Ultrasonic Healing of Plastrons.

Superhydrophobic surfaces (SHS) exhibit a pronounced ability to resist wetting. When immersed in water, water does not penetrate between the microstructures of the SHS. Instead, a thin layer of trapped gas remains, i.e., plastron. This fractional wetting is also known as the Cassie-Baxter state (CB). Impairment of superhydrophobicity occurs when water penetrates the plastron and, when complete wetting is achieved, a Wenzel state (W) results. Subsequent recovery back to CB state is one of the main challenges in the field of SHS wetting. Current methods for plastron recovery require complex mechanical or chemical integration, are time-consuming or lack spatial control. Here an on-demand, contact-less approach for performing facile transitions between these wetting states at micrometer length scales is proposed. This is achieved by the use of acoustic radiation force (ARF) produced by high-intensity focused ultrasound (HIFU). Switching from CB to W state takes <100 µs, while the local recovery back to CB state takes <45 s. To the best of authors knowledge, this is the first demonstration of ARF-induced manipulation of the plastron enabling facile two-way controlled switching of wetting states.

Development of Focused Ultrasound-Assisted Nanoplexes for RNA Delivery.

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 µL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100-200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.

Ultrasound-Enhanced Fine-Needle Biopsy Improves Yield in Human Epithelial and Lymphoid Tissue.

OBJECTIVE: Needle biopsy is a common technique used to obtain cell and tissue samples for diagnostics. Currently, two biopsy methods are widely used: (i) fine-needle aspiration biopsy (FNAB) and (ii) core needle biopsy (CNB). However, these methods have limitations. Recently, we developed ultrasound-enhanced fine-needle aspiration biopsy (USeFNAB), which employs a needle that flexurally oscillates at an ultrasonic frequency of ∼32 kHz. The needle motion contributes to increased tissue collection while preserving cells and tissue constructs for pathological assessment. Previously, USeFNAB has been investigated only in ex vivo animal tissue. The present study was aimed at determining the feasibility of using USeFNAB in human epithelial and lymphoid tissue. METHODS: Needle biopsy samples were acquired using FNAB, CNB and USeFNAB on ex vivo human tonsils (N = 10). The tissue yield and quality were quantified by weight measurement and blinded pathologists' assessments. The biopsy methods were then compared. RESULTS: The results revealed sample mass increases of, on average, 2.3- and 5.4-fold with USeFNAB compared with the state-of-the-art FNAB and CNB, respectively. The quality of tissue fragments collected by USeFNAB was equivalent to that collected by the state-of-the-art methods in terms of morphology and immunohistochemical stainings made from cell blocks as judged by pathologists. CONCLUSION: Our study indicates that USeFNAB is a promising method that could improve tissue yield to ensure sufficient material for ancillary histochemical and molecular studies for diagnostic pathology, thereby potentially increasing diagnostic accuracy.

Multi-modal transducer-waveguide construct coupled to a medical needle.

Annually, more than 16 × 109 medical needles are consumed worldwide. However, the functions of the medical needle are still limited mainly to cutting and delivering material to or from a target site. Ultrasound combined with a hypodermic needle could add value to many medical applications, for example, by reducing the penetration force needed during the intervention, adding precision by limiting the needle deflection upon insertion into soft tissues, and even improving tissue collection in fine-needle biopsy applications. In this study, we develop a waveguide construct able to operate a longitudinal-flexural conversion of a wave when transmitted from a Langevin transducer to a conventional medical needle, while maintaining high electric-to-acoustic power efficiency. The optimization of the waveguide structure was realized in silico using the finite element method followed by prototyping the construct and characterizing it experimentally. The experiments conducted at low electrical power consumption (under 5 W) show a 30 kHz flexural needle tip displacement up to 200 µm and 73% electric-to-acoustic power efficiency. This, associated with a small sized transducer, could facilitate the design of ultrasonic medical needles, enabling portability, batterization, and improved electrical safety, for applications such as biopsy, drug and gene delivery, and minimally invasive interventions.

Evaluation of bone growth around bioactive glass S53P4 by scanning acoustic microscopy co-registered with optical interferometry and elemental analysis.

Bioactive glass (BAG) is a bone substitute that can be used in orthopaedic surgery. Following implantation, the BAG is expected to be replaced by bone via bone growth and gradual degradation of the BAG. However, the hydroxyapatite mineral forming on BAG resembles bone mineral, not providing sufficient contrast to distinguish the two in X-ray images. In this study, we co-registered coded-excitation scanning acoustic microscopy (CESAM), scanning white light interferometry (SWLI), and scanning electron microscopy with elemental analysis (Energy Dispersive X-ray Spectroscopy) (SEM-EDX) to investigate the bone growth and BAG reactions on a micron scale in a rabbit bone ex vivo. The acoustic impedance map recorded by the CESAM provides high elasticity-associated contrast to study materials and their combinations, while simultaneously producing a topography map of the sample. The acoustic impedance map correlated with the elemental analysis from SEM-EDX. SWLI also produces a topography map, but with higher resolution than CESAM. The two topography maps (CESAM and SWLI) were in good agreement. Furthermore, using information from both maps simultaneously produced by the CESAM (acoustic impedance and topography) allowed determining regions-of-interest related to bone formation around the BAG with greater ease than from either map alone. CESAM is therefore a promising tool for evaluating the degradation of bone substitutes and the bone healing process ex vivo.

Needle bevel geometry influences the flexural deflection magnitude in ultrasound-enhanced fine-needle biopsy.

It has been recently demonstrated that use of ultrasound increases the tissue yield in ultrasound-enhanced fine-needle aspiration biopsy (USeFNAB) as compared to conventional fine-needle aspiration biopsy (FNAB). To date, the association between bevel geometry and needle tip action has not been widely explored. In this study, we studied the needle resonance characteristics and deflection magnitude of various needle bevel geometries with varying bevel lengths. With a conventional lancet, having a 3.9 mm long bevel, the tip deflection-to-power ratio (DPR) in air and water was 220 and 105 µm/W, respectively. This was higher in comparison to an axi-symmetric tip, having a bevel length of 4 mm, which achieved a DPR of 180 and 80 µm/W in air and water, respectively. This study emphasised the importance of relationship between flexural stiffness of bevel geometry in the context of various insertion media and, thus, could provide understanding on approaches to control post-puncture cutting action by modifying the needle bevel geometry, essential for the USeFNAB application.

An ultrasonically actuated needle promotes the transport of nanoparticles and fluids.

Non-invasive therapeutic ultrasound (US) methods, such as high-intensity focused ultrasound (HIFU), have limited access to tissue targets shadowed by bones or presence of gas. This study demonstrates that an ultrasonically actuated medical needle can be used to translate nanoparticles and fluids under the action of nonlinear phenomena, potentially overcoming some limitations of HIFU. A simulation study was first conducted to study the delivery of a tracer with an ultrasonically actuated needle (33 kHz) inside a porous medium acting as a model for soft tissue. The model was then validated experimentally in different concentrations of agarose gel showing a close match with the experimental results, when diluted soot nanoparticles (diameter < 150 nm) were employed as delivered entity. An additional simulation study demonstrated a threefold increase in the volume covered by the delivered agent in liver under a constant injection rate, when compared to without US. This method, if developed to its full potential, could serve as a cost effective way to improve safety and efficacy of drug therapies by maximizing the concentration of delivered entities within, e.g., a small lesion, while minimizing exposure outside the lesion.

An ultrasonically actuated fine-needle creates cavitation in bovine liver.

Ultrasonic cavitation is being used in medical applications as a way to influence matter, such as tissue or drug vehicles, on a micro-scale. Oscillating or collapsing cavitation bubbles provide transient mechanical force fields, which can, e.g., fractionate soft tissue or even disintegrate solid objects, such as calculi. Our recent study demonstrates that an ultrasonically actuated medical needle can create cavitation phenomena inside water. However, the presence and behavior of cavitation and related bioeffects in diagnostic and therapeutic applications with ultrasonically actuated needles are not known. Using simulations, we demonstrate numerically and experimentally the cavitation phenomena near ultrasonically actuated needles. We define the cavitation onset within a liver tissue model with different total acoustic power levels. We directly visualize and quantitatively characterize cavitation events generated by the ultrasonic needle in thin fresh bovine liver sections enabled by high-speed imaging. On a qualitative basis, the numerical and experimental results show a close resemblance in threshold and spatial distribution of cavitation. These findings are crucial for developing new methods and technologies employing ultrasonically actuated fine needles, such as ultrasound-enhanced fine-needle biopsy, drug delivery, and histotripsy.

Ultrasonic actuation of a fine-needle improves biopsy yield.

Despite the ubiquitous use over the past 150 years, the functions of the current medical needle are facilitated only by mechanical shear and cutting by the needle tip, i.e. the lancet. In this study, we demonstrate how nonlinear ultrasonics (NLU) extends the functionality of the medical needle far beyond its present capability. The NLU actions were found to be localized to the proximity of the needle tip, the SonoLancet, but the effects extend to several millimeters from the physical needle boundary. The observed nonlinear phenomena, transient cavitation, fluid streams, translation of micro- and nanoparticles and atomization, were quantitatively characterized. In the fine-needle biopsy application, the SonoLancet contributed to obtaining tissue cores with an increase in tissue yield by 3-6× in different tissue types compared to conventional needle biopsy technique using the same 21G needle. In conclusion, the SonoLancet could be of interest to several other medical applications, including drug or gene delivery, cell modulation, and minimally invasive surgical procedures.

Modelling aerosol transport and virus exposure with numerical simulations in relation to SARS-CoV-2 transmission by inhalation indoors.

We provide research findings on the physics of aerosol and droplet dispersion relevant to the hypothesized aerosol transmission of SARS-CoV-2 during the current pandemic. We utilize physics-based modeling at different levels of complexity, along with previous literature on coronaviruses, to investigate the possibility of airborne transmission. The previous literature, our 0D-3D simulations by various physics-based models, and theoretical calculations, indicate that the typical size range of speech and cough originated droplets ( d ⩽ 20 µ m ) allows lingering in the air for O ( 1 h ) so that they could be inhaled. Consistent with the previous literature, numerical evidence on the rapid drying process of even large droplets, up to sizes O ( 100 µ m ) , into droplet nuclei/aerosols is provided. Based on the literature and the public media sources, we provide evidence that the individuals, who have been tested positive on COVID-19, could have been exposed to aerosols/droplet nuclei by inhaling them in significant numbers e.g. O ( 100 ) . By 3D scale-resolving computational fluid dynamics (CFD) simulations, we give various examples on the transport and dilution of aerosols ( d ⩽ 20 µ m ) over distances O ( 10 m ) in generic environments. We study susceptible and infected individuals in generic public places by Monte-Carlo modelling. The developed model takes into account the locally varying aerosol concentration levels which the susceptible accumulate via inhalation. The introduced concept, 'exposure time' to virus containing aerosols is proposed to complement the traditional 'safety distance' thinking. We show that the exposure time to inhale O ( 100 ) aerosols could range from O ( 1 s ) to O ( 1 min ) or even to O ( 1 h ) depending on the situation. The Monte-Carlo simulations, along with the theory, provide clear quantitative insight to the exposure time in different public indoor environments.

Non-contact determination of intra-ocular pressure in an ex vivo porcine model.

People suffering from glaucoma often endure high intra-ocular pressure (IOP). Methods for determining IOP either contact the eye or are unpleasant to some patients. There is therefore a need for a rapid and patient friendly non-contacting method to determine IOP. To address this need, we developed a tonometer prototype that employs spark-gap induced shock waves and a laser Doppler vibrometer (LDV) that reads the amplitude of membrane waves. The IOP was first identified from the membrane wave propagation velocity first in a custom-made ocular phantom and was then verified in ex vivo porcine eyes. The time-of-flight (TOF) of the membrane wave travelling on a hemispherical membrane was compared to reference IOP values in the sample obtained with an iCare TA01 tonometer. The shock front was characterized by high speed photography. Within one eye, the method achieved an agreement of 5 mmHg (1.96 standard deviation between the shock wave tonometer and the commercial manometer) and high method-to-method association (Pearson correlation, R2 = 0.98). The results indicate that the presented method could potentially be developed into a non-contacting technique for measuring IOP in vivo.

Localized delivery of compounds into articular cartilage by using high-intensity focused ultrasound.

Localized delivery of drugs into an osteoarthritic cartilaginous lesion does not yet exist, which limits pharmaceutical management of osteoarthritis (OA). High-intensity focused ultrasound (HIFU) provides a means to actuate matter from a distance in a non-destructive way. In this study, we aimed to deliver methylene blue locally into bovine articular cartilage in vitro. HIFU-treated samples (n = 10) were immersed in a methylene blue (MB) solution during sonication (f = 2.16 MHz, peak-positive-pressure = 3.5 MPa, mechanical index = 1.8, pulse repetition frequency = 3.0 kHz, cycles per burst: 50, duty cycle: 7%). Adjacent control 1 tissue (n = 10) was first pre-treated with HIFU followed by immersion into MB; adjacent control 2 tissue (n = 10) was immersed in MB without ultrasound exposure. The MB content was higher (p < 0.05) in HIFU-treated samples all the way to a depth of 600 µm from AC surface when compared to controls. Chondrocyte viability and RNA expression levels associated with cartilage degeneration were not different in HIFU-treated samples when compared to controls (p > 0.05). To conclude, HIFU delivers molecules into articular cartilage without major short-term concerns about safety. The method is a candidate for a future approach for managing OA.

Comparison of Traditional and Ultrasound-Enhanced Electrospinning in Fabricating Nanofibrous Drug Delivery Systems.

We investigated nozzleless ultrasound-enhanced electrospinning (USES) as means to generate nanofibrous drug delivery systems (DDSs) for pharmaceutical and biomedical applications. Traditional electrospinning (TES) equipped with a conventional spinneret was used as a reference method. High-molecular polyethylene oxide (PEO) and chitosan were used as carrier polymers and theophylline anhydrate as a water-soluble model drug. The nanofibers were electrospun with the diluted mixture (7:3) of aqueous acetic acid (90% v/v) and formic acid solution (90% v/v) (with a total solid content of 3% w/v). The fiber diameter and morphology of the nanofibrous DDSs were modulated by varying ultrasonic parameters in the USES process (i.e., frequency, pulse repetition frequency and cycles per pulse). We found that the USES technology produced nanofibers with higher fiber diameter (402 ± 127 nm) than TES (77 ± 21 nm). An increase of a burst count in USES increased the fiber diameter (555 ± 265 nm) and the variation in fiber size. The slight-to-moderate changes in a solid state (crystallinity) were detected when compared the nanofibers generated by TES and USES. In conclusion, USES provides a promising alternative for aqueous-based fabrication of nanofibrous DDSs for pharmaceutical and biomedical applications.

Needle Biopsy Adequacy in the Era of Precision Medicine and Value-Based Health Care.

CONTEXT.­: Needle biopsy of diseased tissue is an essential diagnostic tool that is becoming even more important as precision medicine develops. However, the capability of this modality to efficiently provide samples adequate for diagnostic and prognostic analysis remains quite limited relative to current diagnostic needs. For physicians and patients, inadequate biopsy frequently leads to diagnostic delay, procedure duplication, or insufficient information about tumor biology leading to delay in treatment; for health systems, this results in substantial incremental costs and inefficient use of scarce specialized diagnostic resources. OBJECTIVE.­: To review current needle biopsy technology, devices, and practice with a perspective to identify current limitations and opportunities for improvement in the context of advancing precision medicine. DATA SOURCES.­: PubMed searches of fine-needle aspiration and core needle biopsy devices and similar technologies were made generally, by tissue site, and by adequacy as well as by health economics of these technologies. CONCLUSIONS.­: Needle biopsy adequacy can be improved by recognizing the importance of this diagnostic tool by promoting common criteria for needle biopsy adequacy; by optimizing needle biopsy procedural technique, technologies, clinical practice, professional education, and quality assurance; and by bundling biopsy procedure costs with downstream diagnostic modalities to provide better accountability and incentives to improve the diagnostic process.

Practical realization of a sub-λ/2 acoustic jet.

Studies in optics and acoustics have employed metamaterial lenses to achieve sub-wavelength localization, e.g. a recently introduced concept called 'acoustojet' which in simulations localizes acoustic energy to a spot smaller than λ/2. However previous experimental results on the acoustojet have barely reached λ/2-wide localization. Here we show, by simulations and experiments, that a sub-λ/2 wide localization can be achieved by translating the concept of a photonic jet into the acoustic realm. We performed nano- to macroscale molecular dynamics (MD) and finite element method (FEM) simulations as well as macroscale experiments. We demonstrated that by choosing a suitable size cylindrical lens, and by selecting the speed-of-sound ratio between the lens material(s) and the surrounding medium, an acoustic jet ('acoustic sheet') is formed with a full width at half maximum (FWHM) less than λ/2. The results show, that the acoustojet approach can be experimentally realized with easy-to-manufacture acoustic lenses at the macroscale. MD simulations demonstrate that the concept can be extended to coherent phonons at nanoscale. Finally, our FEM simulations identify some micrometer size structures that could be realized in practice. Our results may contribute to starting a new era of super resolution acoustic imaging: We foresee that jet generating constructs can be readily manufactured, since suitable material combinations can be found from nanoscale to macroscale. Tight focusing of mechanical energy is highly desirable in e.g. electronics, materials science, medicine, biosciences, and energy harvesting.

Quantifying Complex Micro-Topography of Degenerated Articular Cartilage Surface by Contrast-Enhanced Micro-Computed Tomography and Parametric Analyses.

One of the earliest changes in osteoarthritis (OA) is a surface discontinuity of the articular cartilage (AC), and these surface changes become gradually more complex with OA progression. We recently developed a contrast enhanced micro-computed tomography (µCT) method for visualizing AC surface in detail. The present study aims to introduce a µCT analysis technique to parameterize these complex AC surface features and to demonstrate the feasibility of using these parameters to quantify degenerated AC surface. Osteochondral plugs (n = 35) extracted from 19 patients undergoing joint surgery were stained with phosphotungstic acid and imaged using µCT. The surface micro-topography of AC was analyzed with developed method. Standard root mean square roughness (Rq ) was calculated as a reference, and the Area Under Curve (AUC) for receiver operating characteristic analysis was used to compare the acquired quantitative parameters with semi-quantitative visual grading of µCT image stacks. The parameters quantifying the complex micro-topography of AC surface exhibited good sensitivity and specificity in identifying surface continuity (AUC: 0.93, [0.80 0.99]), fissures (AUC: 0.94, [0.83 0.99]) and fibrillation (AUC: 0.98, [0.88 1.0]). Standard Rq was significantly smaller compared with the complex roughness (CRq ) already with mild surface changes with all surface reference parameters - continuity, fibrillation, and fissure sum. Furthermore, only CRq showed a significant difference when comparing the intact surface with lowest fissure sum score. These results indicate that the presented method for evaluating complex AC surfaces exhibit potential to identify early OA changes in superficial AC and is dynamic throughout OA progression. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. Society. 9999:1-12, 2019.

Ultrasound-enhanced electrospinning.

Electrospinning is commonly used to produce polymeric nanofibers. Potential applications for such fibers include novel drug delivery systems, tissue engineering scaffolds, and filters. Electrospinning, however, has shortcomings such as needle clogging and limited ability to control the fiber-properties in a non-chemical manner. This study reports on an orifice-less technique that employs high-intensity focused ultrasound, i.e. ultrasound-enhanced electrospinning. Ultrasound bursts were used to generate a liquid protrusion with a Taylor cone from the surface of a polymer solution of polyethylene oxide. When the polymer was charged with a high negative voltage, nanofibers jetted off from the tip of the protrusion landed on an electrically grounded target held at a constant distance from the tip. Controlling the ultrasound characteristics permitted physical modification of the nanofiber topography at will without using supplemental chemical intervention. Possible applications of tailor-made fibers generated by ultrasound-enhanced electrospinning include pharmaceutical controlled-release applications and biomedical scaffolds with spatial gradients in fiber thickness and mechanical properties.

Laser-ultrasonic delivery of agents into articular cartilage.

Research is ongoing to develop drug therapies to manage osteoarthritis (OA) and articular cartilage (AC) injuries. However, means to deliver drug to localized AC lesions are highly limited and not clinically available. This study investigates the capability of laser ultrasound (laser-induced plasma sound source) to deliver agents (methylene blue, MB, in PBS) into bovine AC. Treatment samples (n = 10) were immersed in MB solution simultaneously with LU exposure, while adjacent control 1 tissue (n = 10) was pre-treated with LU followed by immersion in MB and adjacent control 2 tissue (n = 10) was only immersed in MB. AC exposed (n = 22) or not exposed (n = 27) to LU were characterized for anomalies in structure, composition, viability or RNA expression. Optically detected MB content was significantly (p < 0.01) higher in treatment samples up to a depth of 500 µm from AC surface as compared to controls. No major unwanted short-term effects on AC structure, proteoglycan or collagen contents, chondrocyte viability or RNA expression levels were detected. In conclusion, LU can deliver agents into AC without major short-term concerns on safety. LU could reveal new strategies for the development of localized drug therapies in AC.

Effects of Articular Cartilage Constituents on Phosphotungstic Acid Enhanced Micro-Computed Tomography.

Contrast-enhanced micro-computed tomography (CEµCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEµCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0-13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13-39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEµCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEµCT method for 3D characterization of articular cartilage.

Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos.

Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2-14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development.