Comparative Study of Histotripsy Pulse Parameters Used to Inactivate Escherichia coli in Suspension.

OBJECTIVE: Bacterial loads can be effectively reduced using cavitation-mediated focused ultrasound, or histotripsy. In this study, gram-negative bacteria (Escherichia coli) in suspension were used as model bacteria to evaluate the effectiveness of two regimens of histotripsy treatments: cavitation histotripsy (CH) and boiling histotripsy (BH). METHODS: Ten-milliliter volumes of Escherichia coli were treated at different negative focal pressure amplitudes and over time periods up to 40 min. Cavitation activity was characterized with coaxial passive cavitation detection (PCD) and synchronized plane wave B-mode imaging. RESULTS: CH treatments exhibited a threshold behavior that was consistent with PCD metrics of cavitation. Above the threshold, bacterial inactivation followed a monotonically increasing log-linear relationship that indicated an exponential inactivation rate. BH exhibited no threshold, but instead followed a different monotonically increasing inactivation rate. Inactivation rates were larger for BH at or below the CH threshold, and larger for CH substantially above the threshold. CH studies performed at different pulse lengths at the same duty cycle had similar inactivation rates, suggesting that at any given pressure amplitude, the "on time" was the most important variable for inactivating E. coli. The maximum inactivation was produced by CH at the highest pressure amplitudes used, leading to a log reduction >4.2 for a 40 min treatment. CONCLUSION: The results of this study suggest that both CH and BH can be used to inactivate E. coli in suspension, with the optimal regimen depending on the attainable peak negative focal pressure at the target.

CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Sonographic Features of Abscess Maturation in a Porcine Model.

Abscesses are walled-off collections of infected fluids that often develop as complications in the setting of surgery and trauma. Treatment is usually limited to percutaneous catheterization with a course of antibiotics. As an alternative to current treatment strategies, a histotripsy approach was developed and tested in a novel porcine animal model. The goal of this article is to use advanced ultrasound imaging modes to extract sonographic features associated with the progression of abscess development in a porcine model. Intramuscular or subcutaneous injections of a bi-microbial bacteria mixture plus dextran particles as an irritant led to identifiable abscesses over a 2 to 3 wk period. Selected abscesses were imaged at least weekly with B-mode, 3-D B-mode, shear-wave elastography and plane-wave Doppler imaging. Mature abscesses were characterized by a well-defined core of varying echogenicity surrounded by a hypoechoic capsule that was highly vascularized on Doppler imaging. 3-D imaging demonstrated the natural history of abscess morphology, with the abscess becoming less complex in shape and increasing in volume. Furthermore, shear-wave elastography demonstrated variations in stiffness as phlegmon becomes abscess and then liquefies, over time. These ultrasound features potentially provide biomarkers to aid in selection of treatment strategies for abscesses.

Treating Porcine Abscesses with Histotripsy: A Pilot Study.

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical washout when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

Novel ultrasmall multifunctional nanodots for dual-modal MR/NIR-II imaging-guided photothermal therapy.

Encouraging progress in multifunctional nanotheranostic agents that combine photothermal therapy (PTT) and different imaging modalities has been made. However, rational designed and biocompatible multifunctional agents that suitfable for in vivo application is highly desired but still challenging. In this work, we rationally designed novel ultrasmall multifunctional nanodots (FS-GdNDs) by combining the bovine serum albumin (BSA)-based gadolinium oxide nanodots (GdNDs) obtained through a biomineralization process with a small-molecule NIR-II fluorophore (FS). The as-prepared FS-GdNDs with an ultrasmall hydrodynamic diameter of 9.3 nm exhibited prominent NIR-II fluorescence properties, high longitudinal relaxivity (10.11 mM-1 s-1), and outstanding photothermal conversion efficiency (43.99%) and photothermal stability. In vivo studies showed that the FS-GdNDs with enhanced multifunctional characteristics diaplayed satisfactory dual-modal MR/NIR-II imaging performance with a quite low dose. The imaging-guided PTT achieved successful ablation of tumors and effectively extended the survival of mice. Cytotoxicity studies and histological assay demonstrated excellent biocompatibility of the nanodots. Importantly, this novel FS-GdNDs can undergo efficient body clearance through both hepatobiliary and renal excretion pathways. The novel ultrasmall multifunctional FS-GdNDs with excellent features hold tremendous potential in biomedical and clinical applications.

Effect of Stiffness of Large Extravascular Hematomas on Their Susceptibility to Boiling Histotripsy Liquefaction in Vitro.

Large intra-abdominal, retroperitoneal and intramuscular hematomas are common consequences of sharp and blunt trauma and post-surgical bleeds, and often threaten organ failure, compartment syndrome or spontaneous infection. Current therapy options include surgical evacuation and placement of indwelling drains that are not effective because of the viscosity of the organized hematoma. We have previously reported the feasibility of using boiling histotripsy (BH)-a pulsed high-intensity focused ultrasound method-for liquefaction of large volumes of freshly coagulated blood and subsequent fine-needle aspiration. The goal of this work was to evaluate the changes in stiffness of large coagulated blood volumes with aging and retraction in vitro, and to correlate these changes with the size of the BH void and, therefore, the susceptibility of the material to BH liquefaction. Large-volume (55-200 mL) whole-blood clots were fabricated in plastic molds from human and bovine blood, either by natural clotting or by recalcification of anticoagulated blood, with or without addition of thrombin. Retraction of the clots was achieved by incubation for 3 h, 3 d or 8 d. The shear modulus of the samples was measured with a custom-built indentometer and shear wave elasticity (SWE) imaging. Sizes of single liquefied lesions produced with a 1.5-MHz high-intensity focused ultrasound transducer within a 30-s standard BH exposure served as the metric for susceptibility of clot material to this treatment. Neither the shear moduli of naturally clotted human samples (0.52 ± 0.08 kPa), nor their degree of retraction (ratio of expelled fluid to original volume 50%-58%) depended on the length of incubation within 0-8 d, and were significantly lower than those of bovine samples (2.85 ± 0.17 kPa, retraction 5%-38%). In clots made from anticoagulated bovine blood, the variation of calcium chloride concentration within 5-40 mmol/L did not change the stiffness, whereas lower concentrations and the addition of thrombin resulted in significantly softer clots, similar to naturally clotted human samples. Within the achievable shear modulus range (0.4-1.6 kPa), the width of the BH-liquefied lesion was more affected by the changes in stiffness than the length of the lesion. In all cases, however, the lesions were larger compared with any soft tissue liquefied with the same BH parameters, indicating higher susceptibility of hematomas to BH damage. These results suggest that clotted bovine blood with added thrombin is an acceptable in vitro model of both acute and chronic human hematomas for assessing the efficiency of BH liquefaction strategies.

Recent developments on foaming mechanical and electronic techniques for the management of varicose veins.

Introduction: Varicose veins are a common disease, causing significant impairment of quality of life to afflicted individuals. Conventional surgery has represented the traditional treatment for years, with significant post-operative complications. By the end of the 20th century, novel approaches had been developed to induce biochemical sclerosis into the treated vein in order to exclude it from blood circulation.Areas covered: Foaming techniques for treatment of varicose veins, both clinically-approved methods and those under experimental studies. A brief description of cavitation, which is the basis of microbubbles formation, and an overview of foam properties have been also provided, including a discussion on clinical efficacy and safety profile.Expert commentary: Foam sclerotherapy has rapidly gained popularity since it represents the most minimally invasive and cost-effective procedure in the short term. Several different methods of foam preparation have been described in literature. In general, the foam generation method may affect characteristics such as stability and bubble size distribution, which in turn affect the therapeutic action of foam itself. Therefore, the selection of a suitable foaming technique is of importance for treatment success. Future developments on foaming techniques are expected to make sclerotherapy, already an effective treatment, even safer and more versatile therapeutic procedure.

PIXUL-ChIP: integrated high-throughput sample preparation and analytical platform for epigenetic studies.

Chromatin immunoprecipitation (ChIP) is the most widely used approach for identification of genome-associated proteins and their modifications. We have previously introduced a microplate-based ChIP platform, Matrix ChIP, where the entire ChIP procedure is done on the same plate without sample transfers. Compared to conventional ChIP protocols, the Matrix ChIP assay is faster and has increased throughput. However, even with microplate ChIP assays, sample preparation and chromatin fragmentation (which is required to map genomic locations) remains a major bottleneck. We have developed a novel technology (termed 'PIXUL') utilizing an array of ultrasound transducers for simultaneous shearing of samples in standard 96-well microplates. We integrated PIXUL with Matrix ChIP ('PIXUL-ChIP'), that allows for fast, reproducible, low-cost and high-throughput sample preparation and ChIP analysis of 96 samples (cell culture or tissues) in one day. Further, we demonstrated that chromatin prepared using PIXUL can be used in an existing ChIP-seq workflow. Thus, the high-throughput capacity of PIXUL-ChIP provides the means to carry out ChIP-qPCR or ChIP-seq experiments involving dozens of samples. Given the complexity of epigenetic processes, the use of PIXUL-ChIP will advance our understanding of these processes in health and disease, as well as facilitate screening of epigenetic drugs.

Ultrasound-based cell sorting with microbubbles: A feasibility study.

The isolation and sorting of cells is an important process in research and hospital labs. Most large research and commercial labs incorporate fluorescently or magnetically labeled antibodies adherent to cell surface antigens for cell identification and separation. In this paper, a process is described that merges biochemical labeling with ultrasound-based separation. Instead of lasers and fluorophore tags, or magnets and magnetic particle tags, the technique uses ultrasound and microbubble tags. Streptavidin-labeled microbubbles were mixed with a human acute lymphoblastic leukemia cell line, CCL 119, conjugated with biotinylated anti-CD7 antibodies. Tagged cells were forced under ultrasound, and their displacement and velocity quantified. Differential displacement in a flow stream was quantified against erythrocytes, which showed almost no displacement under ultrasound. A model for the acoustic radiation force on the conjugated pairs compares favorably with observations. This technology may improve on current time-consuming and costly purification procedures.

Inactivation of Planktonic Escherichia coli by Focused 1-MHz Ultrasound Pulses with Shocks: Efficacy and Kinetics Upon Volume Scale-Up.

This study addresses inactivation of E. coli in either 5- or 10-mL volumes, which were 50- to 100-fold greater than used in an earlier study (Brayman et al. 2017). Cells were treated with 1-MHz pulsed high-intensity focused ultrasound (10 cycles, 2-kHz repetition frequency, +65/-12.8 MPa focal pressures). The surviving fraction was assessed by coliform assay, and inactivation demonstrated curvilinear kinetics. The reduction of surviving fraction to 50% required 2.5 or 6 min in 5- or 10-mL samples, respectively. Exposure of 5 mL for 20 min reduced the surviving fraction to ∼1%; a similar exposure of 10-mL samples reduced the surviving fraction to ∼10%. Surviving cells from 5-min exposures appeared normal under light microscopy, with minimal debris; after 20 min, debris dominated. Transmission electron microscopy images of insonated samples showed some undamaged cells, a few damaged but largely intact cells and comminuted debris. Cellular damage associated with substantive but incomplete levels of inactivation can be variable, ranging from membrane holes tens of nanometers in diameter to nearly complete comminution.

Inactivation of Planktonic Escherichia coli by Focused 2-MHz Ultrasound.

This study was motivated by the desire to develop a non-invasive means to treat abscesses, and represents the first steps toward that goal. Non-thermal, high-intensity focused ultrasound (HIFU) was used to inactivate Escherichia coli (∼1 × 109 cells/mL) in suspension. Cells were treated in 96-well culture plate wells using 1.95-MHz ultrasound and incident focal acoustic pressures as high as 16 MPa peak positive and 9.9 MPa peak negative (free field measurements). The surviving fraction was assessed by coliform culture and the alamarBlue assay. No biologically significant heating was associated with ultrasound exposure. Bacterial inactivation kinetics were well described by a half-life model, with a half-time of 1.2 min. At the highest exposure levels, a 2log inactivation was typically achieved within 10 min. The free field-equivalent peak negative acoustic pressure threshold for inactivation was ∼7 MPa. At the highest acoustic pressures used, inactivation efficacy was insensitive to reciprocal changes in pulse length and pulse repetition frequency at constant duty factor. Although treated volumes were very small, proof of principle was provided by these experiments.

Size of Sclerosing Foams Prepared by Ultrasound, Mechanical Agitation, and the Handmade Tessari Method for Treatment of Varicose Veins.

OBJECTIVES: Sclerotherapy is a therapeutic method used in the treatment of varicose veins and works by occluding damaged blood vessels with a chemical solution. Foam sclerotherapy is an attractive treatment because the results are more effective than those obtained by using liquid sclerosants. However, serious neurologic complications, which are likely related to air embolism, have been reported after treatment with foams generated by the handmade method (Tessari technique) most often used clinically. We present an alternative ultrasonic technique for preparation of sclerosing foams to treat varicose veins. METHODS: Three methods of foam generation were compared: ultrasound, mechanical agitation, and Tessari techniques. RESULTS: Optical microscopic analyses showed that low-frequency ultrasound can generate foams with smaller bubble distributions compared to those produced by handmade and mechanical agitation methods: 98% of the bubble population was less than 55 ± 10 µm for sonicated foams (mean ± SD, 19 ± 1.8 µm; maximum bubble size, <138.3 ± 32.5 µm), 196.7 ± 38.2 µm for mechanically agitated foams (mean, 37.1 ± 10.6 µm; maximum bubble size, <350 ± 70.9 µm), and 211.7 ± 20.8 µm for handmade foams (mean, 30.8 ± 3.8 µm; maximum bubble size, <445 ± 32.8 µm). CONCLUSIONS: Low-frequency ultrasonic foam generation yields smaller bubbles and more uniform size distributions than other investigated methods. These properties may reduce serious adverse events reported for sclerotherapy of varicose veins, increasing the safety of foam treatment.

Histotripsy Liquefaction of Large Hematomas.

Intra- and extra-muscular hematomas result from repetitive injury as well as sharp and blunt limb trauma. The clinical consequences can be serious, including debilitating pain and functional deficit. There are currently no short-term treatment options for large hematomas, only lengthy conservative treatment. The goal of this work was to evaluate the feasibility of a high intensity focused ultrasound (HIFU)-based technique, termed histotripsy, for rapid (within a clinically relevant timeframe of 15-20 min) liquefaction of large volume (up to 20 mL) extra-vascular hematomas for subsequent fine-needle aspiration. Experiments were performed using in vitro extravascular hematoma phantoms-fresh bovine blood poured into 50 mL molds and allowed to clot. The resulting phantoms were treated by boiling histotripsy (BH), cavitation histotripsy (CH) or a combination in a degassed water tank under ultrasound guidance. Two different transducers operating at 1 MHz and 1.5 MHz with f-number = 1 were used. The liquefied lysate was aspirated and analyzed by histology and sized in a Coulter Counter. The peak instantaneous power to achieve BH was lower than (at 1.5 MHz) or equal to (at 1 MHz) that which was required to initiate CH. Under the same exposure duration, BH-induced cavities were one and a half to two times larger than the CH-induced cavities, but the CH-induced cavities were more regularly shaped, facilitating easier aspiration. The lysates contained a small amount of debris larger than 70 µm, and 99% of particulates were smaller than 10 µm. A combination treatment of BH (for initial debulking) and CH (for liquefaction of small residual fragments) yielded 20 mL of lysate within 17.5 minutes of treatment and was found to be most optimal for liquefaction of large extravascular hematomas.

Microbubbles and ultrasound increase intraventricular polyplex gene transfer to the brain.

Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits further research.

Stem Cell Labeling with Superparamagnetic Iron Oxide Nanoparticles Using Focused Ultrasound and Magnetic Resonance Imaging Tracking.

Magnetosonoporation (MSP) is a relatively safe and efficient approach for instant MR stem cell labeling. In this study, the physical and magnetic properties of different formulations of synthesized superparamagnetic iron oxide nanoparticles (SPION) were characterized. Then, a "closed" MSP apparatus using focused ultrasound was designed and the feasibility of MSP stem cell labeling using focused ultrasound was validated by evaluating the proliferation, migration and differentiation of the magnetically labeled cells. Subsequently, MSP/SPION labeled neural stem cells (NSCs) were transplanted into the contralateral striatum of glioma-bearing nude mice, and their migration was monitored using magnetic resonance imaging (MRI) in vivo. The results indicated that SPION-1 with the largest size (28.43 ± 9.55 nm) had the highest T2 relaxivity (136.62 Fe mM(-1) S(-1)) and the best MRI contrast effect. Without additional transfection reagents, NSCs were labeled with SPION using focused ultrasound in vitro and the safety of MSP stem cell labeling was validated with the optimized MSP technique. Finally, confirmed by histological evaluation, pronounced signal attenuation on T2-weighted images demonstrated the intracranial tumor tropism of NSCs could be monitored non-invasively by MRI. In conclusion, MSP cell labeling using focused ultrasound is a promising technique and the "closed" device is feasible, convenient and safe for instant magnetic stem cell labeling and MRI cell tracking.

Sono-photoacoustic imaging of gold nanoemulsions: Part I. Exposure thresholds.

Integrating high contrast bubbles from ultrasound imaging with plasmonic absorbers from photoacoustic imaging is investigated. Nanoemulsion beads coated with gold nanopsheres (NEB-GNS) are excited with simultaneous light (transient heat at the GNS's) and ultrasound (rarefactional pressure) resulting in a phase transition achievable under different scenarios, enhancing laser-induced acoustic signals and enabling specific detection of nanoprobes at lower concentration. An automated platform allowed dual parameter scans of both pressure and laser fluence while recording broadband acoustic signals. Two types of NEB-GNS and individual GNS were investigated and showed the great potential of this technique to enhance photoacoustic/acoustic signals. The NEB-GNS size distribution influences vaporization thresholds which can be reached at both permissible ultrasound and light exposures at deep penetration and at low concentrations of targets. This technique, called sono-photoacoustics, has great potential for targeted molecular imaging and therapy using compact nanoprobes with potentially high-penetrability into tissue.

Laser-induced cavitation in nanoemulsion with gold nanospheres for blood clot disruption: in vitro results.

Optically activated cavitation in a nanoemulsion contrast agent is proposed for therapeutic applications. With a 56°C boiling point perfluorohexane core and highly absorptive gold nanospheres at the oil-water interface, cavitation nuclei in the core can be efficiently induced with a laser fluence below medical safety limits (70 mJ/cm2 at 1064 nm). This agent is also sensitive to ultrasound (US) exposure and can induce inertial cavitation at a pressure within the medical diagnostic range. Images from a high-speed camera demonstrate bubble formation in these nanoemulsions. The potential of using this contrast agent for blood clot disruption is demonstrated in an in vitro study. The possibility of simultaneous laser and US excitation to reduce the cavitation threshold for therapeutic applications is also discussed.

Nonlinear contrast enhancement in photoacoustic molecular imaging with gold nanosphere encapsulated nanoemulsions.

A composite contrast agent, a nanoemulsion bead with assembled gold nanospheres at the interface, is proposed to improve the specific contrast of photoacoustic molecular imaging. A phase transition in the bead's core is induced by absorption of a nanosecond laser pulse with a fairly low laser fluence (∼3.5 mJ/cm2), creating a transient microbubble through dramatically enhanced thermal expansion. This generates nonlinear photoacoustic signals with more than 10 times larger amplitude compared to that of a linear agent with the same optical absorption. By applying a differential scheme similar to ultrasound pulse inversion, more than 40 dB contrast enhancement is demonstrated with suppression of background signals.

Cardiovascular applications of therapeutic ultrasound.

Ultrasound (US) has gained widespread use in diagnostic cardiovascular applications. At amplitudes and frequencies typical of diagnostic use, its biomechanical effects on tissue are largely negligible. However, these parameters can be altered to harness US's thermal and non-thermal effects for therapeutic indications. High-intensity focused ultrasound (HIFU) and extracorporeal shock wave therapy (ECWT) are two therapeutic US modalities which have been investigated for treating cardiac arrhythmias and ischemic heart disease, respectively. Here, we review the biomechanical effects of HIFU and ECWT, their potential therapeutic mechanisms, and pre-clinical and clinical studies demonstrating their efficacy and safety limitations. Furthermore, we discuss other potential clinical applications of therapeutic US and areas in which future research is needed.

Mechanical characterization of ultrasonically synthesized microbubble shells by flow cytometry and AFM.

The mechanical properties of the shell of ultrasonically synthesized lysozyme microbubbles, LSMBs, were evaluated by acoustic interrogation and nanoindentation techniques. The Young's modulus of LSMBs was found to be 1.0 ± 0.3 MPa and 0.6 ± 0.1 MPa when analyzed by flow cytometry and AFM, respectively. The shell elasticity and Young's modulus were not affected by the size of the microbubbles (MBs). The hydrogel-like protein shell of LSMBs offers a softer, more elastic and viscous interface compared to lipid-shelled MBs. We show that the acoustic interrogation technique is a real-time, fast, and high-throughput method to characterize the mechanical characteristics of air-filled microbubbles coated by a variety of materials.