Validation of ultrasound velocimetry and computational fluid dynamics for flow assessment in femoral artery stenotic disease.

Purpose: To investigate the accuracy of high-framerate echo particle image velocimetry (ePIV) and computational fluid dynamics (CFD) for determining velocity vectors in femoral bifurcation models through comparison with optical particle image velocimetry (oPIV). Approach: Separate femoral bifurcation models were built for oPIV and ePIV measurements of a non-stenosed (control) and a 75%-area stenosed common femoral artery. A flow loop was used to create triphasic pulsatile flow. In-plane velocity vectors were measured with oPIV and ePIV. Flow was simulated with CFD using boundary conditions from ePIV and additional duplex-ultrasound (DUS) measurements. Mean differences and 95%-limits of agreement (1.96*SD) of the velocity magnitudes in space and time were compared, and the similarity of vector complexity (VC) and time-averaged wall shear stress (TAWSS) was assessed. Results: Similar flow features were observed between modalities with velocities up to 110 and 330 cm/s in the control and the stenosed model, respectively. Relative to oPIV, ePIV and CFD-ePIV showed negligible mean differences in velocity (<3 cm/s), with limits of agreement of ±25 cm/s (control) and ±34 cm/s (stenosed). CFD-DUS overestimated velocities with limits of agreements of 13±40 and 16.1±55 cm/s for the control and stenosed model, respectively. VC showed good agreement, whereas TAWSS showed similar trends but with higher values for ePIV, CFD-DUS, and CFD-ePIV compared to oPIV. Conclusions: EPIV and CFD-ePIV can accurately measure complex flow features in the femoral bifurcation and around a stenosis. CFD-DUS showed larger deviations in velocities making it a less robust technique for hemodynamical assessment. The applied ePIV and CFD techniques enable two- and three-dimensional assessment of local hemodynamics with high spatiotemporal resolution and thereby overcome key limitations of current clinical modalities making them an attractive and cost-effective alternative for hemodynamical assessment in clinical practice.

A unifying Rayleigh-Plesset-type equation for bubbles in viscoelastic media.

Understanding the ultrasound pressure-driven dynamics of microbubbles confined in viscoelastic materials is relevant for multiple biomedical applications, ranging from contrast-enhanced ultrasound imaging to ultrasound-assisted drug delivery. The volumetric oscillations of spherical bubbles are analyzed using the Rayleigh-Plesset equation, which describes the conservation of mass and momentum in the surrounding medium. Several studies have considered an extension of the Rayleigh-Plesset equation for bubbles embedded into viscoelastic media, but these are restricted to a particular choice of constitutive model and/or to small deformations. Here, we derive a unifying equation applicable to bubbles in viscoelastic media with arbitrary complex moduli and that can account for large bubble deformations. To derive this equation, we borrow concepts from finite-strain theory. We validate our approach by comparing the result of our model to previously published results and extend it to show how microbubbles behave in arbitrary viscoelastic materials. In particular, we use our viscoelastic Rayleigh-Plesset model to compute the bubble dynamics in benchmarked viscoelastic liquids and solids.

Wall Shear Stress and Pressure Fluctuations under Oscillating Stimulation in Helical Square Ducts with Cochlea-like Geometrical Curvature and Torsion.

Our study aims to provide basic insights on the impact of the spiral shape of the cochlea, i.e., of geometric torsion and curvature, on wall pressure and wall shear stress. We employed computational fluid dynamics in square duct models with curvature and torsion similar to those found in human cochleae. The results include wall pressures and wall shear stresses within the ducts under oscillating axial flow. Our findings indicate that the helical shape generates higher transverse wall shear stresses compared to exclusively curved or twisted ducts. The wall pressures and transverse wall shear stresses we found rise to amounts that may be physiologically relevant in the cochlea.Clinical relevance- The role of the spiral shape of the cochlea in hearing physiology remains, for a large part, elusive. For a better apprehension of hearing and its disorders, it is important to investigate the influence of geometric properties on biofluids motion and emerging phenomena in the cochlea.

Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study.

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 µm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-µm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

Time-resolved absolute radius estimation of vibrating contrast microbubbles using an acoustical camera.

Ultrasound (US) contrast agents consist of microbubbles ranging from 1 to 10 µm in size. The acoustical response of individual microbubbles can be studied with high-frame-rate optics or an "acoustical camera" (AC). The AC measures the relative microbubble oscillation while the optical camera measures the absolute oscillation. In this article, the capabilities of the AC are extended to measure the absolute oscillations. In the AC setup, microbubbles are insonified with a high- (25 MHz) and low-frequency US wave (1-2.5 MHz). Other than the amplitude modulation (AM) from the relative size change of the microbubble (employed in Renaud, Bosch, van der Steen, and de Jong (2012a). "An 'acoustical camera' for in vitro characterization of contrast agent microbubble vibrations," Appl. Phys. Lett. 100(10), 101911, the high-frequency response from individual vibrating microbubbles contains a phase modulation (PM) from the microbubble wall displacement, which is the extension described here. The ratio of PM and AM is used to determine the absolute radius, R0. To test this sizing, the size distributions of two monodisperse microbubble populations ( R = 2.1 and 3.5 µm) acquired with the AC were matched to the distribution acquired with a Coulter counter. As a result of measuring the absolute size of the microbubbles, this "extended AC" can capture the full radial dynamics of single freely floating microbubbles with a throughput of hundreds of microbubbles per hour.

Blood Flow Quantification with High-Frame-Rate, Contrast-Enhanced Ultrasound Velocimetry in Stented Aortoiliac Arteries: In Vivo Feasibility.

Local flow patterns influence stent patency, while blood flow quantification in stents is challenging. The aim of this study was to investigate the feasibility of 2-D blood flow quantification using high-frame-rate, contrast-enhanced ultrasound (HFR-CEUS) and particle image velocimetry (PIV), or echoPIV, in patients with aortoiliac stents. HFR-CEUS measurements were performed at 129 locations in 62 patients. Two-dimensional blood flow velocity fields were obtained using echoPIV. Visual inspection was performed by five observers to evaluate feasibility. The contrast-to-background ratio and average vector correlation were calculated and compared between stented and native vessel segments. Flow quantification with echoPIV was feasible in 128 of 129 locations (99%), with optimal quantification in 40 of 129 locations (31%). Partial quantification was achieved in 88 of 129 locations (68%), where one or multiple limiting issues occurred (not related to the stent) including loss of correlation during systole (57/129), short vessel segments (20/129), loss of contrast during diastole (20/129) and shadow regions (20/129). The contrast-to-background ratio and vector correlation were lower downstream in the imaged blood vessel, independent of the location of the stent. In conclusion, echoPIV was feasible in stents placed in the aortoiliac region, and the stents did not adversely affect flow tracking.

Super-Resolved Microbubble Localization in Single-Channel Ultrasound RF Signals Using Deep Learning.

Recently, super-resolution ultrasound imaging with ultrasound localization microscopy (ULM) has received much attention. However, ULM relies on low concentrations of microbubbles in the blood vessels, ultimately resulting in long acquisition times. Here, we present an alternative super-resolution approach, based on direct deconvolution of single-channel ultrasound radio-frequency (RF) signals with a one-dimensional dilated convolutional neural network (CNN). This work focuses on low-frequency ultrasound (1.7 MHz) for deep imaging (10 cm) of a dense cloud of monodisperse microbubbles (up to 1000 microbubbles in the measurement volume, corresponding to an average echo overlap of 94%). Data are generated with a simulator that uses a large range of acoustic pressures (5-250 kPa) and captures the full, nonlinear response of resonant, lipid-coated microbubbles. The network is trained with a novel dual-loss function, which features elements of both a classification loss and a regression loss and improves the detection-localization characteristics of the output. Whereas imposing a localization tolerance of 0 yields poor detection metrics, imposing a localization tolerance corresponding to 4% of the wavelength yields a precision and recall of both 0.90. Furthermore, the detection improves with increasing acoustic pressure and deteriorates with increasing microbubble density. The potential of the presented approach to super-resolution ultrasound imaging is demonstrated with a delay-and-sum reconstruction with deconvolved element data. The resulting image shows an order-of-magnitude gain in axial resolution compared to a delay-and-sum reconstruction with unprocessed element data.

Ultrasound-Mediated Drug Delivery With a Clinical Ultrasound System: In Vitro Evaluation.

Chemotherapy efficacy is often reduced by insufficient drug uptake in tumor cells. The combination of ultrasound and microbubbles (USMB) has been shown to improve drug delivery and to enhance the efficacy of several drugs in vitro and in vivo, through effects collectively known as sonopermeation. However, clinical translation of USMB therapy is hampered by the large variety of (non-clinical) US set-ups and US parameters that are used in these studies, which are not easily translated to clinical practice. In order to facilitate clinical translation, the aim of this study was to prove that USMB therapy using a clinical ultrasound system (Philips iU22) in combination with clinically approved microbubbles (SonoVue) leads to efficient in vitro sonopermeation. To this end, we measured the efficacy of USMB therapy for different US probes (S5-1, C5-1 and C9-4) and US parameters in FaDu cells. The US probe with the lowest central frequency (i.e. 1.6 MHz for S5-1) showed the highest USMB-induced intracellular uptake of the fluorescent dye SYTOX™ Green (SG). These SG uptake levels were comparable to or even higher than those obtained with a custom-built US system with optimized US parameters. Moreover, USMB therapy with both the clinical and the custom-built US system increased the cytotoxicity of the hydrophilic drug bleomycin. Our results demonstrate that a clinical US system can be used to perform USMB therapy as efficiently as a single-element transducer set-up with optimized US parameters. Therefore, future trials could be based on these clinical US systems, including validated US parameters, in order to accelerate successful translation of USMB therapy.

US Velocimetry in Participants with Aortoiliac Occlusive Disease.

Background The accurate quantification of blood flow in aortoiliac arteries is challenging but clinically relevant because local flow patterns can influence atherosclerotic disease. Purpose To investigate the feasibility and clinical application of two-dimensional blood flow quantification using high-frame-rate contrast-enhanced US (HFR-CEUS) and particle image velocimetry (PIV), or US velocimetry, in participants with aortoiliac stenosis. Materials and Methods In this prospective study, participants with a recently diagnosed aortoiliac stenosis underwent HFR-CEUS measurements of the pre- and poststenotic vessel segments (August 2018 to July 2019). Two-dimensional quantification of blood flow was achieved by performing PIV analysis, which was based on pairwise cross-correlation of the HFR-CEUS images. Visual inspection of the entire data set was performed by five observers to evaluate the ability of the technique to enable adequate visualization of blood flow. The contrast-to-background ratio and average vector correlation were calculated. In two participants who showed flow disturbances, the flow complexity and vorticity were calculated. Results Thirty-five participants (median age, 67 years; age range, 56-84 years; 22 men) were included. Visual scoring showed that flow quantification was achieved in 41 of 42 locations. In 25 locations, one or multiple issues occurred that limited optimal flow quantification, including loss of correlation during systole (n = 12), shadow regions (n = 8), a short vessel segment in the image plane (n = 7), and loss of contrast during diastole (n = 5). In the remaining 16 locations, optimal quantification was achieved. The contrast-to-background ratio was higher during systole than during diastole (11.0 ± 2.9 vs 6.9 ± 3.4, respectively; P < .001), whereas the vector correlation was lower (0.58 ± 0.21 vs 0.47 ± 0.13; P < .001). The flow complexity and vorticity were high in regions with disturbed flow. Conclusion Blood flow quantification with US velocimetry is feasible in patients with an aortoiliac stenosis, but several challenges must be overcome before implementation into clinical practice. Clinical trial registration no. NTR6980 © RSNA, 2021 Online supplemental material is available for this article.

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

Feedback-controlled microbubble generator producing one million monodisperse bubbles per second.

Monodisperse lipid-coated microbubbles are a promising route to unlock the full potential of ultrasound contrast agents for medical diagnosis and therapy. Here, we present a stand-alone lab-on-a-chip instrument that allows microbubbles to be formed with high monodispersity at high production rates. Key to maintaining a long-term stable, controlled, and safe operation of the microfluidic device with full control over the output size distribution is an optical transmission-based measurement technique that provides real-time information on the production rate and bubble size. We feed the data into a feedback loop and demonstrate that this system can control the on-chip bubble radius (2.5 µm-20 µm) and the production rate up to 106 bubbles/s. The freshly formed phospholipid-coated bubbles stabilize after their formation to a size approximately two times smaller than their initial on-chip bubble size without loss of monodispersity. The feedback control technique allows for full control over the size distribution of the agent and can aid the development of microfluidic platforms operated by non-specialist end users.

High-Frequency Acoustic Droplet Vaporization is Initiated by Resonance.

Vaporization of low-boiling point droplets has numerous applications in combustion, process engineering, and in recent years, in clinical medicine. However, the physical mechanisms governing the phase conversion are only partly explained. Here, we show that an acoustic resonance can arise from the large speed of sound mismatch between a perfluorocarbon microdroplet and its surroundings. The fundamental resonance mode obeys a unique relationship kR∼0.65 between droplet size and driving frequency that leads to a threefold pressure amplification inside the droplet. Classical nucleation theory shows that this pressure amplification increases the nucleation rate by several orders of magnitude. These findings are confirmed by high-speed imaging performed at a timescale of 10 ns. The optical recordings demonstrate that droplets exposed to intense acoustic waves generated by interdigital transducers nucleate only if they match the theoretical resonance size.

Three-phase vaporization theory for laser-activated microcapsules.

Precision control of vaporization, both in space and time, is critical for numerous applications, including medical imaging and therapy, catalysis and energy conversion, and it can be greatly improved through the use of micro- or nano-sized light absorbers. Ultimately, optimization of these applications also requires a fundamental understanding of the vaporization process. Upon laser irradiation, polymeric microcapsules containing a dye can vaporize, leading to the growth of a vapor bubble that emits a strong acoustic signature. Here, we compare laser-activated capsules containing either a volatile or a non-volatile oil core. We theoretically explore the vaporization of the capsules based on a three-phase thermodynamics model, that accounts for the partial vaporization of both the surrounding fluid and the oil core as well as for the interaction between heat transfer and microbubble growth. The model is compared to ultra-high-speed imaging experiments, where we record the cavitation events. Theory and experiments are in convincing agreement.

Ultrasound Contrast Agent Modeling: A Review.

Ultrasound is extensively used in medical imaging, being safe and inexpensive and operating in real time. Its scope of applications has been widely broadened by the use of ultrasound contrast agents (UCAs) in the form of microscopic bubbles coated by a biocompatible shell. Their increased use has motivated a large amount of research to understand and characterize their physical properties as well as their interaction with the ultrasound field and their surrounding environment. Here we review the theoretical models that have been proposed to study and predict the behavior of UCAs. We begin with a brief introduction on the development of UCAs. We then present the basics of free-gas-bubble dynamics upon which UCA modeling is based. We review extensively the linear and non-linear models for shell elasticity and viscosity and present models for non-spherical and asymmetric bubble oscillations, especially in the presence of surrounding walls or tissue. Then, higher-order effects such as microstreaming, shedding and acoustic radiation forces are considered. We conclude this review with promising directions for the modeling and development of novel agents.

Inverse leidenfrost drop manipulation using menisci.

Drops deposited on an evaporating liquid bath can be maintained in an inverse Leidenfrost state by the vapor emanating from the bath, making them levitate and hover without effective friction. These perfectly non-wetting droplets create a depression in the liquid interface that sustains their weight, which generates repellent forces when they approach a meniscus rising against a wall. Here, we study this reflection in detail, and show that frictionless Leidenfrost drops are a simple and efficient tool to probe the shape of an unknown interface. We then use the menisci to control the motion of the otherwise elusive drops. We create waveguides to direct and accelerate them and use parabolic walls to reflect and focus them. This could be particularly beneficial in the scale up of droplet cryopreservation processes: capillary interactions can be used to transport, gather and collect vitrified biological samples in absence of contact and contamination.

Microbubble Agents: New Directions.

Microbubble ultrasound contrast agents have now been in use for several decades and their safety and efficacy in a wide range of diagnostic applications have been well established. Recent progress in imaging technology is facilitating exciting developments in techniques such as molecular, 3-D and super resolution imaging and new agents are now being developed to meet their specific requirements. In parallel, there have been significant advances in the therapeutic applications of microbubbles, with recent clinical trials demonstrating drug delivery across the blood-brain barrier and into solid tumours. New agents are similarly being tailored toward these applications, including nanoscale microbubble precursors offering superior circulation times and tissue penetration. The development of novel agents does, however, present several challenges, particularly regarding the regulatory framework. This article reviews the developments in agents for diagnostic, therapeutic and "theranostic" applications; novel manufacturing techniques; and the opportunities and challenges for their commercial and clinical translation.

Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization.

Mistletoe lectin-1 (ML1) is a nature-derived macromolecular cytotoxin that potently induces apoptosis in target cells. Non-specific cytotoxicity to normal cells is one of the major risks in its clinical application, and we therefore propose to encapsulate ML1 in a nanocarrier that can specifically release its cargo intratumorally, thus improving the efficacy to toxicity ratio of the cytotoxin. We investigated the encapsulation of ML1 in ultrasound-sensitive liposomes (USL) and studied its release by high-intensity focused ultrasound (HAccessedIFU). USL were prepared by entrapment of perfluorocarbon nanodroplets in pegylated liposomes. The liposomes were prepared with different DPPC/cholesterol/DSPE-PEG2000 lipid molar ratios (60/20/20 for USL20; 60/30/10 for USL10; 65/30/5 for USL5) before combination with perfluorocarbon (PFC) nanoemulsions (composed of DPPC and perfluoropentane). When triggered with HIFU (peak negative pressure, 2-24 MPa; frequency, 1.3 MHz), PFC nanodroplets can undergo phase transition from liquid to gas thus rupturing the lipid bilayer of usl. Small unilamellar liposomes were obtained with appropriate polydispersity and stability. ML1 and the model protein horseradish peroxidase (HRP) were co-encapsulated with the PFC nanodroplets in USL, with 3% and 7% encapsulation efficiency for USL20 and USL10/USL5, respectively. Acoustic characterization experiments indicated that release is induced by cavitation. HIFU-triggered release of HRP from USL was investigated for optimization of liposomal composition and resulted in 80% triggered release for USL with USL10 (60/30/10) lipid composition. ML1 release from the final USL10 composition was also 80%. Given its high stability, suitable release, and ultrasound sensitivity, USL10 encapsulating ML1 was further used to study released ML1 bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging demonstrated its functional activity regarding the interaction with the target cells. We furthermore demonstrated the cytotoxicity of the released ML1 (I.E., After USL were treated with HIFU). The potent cytotoxicity (IC50 400 ng/ml; free ML1 IC50 345 ng/ml) was compared to non-triggered USL loaded with ML1. Our study shows that USL in combination with HIFU hold promise as trigger-sensitive nanomedicines for local delivery of macromolecular cytotoxins.

Plasmonic Bubble Nucleation and Growth in Water: Effect of Dissolved Air.

Under continuous laser irradiation, noble metal nanoparticles immersed in water can quickly heat up, leading to the nucleation of so-called plasmonic bubbles. In this work, we want to further understand the bubble nucleation and growth mechanism. In particular, we quantitatively study the effect of the amount of dissolved air on the bubble nucleation and growth dynamics, both for the initial giant bubble, which forms shortly after switching on the laser and is mainly composed of vapor, and for the final life phase of the bubble, during which it mainly contains air expelled from water. We found that the bubble nucleation temperature depends on the gas concentration: the higher the gas concentration, the lower the bubble nucleation temperature. Also, the long-term diffusion-dominated bubble growth is governed by the gas concentration. The radius of the bubbles grows as R(t) ∝ t 1/3 for air-equilibrated and air-oversaturated water. In contrast, in partially degassed water, the growth is much slower since, even for the highest temperature we achieve, the water remains undersaturated.

Capillary orbits.

Millimeter-sized objects trapped at a liquid surface distort the interface by their weight, which in turn attracts them towards each other. This ubiquitous phenomenon, colloquially called the "Cheerios effect" is seen in the clumping of cereals in a breakfast bowl, and turns out to be a highly promising route towards controlled self-assembly of colloidal particles at the water surface. Here, we study capillary attraction between levitating droplets, maintained in an inverse Leidenfrost state above liquid nitrogen. We reveal that the drops spontaneously orbit around each other - mirroring a miniature celestial system. In this unique situation of negligible friction, the trajectories are solely shaped by the Cheerios-interaction potential, which we obtain directly from the droplet's dynamics. Our findings offer an original perspective on contactless and contamination-free droplet cryopreservation processing, where the Leidenfrost effect and capillarity would be used in synergy to vitrify and transport biological samples.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.