A magnetic phantom technique for investigating structural effects on quantitative ultrasound parameters.

Media that contain ultrasound scatterers arranged in a regular spatial distribution can be considered as structured. Structural effects affect quantitative ultrasound parameters that reflect the microstructure properties. Prior studies examined structural effects using simulations or phantoms with fixed microarchitecture, focusing on a limited set of ultrasound parameters, with limited attention given to their underlying physical significance. This study aims to investigate the concordance of the physical interpretations of multiple quantitative ultrasound parameters experimentally by introducing a phantom type with an adjustable microarchitecture. The phantom consists of an aqueous solution containing superparamagnetic microspheres, acting as scatterers. The spatial arrangement of the magnetic particles is modified by applying an external magnetic field, therefore changing the degree of structure of the phantom. Quantitative ultrasound parameters are estimated in three different configurations: the magnetic field intensity is varied over time, strength, and orientation. In each experiment, the backscatter coefficient and the envelope quantitative ultrasound parameters are successfully extracted (R2 ≈ 0.94). Their physical interpretations are supported by microphotographs and geometrical considerations through concordant hypotheses. This study paves the way for the use of magnetic phantoms. This methodology could be followed to validate theoretical scattering models and the physical meanings of quantitative ultrasound parameters.

Evaluation of Scatterer Parameters From Ultrasound Scattering Models Taking Into Account Scattering From Nuclei and Cells of Cell-Pellet Biophantoms and Ex Vivo Tumors.

The Quantitative Ultrasound backscatter coefficient provides the capability to evaluate tissue microstructure parameters. Tissue-based scatterer parameters are extracted using ultrasound scattering models. It is challenging to correlate ultrasound scatterer parameters of tissue structures from optical-measured histology, possibly because of inappropriate scattering models or the presence of multiple scatterers. The objective of this study is to pursue the quantification of pertinent scatterer parameters with scattering models that consider ultrasound scattering from nuclei and cells. The concentric sphere model (CSM) and the structure factor model adapted for two types of scatterers (SFM2) are evaluated for cell-pellet biophantoms and ex vivo tumors of four cell lines: 4T1, JC, LMTK, and MAT. The structure factor model (SFM) was used for comparison. CSM and SFM2 provided scatterer parameters closer to histology (lower relative errors) for nucleus and cell radii and volume fractions than SFM but were not always accompanied by lower dispersion of the scatterer distribution (lower coefficient of variation). CSM and SFM2 quantified cell and nucleus radius and volume fraction parameters with lower relative error compared to SFM. For tumors, CSM provided better results than SFM2.

Cancer characterization using light backscattering spectroscopy and quantitative ultrasound: an ex vivo study on sarcoma subtypes.

Histological analysis is the gold standard method for cancer diagnosis. However, it is prone to subjectivity and sampling bias. In response to these limitations, we introduce a quantitative bimodal approach that aims to provide non-invasive guidance towards suspicious regions. Light backscattering spectroscopy and quantitative ultrasound techniques were combined to characterize two different bone tumor types from animal models: chondrosarcomas and osteosarcomas. Two different cell lines were used to induce osteosarcoma growth. Histological analyses were conducted to serve as references. Three ultrasound parameters and intensities of the light reflectance profiles showed significant differences between chondrosarcomas and osteosarcomas at the 5% level. Likewise, variations in the same biomarkers were reported for the two types of osteosarcoma, despite their similar morphology observed in the histological examinations. These observations show the sensitivity of our techniques in probing fine tissue properties. Secondly, the ultrasound spectral-based technique identified the mean size of chondrosarcoma cells and nuclei with relative errors of about 22% and 9% respectively. The optical equivalent technique correctly extracted scatterer size distributions that encompass nuclei and cells for chondrosarcomas and osteosarcomas ([Formula: see text] and [Formula: see text] respectively). The optical scattering contributions of nuclei were estimated at 52% for the chondrosarcomas and 69% for the osteosarcomas, probably indicating the abundant and the absent extracellular matrix respectively. Thus, the ultrasound and the optical methods brought complementary parameters. They successfully estimated morphological parameters at the cellular and the nuclear scales, making our bimodal technique promising for tumor characterization.

Ultrasound Scattering From Cell-Pellet Biophantoms and Ex Vivo Tumors Provides Insight Into the Cellular Structure Involved in Scattering.

The histologically identifiable cellular structure(s) involved in ultrasonic scattering is(are) yet to be uniquely identified. The study quantifies six possible cellular scattering parameters, namely, cell and nucleus radii and their respective cell and nucleus volume fractions as well as a combination of cell and nucleus radii and their volume fraction. The six cellular parameters are each derived from four cell lines (4T1, JC, LMTK, and MAT) and two tissue types (cell-pellet biophantom and ex vivo tumor). Optical histology and quantitative ultrasound (QUS), both independent approaches, are used to yield these cellular parameters. QUS scatterer parameters are experimentally determined using two ultrasonic scattering models: the spherical Gaussian model (GM) and the structure factor model (SFM) to yield insight about scattering from nuclei only and cells only. GM is a classical ultrasonic scattering model to evaluate QUS parameters and is well adapted for diluted media. SFM is adapted for dense media to estimate reasonably well scatterer parameters of cellular structures from ex vivo tissue. Nucleus and cell radii and volume fractions are measured optically from histology. They were used as inputs to calculate BSC for scattering from cells, nuclei, and both cells and nuclei. The QUS-derived scatterers (radii and volume fractions) distributions were then compared to the optical histology scatterer parameters derived from these calculated BSCs. The results suggest scattering from cells only (LMTK and MAT) or both cells and nuclei (4T1 and JC) for cell-pellet biophantoms and scattering from nuclei only for tumors.

Ultrafast Radial Modulation Imaging.

Radial modulation imaging improves the detection of microbubbles at high frequency using a dual ultrasonic excitation. However, the synchronization between the imaging pulses is nontrivial because microbubbles need to be interrogated in the compression and the rarefaction phase, and the time-delay difference from dispersion has to be corrected. To address these issues, we propose the use of ultrafast radial modulation imaging (uRMI). In this technique, a beat frequency between the modulation pulse (around 1 MHz) and the ultrafast pulse-repetition frequency was exploited to separate microbubbles from tissue phantom in vitro. This led to a modulated images' set in the spectral domain of the slow time that may then be demodulated through a digital lock-in amplifier to retrieve the contrast image. Ultrafast RMI, applied on a flow phantom with microbubbles, provided a contrast-to-tissue ratio from 7.2 to 14.8 dB at 15 MHz. For flow speed lower than 0.05 mL/min, uRMI (16 dB) provided a better contrast-to-tissue ratio than other techniques: singular value decomposition spatiotemporal filter (11 dB), amplitude modulation (9 dB), or microbubbles disruption (6 dB). This technique may then be suitable to improve the detection of targeted microbubbles, in ultrasound molecular imaging applications, and the detection of extremely slow microbubbles moving in the finest vessels in ultrasound localization microscopy.

Nonlinear ultrasound parameter to monitor cell death in cancer cell samples.

A scaling subtraction method was proposed to analyze the radio frequency data from cancer cell samples exposed to an anti-cancer drug and to estimate a nonlinear parameter. The nonlinear parameter was found to be well correlated (R2 = 0.62) to the percentage of dead cells in apoptosis and necrosis. The origin of the nonlinearity may be related to a change in contacts between cells, since the nonlinear parameter was well correlated to the average total coordination number of binary packings (R2 ≥ 0.77). These results suggest that the scaling subtraction method may be used to early quantify chemotherapeutic treatment efficiency.

Ultrasound Localization Microscopy and Super-Resolution: A State of the Art.

Because it drives the compromise between resolution and penetration, the diffraction limit has long represented an unreachable summit to conquer in ultrasound imaging. Within a few years after the introduction of optical localization microscopy, we proposed its acoustic alter ego that exploits the micrometric localization of microbubble contrast agents to reconstruct the finest vessels in the body in-depth. Various groups now working on the subject are optimizing the localization precision, microbubble separation, acquisition time, tracking, and velocimetry to improve the capacity of ultrasound localization microscopy (ULM) to detect and distinguish vessels much smaller than the wavelength. It has since been used in vivo in the brain, the kidney, and tumors. In the clinic, ULM is bound to improve drastically our vision of the microvasculature, which could revolutionize the diagnosis of cancer, arteriosclerosis, stroke, and diabetes.

Analysis of Two Quantitative Ultrasound Approaches.

There are two well-known ultrasonic approaches to extract sets of quantitative parameters: Lizzi-Feleppa (LF) parameters: slope, intercept, and midband; and quantitative ultrasound (QUS)-derived parameters: effective scatterer diameter (ESD) and effective acoustic concentration (EAC). In this study, the relation between the LF and QUS-derived parameters is studied theoretically and experimentally on ex vivo mouse livers. As expected from the theory, LF slope is correlated to ESD ([Formula: see text]), and from experimental data, LF midband is correlated to EAC ([Formula: see text]). However, LF intercept is not correlated to ESD ([Formula: see text]) nor EAC ([Formula: see text]). The unexpected correlation observed between LF slope and EAC ([Formula: see text]) results likely from the high correlation between ESD and EAC due to the inversion process. For a liver fat percentage estimation, an important potential medical application, the parameters presenting the better correlation are EAC ([Formula: see text]) and LF midband ([Formula: see text]).

High-Frequency Quantitative Ultrasound Spectroscopy of Excised Canine Livers and Mouse Tumors Using the Structure Factor Model.

Three scattering models were examined for characterizing ex vivo canine livers and HT29 mouse tumors in the 10-38- and the 15-42-MHz frequency bandwidth, respectively. The spherical Gaussian model (SGM) and the fluid sphere model (FSM) that were examined are suitable for dealing with sparse media, whereas the structure factor model (SFM) is adapted for characterizing concentrated media. For the canine livers, the scatterer radius and the acoustic concentration estimated with the three models were similar and matched well the nuclear structures obtained from histological analysis (with relative errors less than 7%). These results show that the livers could be considered as a diluted medium and that the nuclei in liver could be a dominant source of scattering. For the homogeneous mouse tumors, containing mostly viable HT29 cells, scatterer radius and volume fraction estimated with the SFM showed good agreement with the whole cell structures obtained from histological analysis (with relative errors less than 15%), whereas the sparse models (the SGM and the FSM) gave no consistent quantitative ultrasound parameters. This suggests that the viable HT29 cell areas have densely packed cellular content and that the whole HT29 cell could be responsible for scattering. For the heterogeneous tumors, the hyperechogenic zones observed in the B-mode images were linked to the presence of small necrotic areas surrounded by viable HT29 cells. Comparison between sparse and concentrated models shows that these hyperechogenic zones could be considered as a concentrated medium.

Hysteresis of inertial cavitation activity induced by fluctuating bubble size distribution.

Amongst the variety of complex phenomena encountered in nonlinear physics, a hysteretic effect can be expected on ultrasound cavitation due to the intrinsic nonlinearity of bubble dynamics. When applying successive ultrasound shots for increasing and decreasing acoustic intensities, a hysteretic behaviour is experimentally observed on inertial cavitation activity, with a loop area sensitive to the inertial cavitation threshold. To get a better insight of the phenomena underlying this hysteretic effect, the evolution of the bubble size distribution is studied numerically by implementing rectified diffusion, fragmentation process, rising and dissolution of bubbles from an initial bubble size distribution. When applying increasing and decreasing acoustic intensities, the numerical distribution exhibits asymmetry in bubble number and distribution. The resulting inertial cavitation activity is assessed through the numerical broadband noise of the emitted acoustic radiation of the bubble cloud dynamics. This approach allows obtaining qualitatively the observed hysteretic effect and its interest in terms of control is discussed.

Jumping acoustic bubbles on lipid bilayers.

In the context of sonoporation, we use supported lipid bilayers as a model for biological membranes and investigate the interactions between the bilayer and microbubbles induced by ultrasound. Among the various types of damage caused by bubbles on the surface, our experiments exhibit a singular dynamic interaction process where bubbles are jumping on the bilayer, forming a necklace pattern of alteration on the membrane. This phenomenon was explored with different time and space resolutions and, based on our observations, we propose a model for a microbubble subjected to the combined action of van der Waals, acoustic and hydrodynamic forces. Describing the repeated jumps of the bubble, this model explains the lipid exchanges between the bubble and bilayer.

Sonoporation of adherent cells under regulated ultrasound cavitation conditions.

A sonoporation device dedicated to the adherent cell monolayer has been implemented with a regulation process allowing the real-time monitoring and control of inertial cavitation activity. Use of the cavitation-regulated device revealed first that adherent cell sonoporation efficiency is related to inertial cavitation activity, without inducing additional cell mortality. Reproducibility is enhanced for the highest sonoporation rates (up to 17%); sonoporation efficiency can reach 26% when advantage is taken of the standing wave acoustic configuration by applying a frequency sweep with ultrasound frequency tuned to the modal acoustic modes of the cavity. This device allows sonoporation of adherent and suspended cells, and the use of regulation allows some environmental parameters such as the temperature of the medium to be overcome, resulting in the possibility of cell sonoporation even at ambient temperature.