High Microbubbles Research Articles

Microbubble tracking based on partial smoothing-based adaptive generalized labelled Multi-Bernoulli filter for super-resolution imaging

Super-resolution ultrasound (SRUS) can image the vasculature at microscopic resolution according to microbubble (MB) localization, with velocity vector maps obtained based on MB tracking information. High MB concentrations can reduce the acquisition time of SRUS imaging, however adjacent and intersecting vessels are difficult to distinguish, thus decreasing resolution. Low acquisition frame rates affect the precision of flow velocity estimation. This study proposes a partial smoothing-based adaptive generalized labeled multi-Bernoulli filter (SAGLMB) to precisely track the MB motion at different flow velocities. SAGLMB employs a generalized labelled multi-Bernoulli filter (GLMB) for MB trajectory allocation to separate adjacent and intersecting vessels. Furthermore, the nonlinear motion of MB was predicted by an unscented Kalman filter, and a cardinalized probability hypothesis density filter was applied to suppress clutter interference. Finally, the trajectories were smoothed by unscented Rauch-Tung-Striebel to improve the resolution of the SRUS image. The simulation results demonstrate that SAGLMB outperforms the conventional bipartite graph-based tracking at high MB concentrations, achieving at least an 8.55 % improvement in the correctly paired precision, with 3 times increase in the structural similarity index measure. Moreover, SAGLMB can obtain more precise flow velocity estimations with a 4 times improvement than the conventional method. The SRUS results of rabbit kidney show that the proposed method significantly improves resolution of adjacent and intersecting vessels at higher MB concentrations and maintains this performance as the acquisition frame rate decreases. Furthermore, the rat brain microvascular network was reconstructed with 9.21 μm (λ/11.1) resolution. Therefore, SAGLMB can achieve robust SRUS imaging at high concentrations and low acquisition frame rates.

Aminolysis-mediated single-step surface functionalization of poly (butyl cyanoacrylate) microbubbles for ultrasound molecular imaging

Molecular ultrasound imaging with actively targeted microbubbles (MB) proved promising in preclinical studies but its clinical translation is limited. To achieve this, it is essential that the actively targeted MB can be produced with high batch-to-batch reproducibility with a controllable and defined number of binding ligands on the surface. In this regard, poly (n-butyl cyanoacrylate) (PBCA)-based polymeric MB have been used for US molecular imaging, however, ligand coupling was mostly done via hydrolysis and carbodiimide chemistry, which is a multi-step procedure with poor reproducibility and low MB yield. Herein, we developed a single-step coupling procedure resulting in high MB yields with minimal batch-to-batch variation. Actively targeted PBCA-MB were generated using an aminolysis protocol, wherein amine-containing cRGD was added to the MB using lithium methoxide as a catalyst. We confirmed the successful conjugation of cRGD on the MB surface, while preserving their structure and acoustic signal. Compared to the conventional hydrolysis protocol, aminolysis resulted in higher MB yields and better reproducibility of coupling efficiency. Optical imaging revealed that under flow conditions, cRGD- and rhodamine-labelled MB, generated by aminolysis, specifically bind to tumor necrosis factor-alpha (TNF-α) activated endothelial cells in vitro. Furthermore, US molecular imaging demonstrated a markedly higher binding of the cRGD-MB than of control MB in TNF-α activated mouse aortas and 4T1 tumors in mice. Thus, using the aminolysis based conjugation approach, important refinements on the production of cRGD-MB could be achieved that will facilitate the production of clinical-scale formulations with excellent binding and ultrasound imaging performance.Graphical

Towards reduced ultrasound localization microscopy acquisition time by means of monodisperse microbubbles uncoupling

Ultrasound localization microscopy (ULM) unveils the microvascular structures using microbubbles (MBs) flowing in the circulatory system. As ULM relies on the precise localization and tracking of individual MBs, using high MB concentrations yields to high localization errors and ULM failure. ULM is, therefore, constrained to low MBs concentrations, leading to long acquisition times. To tackle this limitation, in this study, we propose an approach based on the injection of distinct monodisperse MBs, each characterized by a specific resonance behavior. As a proof of concept, we acquired and analyzed ultrasound data from a vascular phantom, where we singularly injected two monodisperse MB populations. Data were collected using an ULA-OP equipped with an Esaote LA533 probe. Pulses with a center frequency of 3 MHz and a bandwidth of 1MHz were utilized for imaging. MBs with diameters equal to 2.5 and 4.1 μm were injected in the phantom. MBs uncoupling was then performed exploiting the differences in the second harmonic signal intensity generated by the two MB populations. Results demonstrate the feasibility of monodisperse MBs uncoupling, enabling the use of higher microbubble concentrations for ULM, and thus, reducing acquisition time.

Acceleration-Based Kalman Tracking for Super-Resolution Ultrasound Imaging In Vivo.

Super-resolution ultrasound (SRUS) can image microvascular structure and flow at subwave-diffraction resolution based on localizing and tracking microbubbles (MBs). Currently, tracking MBs accurately under limited imaging frame rates and high MB concentrations remains a challenge, especially under the effect of cardiac pulsatility and in highly curved vessels. In this study, an acceleration-incorporated MB motion model is introduced into a Kalman tracking framework. The tracking performance was evaluated using simulated microvasculature with different MB motion parameters, concentrations, and acquisition frame rates, and in vivo human breast tumor US datasets. The simulation results show that the acceleration-based method outperformed the nonacceleration-based method at different levels of acceleration and acquisition frame rates and achieved significant improvement in true positive rate (TPR; up to 11.3%) and false negative rate (FNR; up to 13.2%). The proposed method can also reduce errors in vasculature reconstruction via the acceleration-based nonlinear interpolation, compared with linear interpolation (up to [Formula: see text]). The tracking results from temporally downsampled low frame rate in vivo datasets from human breast tumors show that the proposed method has better MB tracking performance than the baseline method, if using results from the initial high frame data as a reference. Finally, the acceleration estimated from tracking results also provides a spatial speed gradient map that may contain extra valuable diagnostic information.

Velocity field estimation in transcranial small vessel using super-resolution ultrasound imaging velocimetry

Based on the diameter and position information of small vessels obtained by transcranial super-resolution imaging using 3 MHz low-frequency chirp plane waves, a Gaussian-like non-linear compression was adopted to compress the blood flow signals in spatiotemporal filtering (STF) data to a precise region, and then estimate the blood flow velocity field inside the region over the adjacent time intervals using ultrasound imaging velocimetry (UIV). Imaging parameters, such as the mechanical index (MI), frame rate, and microbubble (MB) concentration, are critical during the estimation of velocity fields over a short time at high MB contrast agent concentrations. These were optimized through experiments and algorithms, in which dividing the connected domain was proposed to calculate MB cluster spot centroid spacing (SCS) and the spot-to-flow area ratio (SFAR) to determine the suitable MB concentration. The results of the in vitro experiments showed that the estimation of the small vessel flow velocity field was consistent with the theoretical results; the velocity field resolution for vessels with diameters of 0.5 mm and 0.3 mm was 36 μm and 21 μm, and the error between the mean velocity and the theoretical value was 0.7 % and 0.67 %, respectively.

Three-Dimensional Ultrasound Localization Microscopy with Bipartite Graph-Based Microbubble Pairing and Kalman-Filtering-Based Tracking on a 256-Channel Verasonics Ultrasound System with a 32 × 32 Matrix Array

PurposeThree-dimensional (3D) ultrasound localization microscopy (ULM) using a 2-D matrix probe and microbubbles (MBs) has recently been proposed to visualize microvasculature in three spatial dimensions beyond the ultrasound diffraction limit. However, 3D ULM has several limitations, including: (1) high system complexity, (2) complex MB flow dynamics in 3D, and (3) extremely long acquisition time that had to be addressed.MethodTo reduce the system complexity while maintaining high image quality, we used a sub-aperture process to reduce received channel counts. To address the second issue, a 3D bipartite graph-based method with Kalman filtering-based tracking was used in this study for MB tracking. An MB separation approach was incorporated to separate high concentration MB data into multiple, sparser MB datasets, allowing better MB localization and tracking for a limited acquisition time.ResultsThe proposed method was first validated in a flow channel phantom, showing improved spatial resolutions compared with the contrasted enhanced power Doppler image. Then the proposed method was evaluated with an in vivo chicken embryo brain dataset. Results showed that the reconstructed 3D super-resolution image achieved a spatial resolution of around 52 μm (smaller than the wavelength of around 200 μm).ConclusionA lower system complexity of 3D ULM has been proposed. In addition, our proposed 3D ULM provided the capability of 3D motion compensation and MB tracking. Microvessels that cannot be resolved clearly using localization only, can be well identified with the proposed method.

Deep learning-based fast and dense microbubble localization for ultrasound localization microscopy

Ultrasound localization microscopy (ULM) has gained substantial attention owing to its ability to super-resolve minute blood vessels at clinically relevant imaging depth. However, accurate localization of individual microbubbles (MBs) in areas with high MB concentration and overlapping point spread functions (PSFs) remains a challenge. Furthermore, existing localization methods based on pre-determined MB PSFs cannot reflect the highly non-stationary PSFs that vary spatially with MB concentration and nonlinear responses, ultrasound diffraction, and imaging settings. To address these limitations, we implemented the DECODE (DEep COntext Dependent) neural network that was recently developed for optical imaging on ULM. DECODE constructs a Gaussian mixture model with a mixed MB count and localization loss to output the probability, uncertainty, and sub-pixel location corresponding to each MB detection, achieving accurate identification and localization of MBs. Notably, DECODE was trained with realistic simulation data that incorporates MB brightness, movement, ultrasound system noise, and PSFs produced by a generative adversarial network that encodes the internal distribution of MB PSFs obtained from in vivo ultrasound imaging. In high MB-density regime, simulation studies demonstrated that DECODE improved MB detection rate from 41% to 95%, and reduced localization error from 109.4 μm to 32.5 μm (20 MHz) when comparing to conventional MB localization techniques. DECODE also demonstrated improved in vivo ULM imaging in mouse brain.

Deep Learning-Based Microbubble Localization for Ultrasound Localization Microscopy.

Ultrasound localization microscopy (ULM) is an emerging vascular imaging technique that overcomes the resolution-penetration compromise of ultrasound imaging. Accurate and robust microbubble (MB) localization is essential for successful ULM. In this study, we present a deep learning (DL)-based localization technique that uses both Field-II simulation and in vivo chickenembryo chorioallantoic membrane (CAM) data for training. Both radio frequency (RF) and in-phase and quadrature (IQ) data were tested in this study. The simulation experiment shows that the proposed DL-based localization was able to reduce both missing MB localization rate and MB localization error. In general, RF data showed better performance than IQ. For the in vivo CAM study with high MB concentration, DL-based localization was able to reduce the vessel MB saturation time by more than 50% compared to conventional localization. In addition, we propose a DL-based framework for real-time visualization of the high-resolution microvasculature. The findings of this article support the use of DL for more robust and faster MB localization, especially under high MB concentrations. The results indicate that further improvement could be achieved by incorporating temporal information of the MB data.

Improved Ultrasound Localization Microscopy Based on Microbubble Uncoupling via Transmit Excitation.

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on MB uncoupling via transmit excitation (MUTE). MUTE “silences” MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then, an in-vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half, thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

Super-resolution ultrasound localization microscopy based on a high frame-rate clinical ultrasound scanner: an in-human feasibility study

Non-invasive detection of microvascular alterations in deep tissues in vivo provides critical information for clinical diagnosis and evaluation of a broad-spectrum of pathologies. Recently, the emergence of super-resolution ultrasound localization microscopy (ULM) offers new possibilities for clinical imaging of microvasculature at capillary level. Currently, the clinical utility of ULM on clinical ultrasound scanners is hindered by the technical limitations, such as long data acquisition time, high microbubble (MB) concentration, and compromised tracking performance associated with low imaging frame-rate. Here we present a robust in-human ULM on a high frame-rate (HFR) clinical ultrasound scanner to achieve super-resolution microvessel imaging using a short acquisition time (<10 s). Ultrasound MB data were acquired from different human tissues, including a healthy liver and a diseased liver with acute-on-chronic liver failure, a kidney, a pancreatic tumor, and a breast mass using an HFR clinical scanner. By leveraging the HFR and advanced processing techniques including sub-pixel motion registration, MB signal separation, and Kalman filter-based tracking, MBs can be robustly localized and tracked for ULM under the circumstances of relatively high MB concentration associated with standard clinical MB administration and limited data acquisition time in humans. Subtle morphological and hemodynamic information in microvasculature were shown based on data acquired with single breath-hold and free-hand scanning. Compared with contrast-enhanced power Doppler generated based on the same MB dataset, ULM showed a 5.7-fold resolution improvement in a vessel based on a linear transducer, and provided a wide-range blood flow speed measurement that is Doppler angle-independent. Microvasculatures with complex hemodynamics can be well-differentiated at super-resolution in both normal and pathological tissues. This preliminary study implemented the ultrafast in-human ULM in various human tissues based on a clinical scanner that supports HFR imaging, indicating the potentials of the technique for various clinical applications. However, rigorous validation of the technique in imaging human microvasculature (especially for those tiny vessel structure), preferably with a gold standard, is still required.

Short Acquisition Time Super-Resolution Ultrasound Microvessel Imaging via Microbubble Separation

Super-resolution ultrasound localization microscopy (ULM), based on localization and tracking of individual microbubbles (MBs), offers unprecedented microvascular imaging resolution at clinically relevant penetration depths. However, ULM is currently limited by the requirement of dilute MB concentrations to ensure spatially sparse MB events for accurate localization and tracking. The corresponding long imaging acquisition times (tens of seconds or several minutes) to accumulate sufficient isolated MB events for full reconstruction of microvasculature preclude the clinical translation of the technique. To break this fundamental tradeoff between acquisition time and MB concentration, in this paper we propose to separate spatially overlapping MB events into sub-populations, each with sparser MB concentration, based on spatiotemporal differences in the flow dynamics (flow speeds and directions). MB localization and tracking are performed for each sub-population separately, permitting more robust ULM imaging of high-concentration MB injections. The superiority of the proposed MB separation technique over conventional ULM processing is demonstrated in flow channel phantom data, and in the chorioallantoic membrane of chicken embryos with optical imaging as an in vivo reference standard. Substantial improvement of ULM is further demonstrated on a chicken embryo tumor xenograft model and a chicken brain, showing both morphological and functional microvasculature details at super-resolution within a short acquisition time (several seconds). The proposed technique allows more robust MB localization and tracking at relatively high MB concentrations, alleviating the need for dilute MB injections, and thereby shortening the acquisition time of ULM imaging and showing great potential for clinical translation.

Histologic evaluation of activation of acute inflammatory response in a mouse model following ultrasound-mediated blood-brain barrier using different acoustic pressures and microbubble doses.

Rationale: Localized blood-brain barrier (BBB) opening can be achieved with minimal to no tissue damage by applying pulsed focused ultrasound alongside a low microbubble (MB) dose. However, relatively little is known regarding how varying treatment parameters affect the degree of neuroinflammation following BBB opening. The goal of this study was to evaluate the activation of an inflammatory response following BBB opening as a function of applied acoustic pressure using two different microbubble doses.Methods: Mice were treated with 650 kHz ultrasound using varying acoustic peak negative pressures (PNPs) using two different MB doses, and activation of an inflammatory response, in terms of microglial and astrocyte activation, was assessed one hour following BBB opening using immunohistochemical staining. Harmonic and subharmonic acoustic emissions (AEs) were monitored for all treatments with a passive cavitation detector, and contrast-enhanced magnetic resonance imaging (CE-MRI) was performed following BBB opening to quantify the degree of opening. Hematoxylin and eosin-stained slides were assessed for the presence of microhemorrhage and edema.Results: For each MB dose, BBB opening was achieved with minimal activation of microglia and astrocytes using a PNP of 0.15 MPa. Higher PNPs were associated with increased activation, with greater increases associated with the use of the higher MB dose. Additionally, glial activation was still observed in the absence of histopathological findings. We found that CE-MRI was most strongly correlated with the degree of activation. While acoustic emissions were not predictive of microglial or astrocyte activation, subharmonic AEs were strongly associated with marked and severe histopathological findings.Conclusions: Our study demonstrated that there were mild histologic changes and activation of the acute inflammatory response using PNPs ranging from 0.15 MPa to 0.20 MPa, independent of MB dose. However, when higher PNPs of 0.25 MPa or above were applied, the same applied PNP resulted in more severe and widespread histological findings and activation of the acute inflammatory response when using the higher MB dose. The potential activation of the inflammatory response following ultrasound-mediated BBB opening should be considered when treating patients to maximize therapeutic benefit.

Acute Inflammatory Response Following Increased Blood-Brain Barrier Permeability Induced by Focused Ultrasound is Dependent on Microbubble Dose.

Rationale: Focused ultrasound (FUS), in conjunction with circulating microbubbles (MBs), can be used to transiently increase the permeability of the blood-brain barrier (BBB) in a targeted manner, allowing therapeutic agents to enter the brain from systemic circulation. While promising preclinical work has paved the way for the initiation of 3 human trials, there remains concern regarding neuroinflammation following treatment. The aim of this study was to assess the magnitude of this response following sonication and explore the influence of MB dose.Methods: Differential expression of NFκB signaling pathway genes was assessed in rats at 6 h and 4 days following a FUS-mediated increase in BBB permeability. Three sonication schemes were tested: (1) a clinical imaging dose of MBs + peak negative pressure (PNP) controlled by acoustic feedback, (2) 10x clinical imaging dose of MBs + constant PNP of 0.290 MPa, and (3) 10x clinical imaging dose of MBs + PNP controlled by acoustic feedback. Follow-up magnetic resonance imaging (MRI) was performed to assess edema and hemorrhage. Hematoxylin and eosin histology was used to evaluate general tissue health.Results: MB dose has a significant impact on the expression of several key genes involved in acute inflammation and immune activation, including Tnf, Birc3, and Ccl2. At a clinical imaging dose of MBs, there were no significant changes detected in the expression of any NFκB signaling pathway genes. Conversely, a high MB dose resulted in a clear activation of the NFκB signaling pathway, accompanied by edema, neuronal degeneration, neutrophil infiltration, and microhemorrhage. Results also suggest that post-FUS gadolinium enhancement may hold predictive value in assessing the magnitude of inflammatory response.Conclusion: While a significant and damaging inflammatory response was observed at high MB doses, it was demonstrated that FUS can be used to induce increased BBB permeability without an associated upregulation of NFκB signaling pathway gene expression. This emphasizes the importance of employing optimized FUS parameters to mitigate the chances of causing injury to the brain at the targeted locations.

A Statistical Model for the Quantification of Microbubbles in Destructive Imaging

Quantification of targeted ultrasound contrast agents allows for the monitoring of endothelial marker expressions on a molecular level. In this study, a statistical correction is provided, which allows for improved precision in estimating the concentration of microbubbles (MBs) from Doppler images. Doppler imaging can be used to display the destruction of single MBs. However, concentrations will generally be too high to distinguish the individual events resulting in an inaccurate microbubble (MB) quantification. Therefore, a mathematical description of destruction events in Doppler images is developed which yields a correction formula for the concentration estimate from the color pixel density. The mathematical model is experimentally verified in gelatin phantoms using a high resolution imaging system (Vevo 770) and experimental cyanoacrylate MBs. Sensitive Particle Acoustic Quantification (SPAQ) is used to quantify MB in a defined volume. The SPAQ step size is varied from 32 to 127 μm to demonstrate the validity of the model for high color pixel densities. The corrected acoustic quantification shows the expected linear dependence on the step size and thus the amount of MBs in the images (R = 0.95). At SPAQ step sizes up to 127 μm, a MB concentration of 2.7 × 10 MBs/mL can be quantified. The results demonstrate the validity of the proposed correction. Quantification results of the SPAQ technique were considerably improved. The resulting formula is readily applied to SPAQ measurements at no additional expense.

Acoustic Attenuation by Contrast Agent Microbubbles in Superficial Tissue Markedly Diminishes Petechiae Bioeffects in Deep Tissue

To measure how ultrasound attenuation by contrast agent microbubbles (MBs) in superficial tissue affects petechiae creation in underlying deep tissue. Studies using Sprague-Dawley rats were approved by the Animal Care and Use Committee. MBs were injected intravenously, and 12 ultrasound pulses (100 sinusoids of 1 MHz ultrasound per pulse) were applied through the skin overlying the hindlimb adductors at intervals of 10 or 60 seconds. In some groups, the skin was resected and immediately returned without re-establishing vascular connections. Muscle petechiae were counted. Applying ultrasound through unperfused skin after bolus and continuous intravenous MB injection yielded, respectively, 30-fold and 3.5-fold more petechiae than for perfused skin. Surprisingly, petechiae/mm2 decreased with a higher MB dosage [0.12 +/- 0.05 (1 x 10 MBs/g) vs. 0.04 +/- 0.02 (3 x 10 MBs/g)] when ultrasound was applied through perfused skin. In contrast, petechiae/mm2 was approximately proportional to MB dosage for unperfused skin [0.17 +/- 0.10(5) (1 x 10 MBs/g) vs. 0.42 + 0.14 (3 x 10(5) MBs/g)]. In comparison to MB-free controls, MB solutions in this concentration range reduced the peak-negative pressure of ultrasound by 65% to 85%. Acoustic attenuation by MBs in skin markedly reduces petechiae creation in deep muscle. Petechiae inhibition is dependent on [MB]2.1 and, therefore, dominates the otherwise proportional relationship between petechiae and [MB] in muscle. The drop of peak-negative pressure below a critical microvessel rupturing threshold is the probable mechanism for petechiae inhibition. These results indicate that high MB doses could, paradoxically, reduce the potential for petechiae creation and may have important bearing on the design of contrast ultrasound-based therapeutics.

Selective surfactant prophylaxis in preterm infants born at ≤31 weeks' gestation using the stable microbubble test in gastric aspirates

To evaluate the stable microbubble test (SMT) ability to select candidates for surfactant prophylaxis for respiratory distress syndrome (RDS). We followed patients treated according to a new routine for surfactant prophylaxis based on the SMT to determine timing of the initial dose of surfactant, proportion of infants using surfactant, and the predictive value of the SMT. Gastric secretions were collected after birth. Newborns with < 25 microbubbles (MB)/mm(2) received prophylactic surfactant. Surfactant was given only after confirmation of RDS (rescue therapy) to newborns with > or =25 MB/mm(2). Fifty-four (55%) had a low MB count and received prophylactic surfactant. Three out of 44 infants with a high MB count required rescue therapy (negative predictive value 93%; CI:81.3-98.6%). The median interval and interquartile range between surfactant administration and birth in the prophylaxis group was 20 (17-27) minutes. Surfactant was used in 23 of 28 (82%) infants born at < 28 weeks of gestation and in 34 of 70 (49%) infants between 28 and 31 weeks. The SMT may be useful to determine surfactant prophylaxis (< 30 min after birth). This approach may reduce costs and the number of unnecessary interventions.