Targeted cell immobilization by ultrasound microbeam

Abstract

Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two-dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K-562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two-dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator.

Figure 1

Ray acoustics model of acoustic trapping. Two parallel rays (P1 and P2) are incident upon a sphere in a sound field of Gaussian intensity distribution, and their corresponding exiting rays are denoted as P1′ and P2′. The momentum transfers from the rays to the sphere are defined by dP1 and dP2, and redirect the sound propagation at the sphere. Resultant forces acted on the rays are determined by the rate of the momentum change. According to Newton’s third law, the sphere experiences an equal and opposite force that leads to an acoustic radiation force (a dotted arrow). Consequently the sphere is attracted to the beam axis. A thick arrow at the sphere’s center indicates the trapping direction.

Figure 2

Fabricated ZnO transducer (by Fraunhofer IBMT). (A) Schematic structure and (B) Its photograph.

Figure 3

Pulse echo response and lateral beam profile of ZnO transducer (by Fraunhofer IBMT). (A) A received RF waveform (solid line) and its frequency spectrum (solid-dotted line) show the resonant peak at 202 MHz. (B) The measured beam width was 9.5 μm.

Figure 4

Cell immobilization system. (A) Schematic diagram. (B) Experimental setup. (C) View of the measurement chamber along with the transducer.

Figure 5

Test procedure for lateral cell displacement.

Figure 6

Example of cell motion within acoustic trap (×10 objective). The cell location was recorded every 3 seconds. (A) The cell (see an arrow in the middle) is initially positioned at 50 μm from the trap center and (B) immediately directed to it when the transducer is excited. (C) After migrating over the focal plane, (D) the cell becomes finally settled at the focus. A dot is given as a reference point to show the change in the cell location.

Figure 7

Trajectory of trapped cell as a function of image sequence. (A) Horizontal and (B) vertical coordinates are represented with respect to the trap center located at the origin. (C) Thick arrows indicate the moving direction of the cell whose position converged to the focus. (D) The increment of the vertical displacement is found until frame #8 and then decreased afterwards. This illustrates a spring-like pattern when it is released after being stretched beyond the equilibrium point (reviewer #2).