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1. FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound

   https://doi.org/10.3390/s20174998  

   Yoon, Sangpil; Pan, Yijia; Shung, Kirk; Wang, Yingxiao (September 2020, Sensors)

   null (Ed.)

   Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10–15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU. 

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2. A rapid, spatially dispersive frequency comb spectrograph aimed at gas phase chemical reaction kinetics

   https://doi.org/10.1080/00268976.2020.1733116  

   Roberts, Frances C.; Lewandowski, H. J.; Hobson, Billy F.; Lehman, Julia H. (February 2020, Molecular Physics)

   This New Views article will highlight some recent advances in high sensitivity gas detection using direct infrared absorption frequency comb laser spectroscopy, with a focus on frequency comb use in chemical reaction kinetics and our own contribution to this field. Our recently implemented detection technique uses a combination of a 12.9 GHz free spectral range virtually imaged phased array and diffraction grating to spatially disperse the mid-infrared frequency comb onto a camera. Individual frequencies or ‘comb teeth’ of a 250 MHz repetition-rate frequency comb are able to be resolved. High molecular sensitivity is achieved by increasing the interaction path length using a Herriott multipass cell. High spectral resolution, broadband spectral coverage, and high molecular sensitivity are all achieved on an adjustable 1–50 µs timescale, making this frequency comb apparatus ideal for measuring chemical reaction kinetics where multiple absorbing species can be monitored simultaneously. This New Views article will also discuss some of the challenges and decisions that chemists might face in implementing this advanced physics technology in their own laboratory. Spatially dispersed 250 MHz mid-infrared frequency comb laser, with absorption of some frequencies by a dilute sample of methane. KEYWORDS: Frequency combs, chemical kinetics, trace gas detection 

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3. Towards High-Resolution Ultrasound Neuromodulation With Crossed-Beam Phased Arrays

   https://doi.org/10.1109/TBCAS.2023.3285724  

   Ilham, Sheikh Jawad; Kiani, Mehdi (June 2023, IEEE Transactions on Biomedical Circuits and Systems)

   Transcranial focused ultrasound stimulation (tFUS) has emerged as a potential noninvasive therapeutic technology. Due to skull attenuations at high ultrasound (US) frequencies, successful tFUS with sufficient penetration depth requires sub-MHz US waves, leading to relatively poor stimulation specificity particularly in the axial direction (perpendicular to the US transducer). This shortcoming can potentially be overcome by utilizing two individual US beams properly crossed in time and space. For large-scale tFUS, a phased array is also required to dynamically steer focused US beams at desired neural targets. This article presents the theoretical foundation and optimization (through a wave-propagation simulator) of crossed-beam formation using two US phased arrays. 

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4. Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers

   https://doi.org/10.1007/s12195-019-00578-z  

   Qian, Weiyi; Chen, Weiqiang (January 2019, Cellular and Molecular Bioengineering)

   Introduction— In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an ‘ultrasound tweezers’-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed “actinin-sstFRET,” to monitor in situ single-cell allostasis in response to transient stimulation in real time. Methods— The ultrasound tweezers utilize 1 Hz, 10-second transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis. Results—By subjecting a 1 Hz, 10-second physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 minutes that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation. Conclusions— By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging. 

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5. Cold Sintering of PZT 2-2 Composites for High Frequency Ultrasound Transducer Arrays

   https://doi.org/10.3390/act10090235  

   Gupta, Shruti; Wang, Dixiong; Shetty, Smitha; Meddeb, Amira; Dursun, Sinan; Randall, Clive A.; Trolier-McKinstry, Susan (September 2021, Actuators)

   Medical ultrasound and other devices that require transducer arrays are difficult to manufacture, particularly for high frequency devices (>30 MHz). To enable focusing and beam steering, it is necessary to reduce the center-to-center element spacing to half of the acoustic wavelength. Conventional methodologies prevent co-sintering ceramic–polymer composites due to the low decomposition temperatures of the polymer. Moreover, for ultrasound transducer arrays exceeding 30 MHz, methods such as dice-and-fill cannot provide the dimensional tolerances required. Other techniques in which the ceramic is formed in the green state often fail to retain the required dimensions without distortion on firing the ceramic. This paper explores the use of the cold sintering process to produce dense lead zirconate titanate (PZT) ceramics for application in high frequency transducer arrays. PZT–polymer 2-2 composites were fabricated by cold sintering tape cast PZT with Pb nitrate as a sintering aid and ZnO as the sacrificial layer. PZT beams of 35 μm width with ~5.4 μm kerfs were produced by this technique. The ZnO sacrificial layer was also found to serve as a liquid phase sintering aid that led to grain growth in adjacent PZT. This composite produced resonance frequencies of >17 MHz. 

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