Ultrasound Microbeam Research Articles

Recent Advancements in High-Frequency Ultrasound Applications from Imaging to Microbeam Stimulation.

Ultrasound is a versatile and well-established technique using sound waves with frequencies higher than the upper limit of human hearing. Typically, therapeutic and diagnosis ultrasound operate in the frequency range of 500 kHz to 15 MHz with greater depth of penetration into the body. However, to achieve improved spatial resolution, high-frequency ultrasound (>15 MHz) was recently introduced and has shown promise in various fields such as high-resolution imaging for the morphological features of the eye and skin as well as small animal imaging for drug and gene therapy. In addition, high-frequency ultrasound microbeam stimulation has been demonstrated to manipulate single cells or microparticles for the elucidation of physical and functional characteristics of cells with minimal effect on normal cell physiology and activity. Furthermore, integrating machine learning with high-frequency ultrasound enhances diagnostics, including cell classification, cell deformability estimation, and the diagnosis of diabetes and dysnatremia using convolutional neural networks (CNNs). In this paper, current efforts in the use of high-frequency ultrasound from imaging to stimulation as well as the integration of deep learning are reviewed, and potential biomedical and cellular applications are discussed.

Non-Contact High-Frequency Ultrasound Microbeam Stimulation: A Novel Finding and Potential Causes of Cell Responses.

Invasiveness research is an essential step in breast cancer metastasis. The application of high-frequency ultrasound microbeam stimulation (HFUMS) offers a manner of determining the invasion potential of human breast cancer cells by eliciting the elevation of transient cytoplasmic calcium ions (Ca2+). The fluorescent index (FI), which is a composite parameter reflecting calcium elevations elicited by HFUMS, was shown to be higher in invasive breast cancer cells (MDA-MB-231) compared to weakly invasive breast cancer cells (MCF-7) using the low-intensity 50-MHz HFUMS. This novel finding shows significant difference from the reported studies in which MCF-7 cells showed no response to HFUMS. In addition to the negligible response of normal human breast cells (MCF-10A), HFUMS shows the potential to be capable of differentiating the normal cells from the cancer cells. To understand the mechanism of HFUMS worked on mechanotransduction in cells, different channel blockers were used to investigate the roles of specific channels during HFUMS. It was found that GsMTx4 (30 μM), a selective blocker of mechanosensitive Piezo channels, reduces the FI values significantly in MDA-MB-231 cells, while SKF-96365 (40 μM), a general TRP channel blocker, cannot induce the significant inhibition of FI values. The results indicate that Piezo channels may play the main role in invasion and metastatic propagation of cells.

Label-free analysis of the characteristics of a single cell trapped by acoustic tweezers

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.

Ultrasonic scattering measurements of a live single cell at 86 MHz.

Cell separation and sorting techniques have been employed biomedical applications such as cancer diagnosis and cell gene expression analysis. The capability to accurately measure ultrasonic scattering properties from cells is crucial in making an ultrasonic cell sorter a reality if ultrasound scattering is to be used as the sensing mechanism as well. To assess the performance of sensing and identifying live single cells with high-frequency ultrasound, an 86-MHz lithium niobate press-focused single-element acoustic transducer was used in a high-frequency ultrasound scattering measurement system that was custom designed and developed for minimizing noise and allowing better mobility. Peak-to-peak echo amplitude, integrated backscatter (IB) coefficient, spectral parameters including spectral slope and intercept, and midband fit from spectral analysis of the backscattered echoes were measured and calculated from a live single cell of two different types on an agar surface: leukemia cells (K562 cells) and red blood cells (RBCs). The amplitudes of echo signals from K562 cells and RBCs were 48.25 ± 11.98 mV(pp) and 56.97 ± 7.53 mV(pp), respectively. The IB coefficient was -89.39 ± 2.44 dB for K562 cells and -89.00 ± 1.19 dB for RBCs. The spectral slope and intercept were 0.30 ± 0.19 dB/MHz and -56.07 ± 17.17 dB, respectively, for K562 cells and 0.78 ± 0.092 dB/MHz and -98.18 ± 8.80 dB, respectively, for RBCs. Midband fits of K562 cells and RBCs were -31.02 ± 3.04 dB and -33.51 ± 1.55 dB, respectively. Acoustic cellular discrimination via these parameters was tested by Student's t-test. Their values, except for the IB value, showed statistically significant difference (p < 0.001). This paper reports for the first time that ultrasonic scattering measurements can be made on a live single cell with a highly focused high-frequency ultrasound microbeam at 86 MHz. These results also suggest the feasibility of ultrasonic scattering as a sensing mechanism in the development of ultrasonic cell sorters.

Non-contact High-Frequency Ultrasound Microbeam Stimulation for Studying Mechanotransduction in Human Umbilical Vein Endothelial Cells

We describe how contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca2+) elevation in human umbilical vein endothelial cells. The cellular mechanotransduction process, which includes cell sensing and adaptation to the mechanical micro-environment, has been studied extensively in recent years. A variety of tools for mechanical stimulation have been developed to produce cellular responses. We developed a novel tool, a highly focused ultrasound microbeam, for non-contact cell stimulation at a microscale. This tool, at 200 MHz, was applied to human umbilical vein endothelial cells to investigate its potential to elicit an elevation in cytoplasmic Ca2+ levels. It was found that the response was dose dependent, and moreover, extracellular Ca2+ and cytoplasmic Ca2+ stores were involved in the Ca2+ elevation. These results suggest that high-frequency ultrasound microbeam stimulation is potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequencies can be quantified.

Non-contact multi-particle annular patterning and manipulation with ultrasound microbeam

Multiparticle-trapping offers diverse opportunities and applications in biotechnology. It can be applied to creating various functional materials or organizing reactive particles. In this paper, we demonstrate that it is possible to trap and manipulate multi-particles in an annular pattern with a 24 MHz focused ring-type single element ultrasound transducer. Acoustic ring trap can be useful in undertaking biotropism studies due to an equal-distance condition from the center. Also, this ring trap could serve as a force shield to protect analysis area from other cells. The experimental results showed the capability of the proposed method as a multi-cell manipulator in formatting specific patterns of small cells like sperms.

Investigating contactless high frequency ultrasound microbeam stimulation for determination of invasion potential of breast cancer cells

In this article, we investigate the application of contactless high frequency ultrasound microbeam stimulation (HFUMS) for determining the invasion potential of breast cancer cells. In breast cancer patients, the finding of tumor metastasis significantly worsens the clinical prognosis. Thus, early determination of the potential of a tumor for invasion and metastasis would significantly impact decisions about aggressiveness of cancer treatment. Recent work suggests that invasive breast cancer cells (MDA-MB-231), but not weakly invasive breast cancer cells (MCF-7, SKBR3, and BT-474), display a number of neuronal characteristics, including expression of voltage-gated sodium channels. Since sodium channels are often co-expressed with calcium channels, this prompted us to test whether single-cell stimulation by a highly focused ultrasound microbeam would trigger Ca(2+) elevation, especially in highly invasive breast cancer cells. To calibrate the diameter of the microbeam ultrasound produced by a 200-MHz single element LiNbO3 transducer, we focused the beam on a wire target and performed a pulse-echo test. The width of the beam was ∼17 µm, appropriate for single cell stimulation. Membrane-permeant fluorescent Ca(2+) indicators were utilized to monitor Ca(2+) changes in the cells due to HFUMS. The cell response index (CRI), which is a composite parameter reflecting both Ca(2+) elevation and the fraction of responding cells elicited by HFUMS, was much greater in highly invasive breast cancer cells than in the weakly invasive breast cancer cells. The CRI of MDA-MB-231 cells depended on peak-to-peak amplitude of the voltage driving the transducer. These results suggest that HFUMS may serve as a novel tool to determine the invasion potential of breast cancer cells, and with further refinement may offer a rapid test for invasiveness of tumor biopsies in situ.

Ultrahigh frequency lensless ultrasonic transducers for acoustic tweezers application

Similar to optical tweezers, a tightly focused ultrasound microbeam is needed to manipulate microparticles in acoustic tweezers. The development of highly sensitive ultrahigh frequency ultrasonic transducers is crucial for trapping particles or cells with a size of a few microns. As an extra lens would cause excessive attenuation at ultrahigh frequencies, two types of 200-MHz lensless transducer design were developed as an ultrasound microbeam device for acoustic tweezers application. Lithium niobate single crystal press-focused (PF) transducer and zinc oxide self-focused transducer were designed, fabricated and characterized. Tightly focused acoustic beams produced by these transducers were shown to be capable of manipulating single microspheres as small as 5 µm two-dimensionally within a range of hundreds of micrometers in distilled water. The size of the trapped microspheres is the smallest ever reported in the literature of acoustic PF devices. These results suggest that these lensless ultrahigh frequency ultrasonic transducers are capable of manipulating particles at the cellular level and that acoustic tweezers may be a useful tool to manipulate a single cell or molecule for a wide range of biomedical applications.

Ultrahigh frequency ultrasonic transducers

Acoustic microscopy and ultrahigh frequency (UHF) ultrasound microbeam applications require high performance single element transducers from a few hundred MHz to a few GHz. Design and fabrication of such transducers differ drastically from lower frequency transducers. A top- down approach i.e. lapping, grinding and polishing a bulk piezoelectric material to the desirable thickness no longer works. Instead a bottom-up approach i.e., depositing thin layers of a piezoelectric material is preferred. Although ZnO has been the piezoelectric material of choice for many years because it can be easily sputtered in thin films onto a substrate, it however suffers from a low electromechanical coupling coefficient. Better design and piezoelectric materials are sought to enhance the performance of UHF transducers. Thin film PZT, PMN-PT and other novel materials have been shown to offer superior performance. Lensless design may be an alternative to that used in conventional acoustic microscopic transducers.

Fluorescence response of human HER2+ cancer- and MCF-12F normal cells to 200 MHz ultrasound microbeam stimulation: A preliminary study of membrane permeability variation

Targeted mechanical cell stimulation has been extensively studied for a better understanding of its effect on cellular mechanotransduction signaling pathways and structures by utilizing a variety of mechanical sources. In this work, an ultrasound-driven single cell stimulation method is thus proposed, and a preliminary study is carried out by comparing the fluorescence intensities representing a change in cell membrane permeability between MDA-MB-435 human HER2+ cancer cells (∼40–50μm in diameter) and MCF-12F normal cells (∼50–60μm) in the presence of ultrasound. A 200MHz single element zinc oxide (ZnO) transducer is employed to generate ultrasound microbeam (UM) whose beamwidth and depth of focus are 9.5 and 60μm, comparable to typical cell size. The cells in tetramethyl rhodamine methyl ester (TMRM) are interrogated with 200MHz sinusoidal bursts. The number of cycles per burst is 5 and the pulse repetition frequency (PRF) is 1kHz. The temporal variation of fluorescence intensity in each cell is measured as a function of input voltage to the transducer (16, 32, and 47V), and its corresponding fluorescence images are obtained via a confocal microscope. A systematic method for visualizing UM’s focus by adding Rhodamine B to the immersion medium is also proposed to enhance the precision in aiming the beam at an individual cell.Both types of cells exhibit a decrease in the intensity upon UM irradiation. In particular, normal cells show more fluorescence reduction (down to 0.7 in normalized intensity) than cancer cells (∼0.9) under the same excitation condition of the transducer. With UM being turned off, the normalized intensity level in normal cells is slowly increased to 1.1. The cell images taken before and after UM exposure indicate that the intensity reduction is more pronounced in those cells after exposure. Hence the results show the potential of UM as a non-invasive in vitro stimulation tool for facilitating targeted drug delivery and gene transfection as well as for studying cellular mechanotransduction.

Targeted cell immobilization by ultrasound microbeam

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.