Significance: There is a lack of a general method to noninvasively and remotely manipulate cells with high spatiotemporal precisions. We developed an ultrasound-based mechanogenetics system to achieve this goal. Cells were engineered with the mechanosensor Piezo1 and genetic transducing modules to perceive the mechanical perturbation generated by the ultrasound wave and transduce it into genetic activities. Mechanosensitive and ultrasound-controllable T cells were further engineered to target and eradicate tumor cells with inducible chimeric antigen receptors. This mechanogenetics approach can be extended to remotely control, in principle, any gene activity in live cells for the reprogramming of cellular functions. This method should also provide a general approach to remotely control molecular functions for biological studies and clinical applications, particularly cell-based cancer immunotherapy.

Single-cell analysis is essential to understand the physical and functional characteristics of cells. The basic knowledge of these characteristics is important to elucidate the unique features of various cells and causative factors of diseases and determine the most effective treatments for diseases. Recently, acoustic tweezers based on tightly focused ultrasound microbeam have attracted considerable attention owing to their capability to grab and separate a single cell from a heterogeneous cell sample and to measure its physical cell properties. However, the measurement cannot be performed while trapping the target cell, because the current method uses long ultrasound pulses for grabbing one cell and short pulses for interrogating the target cell. In this paper, we demonstrate that short ultrasound pulses can be used for generating acoustic trapping force comparable to that with long pulses by adjusting the pulse repetition frequency (PRF). This enables us to capture a single cell and measure its physical properties simultaneously. Furthermore, it is shown that short ultrasound pulses at a PRF of 167 kHz can trap and separate either one red blood cell or one prostate cancer cell and facilitate the simultaneous measurement of its integrated backscattering coefficient related to the cell size and mechanical properties.

Goal: Typical SRUS images are reconstructed by localizing ultrasound microbubbles (MBs) injected in a vessel using normalized 2-dimensional cross-correlation (2DCC) between MBs signals and the point spread function of the system. However, current techniques require isolated MBs in a confined area due to inaccurate localization of densely populated MBs. To overcome this limitation, we developed the ℓ1-homotopy based compressed sensing (L1H-CS) based SRUS imaging technique which localizes densely populated MBs to visualize microvasculature in vivo. Methods: To evaluate the performance of L1H-CS, we compared the performance of 2DCC, interior-point method based compressed sensing (CVX-CS), and L1H-CS algorithms. Localization efficiency was compared using axially and laterally aligned point targets (PTs) with known distances and randomly distributed PTs generated by simulation. We developed post-processing techniques including clutter reduction, noise equalization, motion compensation, and spatiotemporal noise filtering for in vivo imaging. We then validated the capabilities of L1H-CS based SRUS imaging technique with high-density MBs in a mouse tumor model, kidney, and zebrafish dorsal trunk, and brain. Results: Compared to 2DCC and CVX-CS algorithms, L1H-CS achieved faster data acquisition time and considerable improvement in SRUS image quality. Conclusions and Significance: These results demonstrate that the L1H-CS based SRUS imaging technique has the potential to examine microvasculature with reduced acquisition and reconstruction time to acquire enhanced SRUS image quality, which may be necessary to translate it into clinics.

A high pulse repetition frequency ultrasound system for an ex vivo measurement of mechanical properties of an animal crystalline lens was developed and validated. We measured the bulk displacement of laser-induced microbubbles created at different positions within the lens using nanosecond laser pulses. An impulsive acoustic radiation force was applied to the microbubble, and spatio-temporal measurements of the microbubble displacement were assessed using a custom-made high pulse repetition frequency ultrasound system consisting of two 25 MHz focused ultrasound transducers. One of these transducers was used to emit a train of ultrasound pulses and another transducer was used to receive the ultrasound echoes reflected from the microbubble. The developed system was operating at 1 MHz pulse repetition frequency. Based on the measured motion of the microbubble, Young's moduli of surrounding tissue were reconstructed and the values were compared with those measured using the indentation test. Measured values of Young's moduli of four bovine lenses ranged from 2.6 ± 0.1 to 26 ± 1.4 kPa, and there was good agreement between the two methods. Therefore, our studies, utilizing the high pulse repetition frequency ultrasound system, suggest that the developed approach can be used to assess the mechanical properties of ex vivo crystalline lenses. Furthermore, the potential of the presented approach for in vivo measurements is discussed.

Even though ultrahigh frequency ultrasonic transducers over 60 MHz have been used for single-cell-level manipulation such as intracellular delivery, acoustic tweezers, and stimulation to investigate cell phenotype and cell mechanics, no techniques have been available to measure the actual acoustic radiation force (ARF) applied to target cells. Therefore, we have developed an approach to measure the ARF of ultrahigh frequency ultrasonic transducers using a theoretical model of the dynamics of a solid sphere in a gelatin phantom. To estimate ARF at the focus of a 130 MHz transducer, we matched measured maximum displacements of a solid sphere with theoretical calculations. We selected appropriate ranges of input voltages and pulse durations for single-cell applications, and the estimated ARF was in the range of tens of μN. To gauge the influence of pulse duration, an impulse of different pulse durations was estimated. Fluorescence resonance energy transfer live cell imaging was demonstrated to visualize calcium transport between cells after a target single cell was stimulated by the developed ultrasonic transducer.