Cytomechanical perturbations during low intensity ultrasound pulsing

Abstract

To establish the therapeutic potential of low-intensity ultrasound, it is fundamentally important to characterize its biophysical interactions with living cells. Here, through a series of single-cell direct observations, we show that low-intensity ultrasound pulsing would give rise to a dynamic course of cytomechanical perturbations at both the membrane and nucleus levels. Our investigation was conducted using a composite platform that coupled a 1 MHz ultrasound exposure hardware to a confocal microscopy system. Short ultrasound pulses (5 cycles; 2 kHz pulse repetition frequency) with 0.24 W/cm2 spatial-peak, time-averaged intensity (0.85 MPa peak positive acoustic pressure) were delivered over a 10 min period to adherent Neuro-2a neuroblastoma cells, and live imaging of cellular dynamics was performed before, during, and after the exposure period. Bright-field imaging results showed progressive shrinkage of cellular cross-sectional area (25-45%; N = 7) during low-intensity ultrasound pulsing; the initial rate of size decrease was estimated to be 8-14% per min. This shrinkage was found to be transient, as the sonicated cells had recovered (at a rate of size increase of 0.4-0.9% per min) to their pre-exposure size within 30 min after the end of exposure. 3-D confocal imaging results further revealed that: (i) ultrasound-induced membrane contraction was volumetric in nature (21-45% reduction); (ii) concomitant decrease in nucleus volume was evident (12-25% reduction). Altogether, these findings demonstrate that low-intensity ultrasound pulsing, if applied on the order of minutes, would reversibly perturb the physical and subcellular structures of living cells.