Sangpil Yoon

Impedance matching network for high frequency ultrasonic transducer for cellular applications

An approach for the design of an impedance matching network (IMN) for high frequency ultrasonic transducers with large apertures based on impedance analysis for cellular applications is presented in this paper. The main objectives were to maximize energy transmission from the excitation source to the ultrasonic transducers for cell manipulation and to achieve low input parameters for the safe operation of an ultrasonic transducer because the piezoelectric material in high frequency ultrasonic transducers is prone to breakage due to its being extremely thin. Two ultrasonic transducers, which were made of lithium niobate single crystal with the thickness of 15 μm, having apertures of 4.3 mm (fnumber=1.23) and 2.6mm (fnumber=0.75) were tested. L-type IMN was selected for high sensitivity and compact design of the ultrasonic transducers. The target center frequency was chosen as the frequency where the electrical admittance (|Y|) and phase angle (θz) from impedance analysis was maximal and zero, respectively. The reference center frequency and reference echo magnitude were selected as the center frequency and echo magnitude, measured by pulse-echo testing, of the ultrasonic transducer without IMN. Initial component values and topology of IMN were determined using the Smith chart, and pulse-echo testing was analyzed to verify the performance of the ultrasonic transducers with and without IMN. After several iterations between changing component values and topology of IMN, and pulse-echo measurement of the ultrasonic transducer with IMN, optimized component values and topology of IMN were chosen when the measured center frequency from pulse-echo testing was comparable to the target frequency, and the measured echo magnitude was at least 30% larger than the reference echo magnitude. Performance of an ultrasonic transducer with and without IMN was tested by observing a tangible dent on the surface of a plastic petridish and single cell response after an acoustic pulse was applied on a target cell.

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

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 (Ca(2+)) 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 Ca(2+) levels. It was found that the response was dose dependent, and moreover, extracellular Ca(2+) and cytoplasmic Ca(2+) stores were involved in the Ca(2+) 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.

Dual-element needle transducer for intravascular ultrasound imaging

Abstract. A dual-element needle transducer for intravascular ultrasound imaging has been developed. A low-frequency element and a high-frequency element were integrated into one device to obtain images which conveyed both low- and high-frequency information from a single scan. The low-frequency element with a center frequency of 48 MHz was fabricated from the single crystal form of lead magnesium niobate-lead titanate solid solution with two matching layers (MLs) and the high frequency element with a center frequency of 152 MHz was fabricated from lithium niobate with one ML. The measured axial and lateral resolutions were 27 and 122  μm, respectively, for the low-frequency element, and 14 and 40  μm, respectively, for the high-frequency element. The performance of the dual-element needle transducer was validated by imaging a tissue-mimicking phantom with lesion-mimicking area, and ex vivo rabbit aortas in water and rabbit whole blood. The results suggest that a low-frequency element effectively provides depth resolved images of the whole vessel and its adjacent tissue, and a high-frequency element visualizes detailed structure near the surface of the lumen wall in the presence of blood within the lumen. The advantages of a dual-element approach for intravascular imaging are also discussed.

Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells

Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities.

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.

Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound

Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.

Mechanogenetics for the remote and noninvasive control of cancer immunotherapy

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. While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

Investigation of Optimized Treatment Conditions for Acoustic-Transfection Technique for Intracellular Delivery of Macromolecules

Manipulation of cellular functions and structures by introduction of genetic materials inside cells has been one of the most prominent research areas in biomedicine. High-frequency ultrasound acoustic-transfection has recently been developed and confirmed by intracellular delivery of small molecules into HeLa cells at the single-cell level with high cell viability. After we proved the concept underlying the acoustic-transfection technique, treatment conditions for different human cancer cell lines have been intensively investigated to further develop acoustic-transfection as a versatile and adaptable transfection method by satisfying the requirements of high-delivery efficiency and cell membrane permeability with minimal membrane disruption. To determine optimal treatment conditions for different cell lines, we developed a quantitative intracellular delivery score based on delivery efficiency, cell membrane permeability and cell viability after 4 and 20 h of treatment. The intracellular delivery of macromolecules and the simultaneous intracellular delivery of two molecules under optimal treatment conditions were successfully achieved. We found that DNA plasmid was delivered by acoustic-transfection technique into epiblast stem cells, which expressed transient mCherry fluorescence.

Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.