset of sonoporation, and the resealing time constant was estimated to be below 20 s; (v) membrane resealing would fail for overly large pores (>100 μm(2)) or in the absence of extracellular calcium ions. These findings serve to underscore the spatiotemporal complexity of membrane-level dynamics in sonoporation.
Keywords: Membrane perforation; Membrane recovery; Real-time confocal imaging; Sonoporation; Spatiotemporal dynamics; Surface topography.
Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.