Influence of the liquid ionic strength on the resonance frequency and shell parameters of lipid-coated microbubbles.

The correct measurement of the resonance frequency and shell properties of coated microbubbles (MBs) is essential in understanding and optimizing their response to ultrasound (US) exposure parameters. In diagnostic and therapeutic ultrasound, MBs are typically surrounded by blood; however, the influence of the medium charges on the MB resonance frequency has not been systematically studied using controlled measurements. This study aims to measure the medium charge interactions on MB behavior by measuring the frequency-dependent attenuation of the same size MBs in mediums with different charge densities. In-house lipid-coated MBs with C3F8 gas core were formulated. The MBs were isolated to a mean size of 2.35 µm using differential centrifugation. MBs were diluted to ≈8×105 MBs/mL in distilled water (DW), Phosphate-Buffered Saline solution (PBS1x) and PBS10x. The frequency-dependent attenuation of the MBs solutions was measured using an aligned pair of PVDF transducers with a center frequency of 10MHz and 100% bandwidth in the linear oscillation regime (7 kPa pressure amplitude). The MB shell properties were estimated by fitting the linear equation to experiments. Using a pendant drop tension meter, the surface tension at the equilibrium of ≈6 mm diameter size drops of the same MB shell was measured inside DW, PBS1x and PBS10x. The surface tension at the C3F8/solution interface was estimated by fitting the Young-Laplace equation from the recorded images. The frequency of the peak attenuation at different salinity levels was 13, 7.5 and 6.25 MHz in DW, PBS1x and PBS-10x, respectively. The attenuation peak increased by ≈140% with increasing ion density. MBs' estimated shell elasticity decreased by 64% between DW and PBS-1x and 36% between PBS-1x and PBS-10x. The drop surface tension reduced by 10.5% between DW and PBS-1x and by 5% between PBS-1x and PBS-10x, respectively. Reduction in the shell stiffness is consistent with the drop surface tension measurements. The shell viscosity was reduced by ≈40% between DW and PBS-1x and 42% between PBS-1x and PBS-10x. The reduction in the fitted stiffness and viscosity is possibly due to the formation of a densely charged layer around the shell, further reducing the effective surface tension on the MBs. The changes in the resonance frequency and estimated shell parameters were significant and may potentially help to better understand and explain bubble behavior in applications.

Scaling of the viscoelastic shell properties of phospholipid encapsulated microbubbles with ultrasound frequency.

Phospholipid encapsulated microbubbles are widely employed as clinical diagnostic ultrasound contrast agents in the 1-5 MHz range, and are increasingly employed at higher ultrasound transmit frequencies. The stiffness and viscosity of the encapsulating "shells" have been shown to play a central role in determining both the linear and nonlinear response of microbubbles to ultrasound. At lower frequencies, recent studies have suggested that shell properties can be frequency dependent. At present, there is only limited knowledge of how the viscoelastic properties of phospholipid shells scale at higher frequencies. In this study, four batches of in-house phospholipid encapsulated microbubbles were fabricated with decreasing volume-weighted mean diameters of 3.20, 2.07, 1.82 and 1.61 µm. Attenuation experiments were conducted in order to assess the frequency-dependent response of each batch, resulting in resonant peaks in response at 4.2, 8.9, 12.6 and 19.5 MHz, respectively. With knowledge of the size measurements, the attenuation spectra were then fitted with a standard linearized bubble model in order to estimate the microbubble shell stiffness Sp and shell viscosity Sf, resulting in a slight increase in Sp (1.53-1.76 N/m) and a substantial decrease in Sf (0.29×10(-6)-0.08×10(-6) kg/s) with increasing frequency. These results performed on a single phospholipid agent show that frequency dependent shell properties persist at high frequencies (up to 19.5MHz).

High-frequency subharmonic pulsed-wave Doppler and color flow imaging of microbubble contrast agents.

A recent study has shown the feasibility of subharmonic (SH) flow imaging at a transmit frequency of 20 MHz. This paper builds on these results by examining the performance of SH flow imaging as a function of transmit pressure. Further, we also investigate the feasibility of SH pulsed-wave Doppler (PWD) imaging. In vitro flow experiments were performed with a 1-mm-diameter wall-less vessel cryogel phantom using the ultrasound contrast agent Definity and an imaging frequency of 20 MHz. The phantom results show that there is an identifiable pressure range where accurate flow velocity and power estimates can be made with SH imaging at 10 MHz (SH10), above which velocity estimates are biased by radiation force effects and unstable bubble behavior, and below which velocity and power estimates are degraded by poor SNR. In vivo validation of SH PWD was performed in an arteriole of a rabbit ear, and blood velocity estimates compared well with fundamental (F20) mode PWD. The ability to suppress tissue signals using SH signals may enable the use of higher frame rates and improve sensitivity to microvascular flow or slow velocities near large vessel walls by reducing or eliminating the need for clutter filters.

High frequency ultrasound imaging of a single-species biofilm.

OBJECTIVE: This study evaluated the feasibility of a high frequency ultrasound scan to examine the 3D morphology of Streptococcus mutans biofilms grown in vitro. METHODS: Six 2-day S. mutans biofilms and six 7-day biofilms were grown on tissue culture membranes and on bovine dentine discs. A sterile growth medium on the membrane and disc were used as controls. Surfaces were rinsed and then immersed in sterile saline. High-frequency ultrasound imaging system was used to scan these surfaces at 55MHz, and a computer program calculated the average thickness of the biofilm layer from the 3D images. RESULTS: 3D pictures of the biofilm layers were obtained. Different cross-sections and plains are easily demonstrated. The average thickness of the 7-day biofilm was significantly bigger than the 2-day on both the membranes and dentinal discs. No structures were observed on the sterile membrane or disc. CONCLUSION: Three-dimensional structural imaging in situ is possible without damaging the biofilm layer in a quick and easy manner and can therefore be used to evaluate biofilms longitudinally as a function of time.

High frequency attenuation measurements of lipid encapsulated contrast agents.

A number of recent studies have indicated the potential of ultrasound contrast agent imaging at high ultrasound frequencies. However, the acoustic properties of microbubbles at frequencies above 10 MHz remain poorly understood at present. In this study we characterize the high frequency attenuation properties of (1) BR14, (2) BR14 that has been mechanically filtered (1 and 2 microm pore sizes) to exclude larger bubbles, and (3) the micron to submicron agent BG2423. A narrowband pulse-echo substitution method is employed with a series of four transducers covering the frequency range from 2 to 50 MHz. For BR14, attenuation decreases rapidly from 2 to 10 MHz and then more gradually from 10 to 50 MHz. For 2 microm filtration, the attenuation peaks between 10 and 15 MHz. For 1 microm filtration, attenuation continues to rise until 50 MHz. The agent BG2423 exhibits a diffuse attenuation peak in the range of 15-25 MHz and remains high until 50 MHz. These results demonstrate a strong influence of bubble size on high frequency attenuation curves, with bubble diameters of 1-2 microm and below having more pronounced acoustic activity at frequencies above 10 MHz.

Intravascular ultrasound tissue harmonic imaging: a simulation study.

Recently, the in vivo feasibility of tissue harmonic imaging (THI) with a mechanically-rotated intravascular ultrasound (IVUS) catheter was experimentally demonstrated. To isolate the second harmonic signal content, both pulse inversion (PI) and analog filtering were used. In the present paper, we report the development of a simulation tool to investigate nonlinear IVUS beams and the influence of rotation on the efficiency of PI signal processing. Nonlinear 20 MHz beams were simulated in a homogeneous tissue-mimicking medium, resulting in second harmonic pressure fields at 40 MHz. The acoustic response from tissue was simulated by summing radio-frequency (RF) pulse-echo responses from many point-scatterers. When the transducer was rotated with respect to the point-scatterers, the fundamental frequency suppression using PI degraded rapidly with increasing inter-pulse angles. The results of this study will aid in the optimization of harmonic IVUS imaging systems.

Reduction of stent artifacts using high-frequency harmonic ultrasound imaging.

Tissue harmonic imaging (THI) has been shown to improve medical ultrasound (US) image quality in the frequency range from 2 to 10 MHz and might, therefore, also be advantageous in high-frequency US applications, like US biomicroscopy and intravascular US (IVUS). In this study, we compared high-frequency THI (40 MHz) with fundamental imaging (20 and 40 MHz) with a distorting reflective metal stent in the near fields of both a spherically-focused US biomicroscopy transducer (aperture 8 mm, focal distance 13 mm) and an unfocused elliptical IVUS element. Hydrophone measurements of the harmonic beam (40 MHz) of both transducers showed relatively low signal strength in the near field compared with both (20 and 40 MHz) fundamental beams. For the focused transducer, THI suppressed the second stent echo up to 14 dB compared with fundamental imaging. No significant reduction in stent artifact imaging was observed for the unfocused IVUS element.

High-frequency color flow imaging of the microcirculation.

The extension of ultrasound (US) color flow imaging (CFI) techniques to high frequencies (> 20 MHz) has the potential to provide valuable noninvasive tools for scientific and clinical investigations of blood flow in the microcirculation. We describe the development of a slow-scan CFI system operating in the 20-100-MHz range that has been optimized to image the microcirculation. The apparatus has incorporated elements of a previously reported pulsed-wave Doppler system and is capable of operating in either CFI or pulsed-wave mode. The performance of the CFI system was evaluated at a center frequency of 50 MHz using two PVDF transducers with -6-dB beam widths of 43 and 60 microm. The -6 dB-axial resolutions were estimated to be 66 and 72 microm, respectively. In vivo validation experiments conducted using the murine ear model demonstrated the detection of flow in vessels down to 15-20 microm in diameter with flow velocities on the order of mm per s. Further experiments examining experimental murine tumors confirmed the successful detection of flow in the tumor microcirculation.

Ultrasound for the visualization and quantification of tumor microcirculation.

Advances in ultrasound based methods for the non-invasive assessment of the tumor microcirculation are described. Two new ultrasound approaches are highlighted. The first method relies on commercially available ultrasound contrast agents in combination with specialized nonlinear imaging sequences. Nonlinear scattering by microbubble contrast agents provides a unique intravascular signature that can be distinguished from the echoes caused by surrounding tissues. Through destruction-reperfusion experiments it is possible to use microbubble contrast agents as a tracer revealing the kinetics of tumor blood flow. The second ultrasound method for examining the microcirculation involves the use of much higher frequencies. At frequencies on the order of 50 MHz, Doppler processing allows the direct assessment of flow dynamics in realtime in arterioles as small as 15 microm. Three dimensional Doppler maps of flow patterning are presented. The strengths and weaknesses of these new methods are discussed and the potential for their use in preclinical animal drug studies, clinical drug trials, and prognostic studies is described.