Investigating the subharmonic response of individual phospholipid encapsulated microbubbles at high frequencies: a comparative study of five agents

There are a range of contrast ultrasound applications above 10 MHz, a frequency regime in which nonlinear microbubble behavior is poorly understood. Lipid-encapsulated microbubbles have considerable potential for use at higher frequencies because they have been shown to exhibit pronounced nonlinear activity at frequencies up to 40 MHz. The objective of this work was to investigate the influence of agent formulation on the subharmonic response of lipid-encapsulated microbubbles at high frequencies with a view to providing information relevant to improving contrast agent design and imaging performance. An optical-acoustical setup was used to measure the subharmonic emissions from small (d < 3 μm) individual lipid-encapsulated microbubbles as a function of transmit pressure, size and composition. In this study, five agent formulations (Definity™, MicroMarker™ and three in-house agents manipulated to exhibit different levels of shell microstructure heterogeneity) were insonified at 25 MHz over a peak negative pressure (P_n) range of 0.02–1.2 MPa. All agents exhibited distinctly different subharmonic behavior, both in terms of amplitude and active sizes. MicroMarker™ exhibited the strongest, broadest and most consistent subharmonic response, 22% greater in power than that of Definity™ and as much as 50% greater than the in-house formulations. No clear relation between in-house agents’ shell microstructure and nonlinear response was found, other than the variability in the nonlinear response itself. An analysis of the response of MicroMarker™ bubbles suggests that these bubbles exhibit “expansion-dominated” oscillations, in contrast to “compression-only” oscillations observed for similar bubbles at lower frequencies (f < 11 MHz).     

In Vivo characterization of ultrasound contrast agents: microbubble spectroscopy in a chicken embryo

The dynamics of coated microbubbles was studied in an in vivo model. Biotinylated lipid-coated microbubbles were prepared in-house and were injected into a chick embryo chorioallantoic membrane (CAM) model on the fifth day of incubation. The microbubbles, ranging between 1.0 and 3.5 μm in diameter, were insonified in the frequency range of 4–7 MHz. Two amplitudes of acoustic pressure were applied: 300 kPa and 400 kPa. The fundamental and subharmonic responses were recorded optically with an ultra-fast camera (Brandaris 128) at 20 million frames per second. A subharmonic response was observed for 44% of the studied bubbles. From the data the frequency of the maximum fundamental and subharmonic response was derived for each individual bubble and resulted in the resonance curves of the microbubbles. All the bubbles showed shell (strain) hardening behavior for a higher acoustic pressure. We conclude that the subharmonic oscillations observed in this study belonged to the transmit at resonance (TR) regime.     

Characterizing the subharmonic response of phospholipid-coated microbubbles for carotid imaging

The subharmonic vibration of BR14 (Bracco Research S.A., Geneva, Switzerland) contrast agent microbubbles is investigated within the preferable frequency range for carotid ultrasound imaging (8-12 MHz). The response of the bubbles was recorded optically with an ultra-fast recording camera (Brandaris 128) at three acoustic pressures (50, 100 and 120 kPa). The vibration of the microbubbles was measured as a function of the excitation frequency and its frequency content was determined. Among 390 recordings, 40% showed subharmonic oscillations. It was observed that for smaller microbubbles (diameter < 3 μm) the frequency of the maximum subharmonic response increases for increasing pressures (shell hardening) opposite to what has been reported for larger microbubbles (3 μm < diameter < 15 μm). These findings are well predicted by the model proposed by Marmottant et al. (2005) after including the dilatational shell viscosity of the microbubbles measured by Van der Meer et al. (2007), which indicates a marked shear-thinning behavior of the phospholipid shell.     

Assessment of the Superharmonic Response of Microbubble Contrast Agents for Acoustic Angiography as a Function of Microbubble Parameters

Acoustic angiography is a superharmonic contrast-enhanced ultrasound imaging technique that enables 3-D high-resolution microvascular visualization. This technique utilizes a dual-frequency imaging strategy, transmitting at a low frequency and receiving at a higher frequency, to detect high-frequency contrast agent signatures and separate them from tissue background. Prior studies have illustrated differences in microbubble scatter dependent on microbubble size and composition; however, most previously reported data have utilized a relatively narrow frequency bandwidth centered around the excitation frequency. To date, a comprehensive study of isolated microbubble superharmonic responses with a broadband dual-frequency system has not been performed. Here, the superharmonic signal production of 14 contrast agents with various gas cores, shell compositions, and bubble diameters at mechanical indices of 0.2 to 1.2 was evaluated using a transmit 4 MHz, receive 25 MHz configuration. Results indicate that perfluorocarbon cores or lipid shells with 18- or 20-carbon acyl chains produce more superharmonic signal than sulfur hexafluoride cores or lipid shells with 16-carbon acyl chains, respectively. As microbubble diameter increases from 1 to 4 µm, superharmonic generation decreases. In a comparison of two clinical agents, Definity and Optison, and one preclinical agent, Micromarker, Optison produced the least superharmonic signal. Overall, this work suggests that microbubbles around 1 μm in diameter with perfluorocarbon cores and longer-chained lipid shells perform best for superharmonic imaging at 4 MHz. Studies have found that microbubble superharmonic response follows trends different from those described in prior studies using a narrower frequency bandwidth centered around the excitation frequency. Future work will apply these results in vivo to optimize the sensitivity of acoustic angiography.     

The Delayed Onset of Subharmonic and Ultraharmonic Emissions from a Phospholipid-Shelled Microbubble Contrast Agent

Characterizing the non-linear response of microbubble contrast agents is important for their efficacious use in imaging and therapy. In this article, we report that the subharmonic and ultraharmonic response of lipid-shelled microbubble contrast agents exhibits a strong temporal dependence. We characterized non-linear emissions from Targestar-p microbubbles (Targeson Inc., San Diego, CA, USA) periodically for 60 min, at 10 MHz excitation frequency. The results revealed a considerable increase in the subharmonic and ultraharmonic response (nearly 12–15 and 5–8 dB) after 5–10 min of agent preparation. However, the fundamental and the harmonic response remained almost unchanged in this period. During the next 50 min, the subharmonic, fundamental, ultraharmonic, and harmonic responses decreased steadily by 2–5 dB. The temporal changes in the non-linear behavior of the agent appeared to be primarily mediated by gas-exchange through the microbubble shell; temperature and prior acoustic excitation based mechanisms were ruled out. Further, there was no measurable change in the agent size distribution by static diffusion. We envisage that these findings will help obtain reproducible measurements from agent characterization, non-linear imaging, and fluid-pressure sensing. These findings also suggest the possibility for improving non-linear imaging by careful design of ultrasound contrast agents.     

Acoustic Sizing of an Ultrasound Contrast Agent

Because the properties of ultrasound contrast agent populations after administration to patients are largely unknown, methods able to study them noninvasively are required. In this study, we acoustically performed a size distribution measurement of the ultrasound contrast agent Definity^®. Single lipid-shelled microbubbles were insonified at 25 MHz, which is considerably higher than their resonance frequency, so that their acoustic responses depended on their geometrical cross sections only. We calculated the size of each microbubble from their measured backscattered pressures. The acoustic size measurements were compared with optical reference size measurements to test their accuracy. Our acoustic sizing method was applied to 88 individual Definity^® bubbles to derive a size distribution of this agent. The size distribution obtained acoustically showed a mean diameter (2.5 μm) and a standard deviation (0.9 μm) in agreement within 8% with the optical reference measurement. At 25 MHz, this method can be applied to bubble sizes larger than 1.2 μm in diameter. It was observed that similar sized bubbles can give different responses (up to a factor 1.5), probably because of shell differences. These limitations should be taken into account when implementing the method in vivo. This acoustic sizing method has potential for estimating the size distribution of an ultrasound contrast agent noninvasively. (E-mail: d.maresca@erasmusmc.nl)     

Acoustical Properties of Individual Liposome-Loaded Microbubbles

A comparison between phospholipid-coated microbubbles with and without liposomes attached to the microbubble surface was performed using the ultra-high-speed imaging camera (Brandaris 128). We investigated 73 liposome-loaded microbubbles (loaded microbubbles) and 41 microbubbles without liposome loading (unloaded microbubbles) with a diameter ranging from 3–10 μm at frequencies ranging from 0.6–3.8 MHz and acoustic pressures ranging from 5–100 kPa. The experimental data showed nearly the same shell elasticity for the loaded and unloaded bubbles, but the shell viscosity was higher for loaded bubbles compared with unloaded bubbles. For loaded bubbles, a higher pressure threshold for the bubble vibrations was noticed. In addition, an “expansion-only” behavior was observed for up to 69% of the investigated loaded bubbles, which mostly occurred at low acoustic pressures (≤30 kPa). Finally, fluorescence imaging showed heterogeneity of liposome distributions of the loaded bubbles.     

High frequency nonlinear scattering from a micrometer to submicrometer sized lipid encapsulated contrast agent

An experimental lipid encapsulated contrast agent comprised substantially of micrometer to submicrometer diameter bubbles was evaluated for its capacity to produce nonlinear scattering in response to high transmit frequencies. Agent characterization experiments were conducted at transmit frequencies of 20 and 30 MHz with bandwidths of 5, 15 and 25% using a broadband focused PVDF transducer. The presence of subharmonic energy was observed for all bandwidths at a wide range of pressures (0.49 to 5.7 MPa and 0.45 to 4.5 MPa for the 20 and 30 MHz cases, respectively). Distinct ultraharmonics were observed only in the 5% bandwidth cases. Second harmonic energy was also present, but this was at least partly due to nonlinear propagation, as indicated by linear scatterer signals. Evidence of destruction was found only at higher peak negative pressures (e.g., >2 MPa for 30 MHz 5% bandwidth pulse). The results suggest that small lipid bubble formulations may be useful for the purposes of high frequency nonlinear contrast imaging.     

Nonlinear Emission from Individual Bound Microbubbles at High Frequencies

Targeted microbubbles detected with high-frequency ultrasound can establish the molecular expression of blood vessels with submillimeter resolution. To improve microbubble-specific imaging at high frequencies, the subharmonic and second harmonic signal from individual microbubbles were measured as a function of size and pressure. Single phospholipid-shell microbubbles (1.1 to 5.0 μm in diameter) bound to gelatin, co-aligned with an optical microscope and transducer, were insonated with 30 MHz Gaussian-enveloped pulses at pressures from 20 kPa to 1 MPa with –6 dB one-way bandwidths of 11%, 20% and 45%. A subharmonic signal (15 MHz) was detected above a pressure threshold of 110 kPa—independent of bandwidth. The signal peaked for microbubbles 1.60 μm in diameter subject to 20% and 11% bandwidth pulses, and 1.80 μm for 45% bandwidth pulses, for pressures up to 400 kPa, agreeing with the notion that microbubbles insonated at twice their resonant frequency preferentially emit a subharmonic component. For pressures between 400 kPa and 1 MPa, a broader range of microbubbles emitted a subharmonic signal, and microbubbles below 1.70 μm in diameter were disrupted. The second harmonic signal measured, within the limited experimental conditions, was consistent with nonlinear propagation. Further, the results shed light on the effect of the shell on the phase of the subharmonic signal with respect to the fundamental signal. (E-mail: michael.sprague@sri.utoronto.ca)     

Real-time texture analysis for identifying optimum microbubble concentration in 2-D ultrasonic particle image velocimetry

Many recent studies on ultrasonic particle image velocimetry (Echo PIV) showed that the accuracy of two-dimensional (2-D) flow velocity measured depends largely on the concentration of ultrasound contrast agents (UCAs) during imaging. This article presents a texture-based method for identifying the optimum microbubble concentration for Echo PIV measurements in real-time. The texture features, standard deviation of gray level, and contrast, energy and homogeneity of gray level co-occurrence matrix were extracted from ultrasound contrast images of rotational and pulsatile flow (10 MHz) in vitro and in vivo mouse common carotid arterial flow (40 MHz) with UCAs at various concentrations. The results showed that, at concentration of 0.8∼2 × 10³ bubbles/mL in vitro and 1∼5 × 10⁵ bubbles/mL in vivo, image texture features had a peak value or trough value, and velocity vectors with high accuracy can be obtained. Otherwise, poor quality velocity vectors were obtained. When the texture features were used as a feature set, the accuracy of K-nearest neighbor classifier can reach 86.4% in vitro and 87.5% in vivo, respectively. The texture-based method is shown to be able to quickly identify the optimum microbubble concentration and improve the accuracy for Echo PIV imaging.     

Subharmonic response of encapsulated microbubbles: conditions for existence and amplification

The response of encapsulated microbubbles at half the ultrasound insonation frequency, termed subharmonic response, may have potential applications in diagnosis and therapy. The subharmonic signal, emitted by Definity microbubble cloud sonicated by ultrasound was studied theoretically and experimentally. The size distribution of the microbubbles was optically analyzed and resonance frequency of 2.7 MHz was determined. An asymptotic model has been developed that generates subharmonic response of a single and of a cloud of bubbles of various sizes. Threshold conditions for existence and the intensity of the subharmonic signal are predicted to depend on microbubbles size distribution and shell properties, as well as on the driving field frequency and pressure. Thin tubes filled with Definity solution were insonated at acoustic pressures from 100 to 630 kPa. The intensities of the emitted fundamental harmonics and subharmonics were measured. At frequency 5.5MHz, twice the resonance frequency, the subharmonic signals were observed only at pressures greater than 190 kPa. The subharmonic to fundamental harmonics intensity ratio was within -12 to -1 dB. The experimental results showed good correlation with the theoretical results in the range of validity of the asymptotic solution, thus supporting the model assumptions.     

Quantitative Guidelines for the Prediction of Ultrasound Contrast Agent Destruction During Injection

Experiments and theory were undertaken on the destruction of ultrasound contrast agent microbubbles on needle injection, with the aim of predicting agent loss during in vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamic calculations determined the pressure drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure drop of 109 ± 7 kPa Two types of lipid-shelled microbubbles were found to have a pressure drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure drop for destruction of 230 ± 10 kPa; for a previously published lipid-shelled agent, this value was 150 ± 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agent during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given.     

Observations of translation and jetting of ultrasound-activated microbubbles in mesenteric microvessels

High-speed photomicrography was used to study the translational dynamics of single microbubbles in microvessels of ex vivo rat mesenteries. The microbubbles were insonated by a single 2 μs ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 0.8-4 MPa. The microvessel diameters ranged from 10-80 μm. The high-speed image sequences show evidence of ultrasound-activated microbubble translation away from the nearest vessel wall; no microbubble showed a net translation toward the nearest vessel wall. Microbubble maximum translation displacements exceeded 20 μm. Microjets with the direction of the jets identifiable were also observed; all microjets appear to have been directed away from the nearest vessel wall. These observations appear to be characteristic of a strong coupling between ultrasound-driven microbubbles and compliant microvessels. Although limited to mesenteric tissues, these observations provide an important step in understanding the physical interactions between microbubbles and microvessels.     

A Method for Differentiating Targeted Microbubbles in Real Time Using Subharmonic Micro-Ultrasound and Interframe Filtering

This study introduces a new method for differentiating targeted microbubbles in the presence of flowing microbubbles and tissue using micro-ultrasound. The method relies on subharmonic (SH) imaging for segmenting microbubble signals from tissue signals, and low-pass interframe filtering for segmenting bound targeted microbubbles from flowing microbubbles. The method is evaluated with 30 frames per second SH B-mode imaging in vitro, using a wall-less vessel flow phantom. The SH B-mode cineloops were postprocessed using an interframe moving average filter to segment the regions of bound microbubbles on the inner surface of the vessel phantom. The bound bubbles were then disrupted with sufficiently high ultrasound pressures, so that the dynamic process of targeted microbubble binding under flowing conditions could be observed. These preliminary results show that the proposed method is a feasible solution to the challenge of differentiating targeted microbubbles in the presence of tissue and freely flowing microbubbles at high frequencies, which in turn should improve the specificity of targeted microbubble detection. (E-mail: aneedles@visualsonics.com)     

Effect of Microbubble Size on Fundamental Mode High Frequency Ultrasound Imaging in Mice

High-frequency ultrasound imaging using microbubble (MB) contrast agents is becoming increasingly popular in pre-clinical and small animal studies of anatomy, flow and vascular expression of molecular epitopes. Currently, in vivo imaging studies rely on highly polydisperse microbubble suspensions, which may provide a complex and varied acoustic response. To study the effect of individual microbubble size populations, microbubbles of 1–2 μm, 4–5 μm and 6–8 μm diameter were isolated using the technique of differential centrifugation. Size-selected microbubbles were imaged in the mouse kidney over a range of concentrations using a Visualsonics Vevo 770 ultrasound imaging system (Visualsonics, Toronto, Ontario, Canada) with a 40-MHz probe in fundamental mode. Results demonstrate that contrast enhancement and circulation persistence are strongly dependent on microbubble size and concentration. Large microbubbles (4–5 and 6–8 μm) strongly enhanced the ultrasound image with positive contrast, while 1–2 μm microbubbles showed little enhancement. For example, the total integrated contrast enhancement, measured by the area under the time-intensity curve (AUC), increased 16-fold for 6–8 μm diameter microbubbles at 5 × 10⁷ MB/bolus compared with 4–5 μm microbubbles at the same concentration. Interestingly, 1–2 μm diameter microbubbles, at any concentration, did not measurably enhance the integrated ultrasound signal at tissue depth, but did noticeably attenuate the signal, indicating that they had a low scattering-to-attenuation ratio. When concentration matched, larger microbubbles were more persistent in circulation. However, when volume matched, all microbubble sizes had a similar circulation half-life. These results indicated that dissolution of the gas core plays a larger role in contrast elimination than filtering by the lungs and spleen. The results of this study show that microbubbles can be tailored for optimal contrast enhancement in fundamental mode imaging. (E-mail: mb2910@columbia.edu)     

Laser Doppler anemometry measurements of the shear stresses on ultrasonic contrast agent microbubbles attached to agar

Ultrasonic contrast agents are currently being developed to target and bind to specific areas of interest such as atheromous plaque. A microbubble has been developed in-house which can be targeted to attach to specific cell-lines. To assess the feasibility of using the microbubble in vivo, the shear stresses which the bound microbubbles can withstand need to be known. A flow chamber was developed for use with intravascular ultrasound (IVUS) and laser Doppler anemometry (LDA). Biotin was incorporated into the microbubble shells and streptavidin was used to attach them to agar. IVUS at 40 MHz was then used to image the attached microbubbles under steady flow at a range of flow rates from 75 to 480 mL min(-1) through a flow area of 9 mm(2). LDA was employed to find high resolution velocity profiles of the flow in the chamber at a selection of these flow rates and the shear stresses on the bubbles were calculated. The bubbles were found to remain attached to the agar for shear stresses of up to 3.4 Pa. This compares with mean physiological arterial shear stresses of less than 1.5 Pa for pulsatile flow.     

Determination of the Interfacial Rheological Properties of a Poly(DL-lactic acid)-Encapsulated Contrast Agent Using In Vitro Attenuation and Scattering

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affects its acoustic properties. Polymers, which behave differently from materials commonly used (i.e., lipids or proteins) for monolayer encapsulation, have the potential for better stability and improved control of encapsulation properties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here using in vitro acoustic experiments and several models of encapsulation. The interfacial rheological properties of the encapsulation are determined according to each model using attenuation of ultrasound through a suspension of microbubbles. Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. For this microbubble population (average diameter, 1.9 μm), the peak in attenuation measurement indicates a weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9 μm). This apparently contradictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reduced surface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. All models considered here are shown to behave similarly even in the non-linear regime because of the low surface dilational elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamental and second-harmonic responses, respectively, for a contrast agent concentration of 1.33 μg/mL in the suspension. Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20 dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with the experimentally measured fundamental response, but not with the experimental second-harmonic response. The characteristic features of sub-harmonic responses and the steady response beyond the threshold are matched well by model predictions. However, prediction of the threshold value depends on estimated properties and size distribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsulation properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonic threshold.     

Ultrasound Directs a Transposase System for Durable Hepatic Gene Delivery in Mice

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1–3 μm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0 MHz, 20-μs pulse duration, 10-Hz pulse repetition frequency and ∼1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 d, compared with 4 d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3 d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro; however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.     

Characterisation of Liposome-Loaded Microbubble Populations for Subharmonic Imaging

Therapeutic microbubbles could make an important contribution to the diagnosis and treatment of cancer. Acoustic characterisation was performed on microfluidic generated microbubble populations that either were bare or had liposomes attached. Through the use of broadband attenuation techniques (3–8 MHz), the shell stiffness was measured to be 0.72 ± 0.01 and 0.78 ± 0.05 N/m and shell friction was 0.37 ± 0.05 and 0.74 ± 0.05 × 10^?6 kg/s for bare and liposome-loaded microbubbles, respectively. Acoustic scatter revealed that liposome-loaded microbubbles had a lower subharmonic threshold, occurring from a peak negative pressure of 50 kPa, compared with 200 kPa for equivalent bare microbubbles. It was found that liposome loading had a negligible effect on the destruction threshold for this microbubble type, because at a mechanical index >0.4 (570 kPa), 80% of both populations were destroyed.     

Dynamics of Targeted Microbubble Adhesion Under Pulsatile Compared with Steady Flow

Hemodynamic flow variations at low fluid shear stress are thought to play a critical role in local atherosclerotic plaque initiation and development and to affect plaque instability. Targeted microbubbles are being developed as intravascular agents for identifying atherosclerotic lesions using ultrasound. How variations in local hydrodynamic flow influence the adhesiveness of targeted microbubbles is not well understood. We postulated that rates of targeted microbubble binding and accumulation differ when subjected to steady flow (SF) as compared with oscillatory or pulsatile flow (PF), because PF imposes non-uniform blood rheology and periodic acceleration and deceleration of blood velocity, when compared with SF. We assessed the binding rates of targeted microbubbles in seven randomly assigned PF and seven matched SF replicate runs at low (<1 Pa) and intermediate (≥1 and <2.5 Pa) wall shear stress (WSS) by drawing 4.8 × 10⁶ microbubbles mL⁻¹ over streptavidin-coated substrates, immobilized within a parallel plate flow chamber at a calculated density of 81 binding sites μm^-2. Selective binding and accumulation of targeted microbubbles was recorded in a single field of view using real-time video microscopy. Microbubble accumulation was modeled to obtain flow-mediated microbubble binding kinetics (amplitude, A, and rate constant, k). PF elicited higher microbubble accumulation rates, in comparison to SF. The rates of microbubble accumulation differed significantly between PF and SF (p < 0.05) at intermediate WSS but not at low WSS (p > 0.05). The rate of microbubble accumulation decreased as WSS increased.