Characterization of dose-dependent Young's modulus for a radiation-sensitive polymer gel.

Radiation-sensitive polymer gels for clinical dosimetry have been intensively investigated with magnetic resonance imaging (MRI) because the transversal magnetic relaxation time is dependent on irradiation dose. MRI is expensive and not easily available in most clinics. For this reason, low-cost, quick and easy-to-use potential alternatives such as optical computed tomography (CT), x-ray CT or ultrasound attenuation CT have also been studied by others. Here, we instead evaluate the dose dependence of the elastic material property, Young's modulus and the dose response of the viscous relaxation of radiation-sensitive gels to discuss their potential for dose imaging. Three batches of a radiation-sensitive polymer gel (MAGIC gel) samples were homogeneously irradiated to doses from 0 Gy to 45.5 Gy. Young's modulus was computed from the measured stress on the sample surface and the strain applied to the sample when compressing it axially, and the viscous relaxation was determined from the stress decay under sustained compression. The viscous relaxation was found not to change significantly with dose. However, Young's modulus was dose dependent; it approximately doubled in the gels between 0 Gy and 20 Gy. By fitting a second-order polynomial to the Young's modulus-versus-dose data, 99.4% of the variance in Young's modulus was shown to be associated with the change in dose. The precision of the gel production, irradiation and Young's modulus measurement combined was found to be 4% at 2 Gy and 3% at 20 Gy. Potential sources of measurement error, such as those associated with the boundary conditions in the compression measurement, inhomogeneous polymerization, temperature (up to 1% error) and the evaporation of water from the sample (up to 1% error), were estimated and discussed. It was concluded that Young's modulus could be used for dose determination. Imaging techniques such as elastography may help to achieve this if they can provide a local measurement of Young's modulus, which may eliminate problems associated with the boundaries (e.g. variation in coefficient of friction) and inhomogeneous polymerization. Elastography combined with a calibration should also be capable of mapping dose in three dimensions.

A Harmonic Dual-Frequency Transducer for Acoustic Cluster Therapy.

Acoustic Cluster Therapy (ACT) is a two-component formulation of commercially available microbubbles (Sonazoid; GE Healthcare, Oslo, Norway) and microdroplets (perfluorated oil) currently under development for cancer treatment. The microbubbles and microdroplets have opposite surface charges to form microbubble/microdroplet clusters, which are administered to patients together with a drug. When the clusters and drug reach the target tumour, two ultrasound (US) exposure regimes are used: First, high-frequency (>2.0 MHz) US evaporates the oil and forms ACT bubbles that lodge at the microvascular level. Second, low-frequency (0.5 MHz) US induces stable mechanical oscillations of the ACT bubbles, causing localized micro-streaming, radiation and shear forces that increase the uptake of the drugs to the target tumour. This report describes the design and testing of a dual-frequency transducer and a laboratory setup for pre-clinical in vivo studies of ACT on murine tumour models. The dual-frequency transducer utilizes the 5th harmonic (2.7 MHz) and fundamental (0.5 MHz) of a single piezoceramic disk for the high-frequency and low-frequency regimes, respectively. Two different aperture radii are used to align the high-frequency and low-frequency beam maxima, and the high-frequency -3 dB beam width diameter is 6 mm, corresponding to the largest tumour sizes we expect to treat. The low-frequency -3 dB beam width extends 6 mm. Although unconventional, the 5th harmonic exhibit a 44% efficiency and can therefore be used for transmission of acoustic energy. Moreover, both in vitro and in vivo measurements demonstrate that the 5th harmonic can be used to evaporate the microbubble/microdroplet clusters. For the in vivo measurements, we used the kidneys of non-tumour-bearing mice as tumour surrogates. Based on this, the transducer is deemed suited for pre-clinical in vivo studies of ACT and replaces a cumbersome test setup consisting of two transducers.

3-D Microvascular Imaging Using High Frame Rate Ultrasound and ASAP Without Contrast Agents: Development and Initial In Vivo Evaluation on Nontumor and Tumor Models.

Three-dimensional imaging is valuable to noninvasively assess angiogenesis given the complex 3-D architecture of vascular networks. The emergence of high frame rate (HFR) ultrasound, which can produce thousands of images per second, has inspired novel signal processing techniques and their applications in structural and functional imaging of blood vessels. Although highly sensitive vascular mapping has been demonstrated using ultrafast Doppler, the detectability of microvasculature from the background noise may be hindered by the low signal-to-noise ratio (SNR) particularly in the deeper region and without the use of contrast agents. We have recently demonstrated a coherence-based technique, acoustic subaperture imaging (ASAP), for super-contrast vascular imaging and illustrated the contrast improvement using HFR contrast-enhanced ultrasound. In this work, we provide a feasibility study for microvascular imaging using ASAP without contrast agents, and extend its capability from 2-D to volumetric vascular mapping. Using an ultrasound research system and a preclinical probe, we demonstrated the improved visibility of microvascular mapping using ASAP in comparison to ultrafast power Doppler (PD) on a mouse kidney, liver, and tumor without contrast agent injection. The SNR of ASAP images improves in average by 10 dB when compared to PD. In addition, directional velocity mappings were also demonstrated by combining ASAP with the phase information extracted from lag-1 autocorrelation. The 3-D vascular and velocity mapping of the mouse kidney, liver, and tumor were demonstrated by stacking the ASAP images acquired using 2-D ultrasound imaging and a trigger-controlled linear translation stage. The 3-D results depicted clear microvasculature morphologies and functional information in terms of flow direction and velocity in two nontumor models and a tumor model. In conclusion, we have demonstrated a new 3-D in vivo ultrasound microvascular imaging technique with significantly improved SNR over existing ultrafast Doppler.

The spatio-temporal strain response of oedematous and nonoedematous tissue to sustained compression in vivo.

Poroelastic theory predicts that compression-induced fluid flow through a medium reveals itself via the spatio-temporal behaviour of the strain field. Such strain behaviour has already been observed in simple poroelastic phantoms using generalised elastographic techniques (Berry et al. 2006a, 2006b). The aim of this current study was to investigate the extent to which these techniques could be applied in vivo to image and interpret the compression-induced time-dependent local strain response in soft tissue. Tissue on both arms of six patients presenting with unilateral lymphoedema was subjected to a sustained compression for up to 500 s, and the induced strain was imaged as a function of time. The strain was found to exhibit time-dependent spatially varying behaviour, which we interpret to be consistent with that of a heterogeneous poroelastic material. This occurred in both arms of all patients, although it was more easily seen in the ipsilateral (affected) arm than in the contralateral (apparently unaffected) arm in five out of the six patients. Further work would appear to be worthwhile to determine if poroelasticity imaging could be used in future both to diagnose lymphoedema and to explore the patho-physiology of the condition.

Characterization of the ultrasonic attenuation coefficient and its frequency dependence in a polymer gel dosimeter.

Research on polymer-gel dosimetry has been driven by the need for three-dimensional dosimetry, and because alternative dosimeters are unsatisfactory or too slow for that task. Magnetic resonance tomography is currently the most well-developed technique for determining radiation-induced changes in polymer structure, but quick low-cost alternatives remain of significant interest. In previous work, ultrasound attenuation and speed of sound were found to change as a function of absorbed radiation dose in polymer-gel dosimeters, although the investigations were restricted to one ultrasound frequency. Here, the ultrasound attenuation coefficient mu in one polymer gel (MAGIC) was investigated as a function of radiation dose D and as a function of ultrasonic frequency f in a frequency range relevant for imaging dose distributions. The nonlinearity of the frequency dependence was characterized, fitting a power-law model mu = af(b); the fitting parameters were examined for potential use as additional dose readout parameters. In the observed relationship between the attenuation coefficient and dose, the slopes in a quasi-linear dose range from 0 to 30 Gy were found to vary with the gel batch but lie between 0.0222 and 0.0348 dB cm(-1) Gy(-1) at 2.3 MHz, between 0.0447 and 0.0608 dB cm(-1) Gy(-1) at 4.1 MHz and between 0.0663 and 0.0880 dB cm(-1) Gy(-1) at 6.0 MHz. The mean standard deviation of the slope for all samples and frequencies was 15.8%. The slope was greater at higher frequencies, but so were the intra-batch fluctuations and intra-sample standard deviations. Further investigations are required to overcome the observed variability, which was largely associated with the sample preparation technique, before it can be determined whether any frequency is superior to others in terms of accuracy and precision in dose determination. Nevertheless, lower frequencies will allow measurements through larger samples. The fit parameter a of the frequency dependence, describing the attenuation coefficient at 1 MHz, was found to be dose dependent, which is consistent with our expectations, as polymerization is known to be associated with increased absorption of ultrasound. No significant dose dependence was found for the fit parameter b, which describes the nonlinearity with frequency. This is consistent with the increased absorption being due to the introduction of new relaxation processes with characteristic frequencies similar to those of existing processes. The data presented here will help with optimizing the design of future 3D dose-imaging systems using ultrasound methods.

Towards an acoustic model-based poroelastic imaging method: II. experimental investigation.

Soft biological tissue contains mobile fluid. The volume fraction of this fluid and the ease with which it may be displaced through the tissue could be of diagnostic significance and may also have consequences for the validity with which strain images can be interpreted according to the traditional idealizations of elastography. In a previous paper, under the assumption of frictionless boundary conditions, the spatio-temporal behavior of the strain field inside a compressed cylindrical poroelastic sample was predicted (Berry et al. 2006). In this current paper, experimental evidence is provided to confirm these predictions. Finite element modeling was first used to extend the previous predictions to allow for the existence of contact friction between the sample and the compressor plates. Elastographic techniques were then applied to image the time-evolution of the strain inside cylindrical samples of tofu (a suitable poroelastic material) during sustained unconfined compression. The observed experimental strain behavior was found to be consistent with the theoretical predictions. In particular, every sample studied confirmed that reduced values of radial strain advance with time from the curved cylindrical surface inwards towards the axis of symmetry. Furthermore, by fitting the predictions of an analytical model to a time sequence of strain images, parametric images of two quantities, each related to one or more of three poroelastic material constants were produced. The two parametric images depicted the Poisson's ratio (nu(s)) of the solid matrix and the product of the aggregate modulus (H(A)) of the solid matrix with the permeability (k) of the solid matrix to the pore fluid. The means of the pixel values in these images, nu(s) = 0.088 (standard deviation 0.023) and H(A)k = 1.449 (standard deviation 0.269) x 10(-7) m(2) s(-1), were in agreement with values derived from previously published data for tofu (Righetti et al. 2005). The results provide the first experimental detection of the fluid-flow-induced characteristic diffusion-like behavior of the strain in a compressed poroelastic material and allow parameters related to the above material constants to be determined. We conclude that it may eventually be possible to use strain data to detect and measure characteristics of diffusely distributed mobile fluid in tissue spaces that are too small to be imaged directly.

Physical parameters affecting ultrasound/microbubble-mediated gene delivery efficiency in vitro.

Ultrasound (US)/microbubble-mediated gene delivery is a technology with many potential advantages suited to clinical application. Previous studies have demonstrated transfection but many are unsatisfactory in respect to the exposure apparatus, lack of definition of the US field or the limitations on parameters that can be explored using clinical diagnostic US machines. We investigated individual exposure parameters using a system minimising experimental artefacts and allowing control of many parameters of the US field. Using a 1-MHz transducer we systematically varied US parameters, the duration of exposure and the microbubble and DNA concentrations to optimise gene delivery. Delivery was achieved, using lipid microbubbles (SonoVue) and clinically acceptable US exposures, to adherent cells at efficiencies of approximately 4%. The acoustic pressure amplitude (0.25 MPa peak-negative), pulse repetition frequency (1-kHz) and duration of exposure (10 s) were important in optimising gene delivery with minimal impact on cell viability. These findings support the hypothesis that varying the physical parameters of US-mediated gene delivery has an affect on both efficiency and cell viability. These data are the first in terms of their thorough exploration of the US parameter space and will be the basis for more-informed approaches to developing clinical applications of this technology.

Vessel orientation-dependent sensitivity of optoacoustic imaging using a linear array transducer.

For clinical optoacoustic imaging, linear probes are preferably used because they allow versatile imaging of the human body with real-time display and free-hand probe guidance. The two-dimensional (2-D) optoacoustic image obtained with this type of probe is generally interpreted as a 2-D cross-section of the tissue just as is common in echo ultrasound. We demonstrate in three-dimensional simulations, phantom experiments, and in vivo mouse experiments that for vascular imaging this interpretation is often inaccurate. The cylindrical blood vessels emit anisotropic acoustic transients, which can be sensitively detected only if the direction of acoustic radiation coincides with the probe aperture. Our results reveal for this reason that the signal amplitude of different blood vessels may differ even if the vessels have the same diameter and initial pressure distribution but different orientation relative to the imaging plane. This has important implications for the image interpretation, for the probe guidance technique, and especially in cases when a quantitative reconstruction of the optical tissue properties is required.

Quantitative ultrasonic elastography for gel dosimetry.

Advanced radiation techniques such as intensity-modulated radiotherapy (IMRT) for complex geometries in which targets are close to organs at risk have been introduced in radiation therapy, creating a need for procedures that allow easy three-dimensional (3-D) measurement of dose for verification purposes. Polymer gels that change their material properties when irradiated have been suggested for such use. For example, the change in their magnetic properties has been thoroughly investigated with magnetic resonance imaging (MRI). Also, we have previously shown that the mechanical stiffness, i.e., Young's modulus, of these gels changes with dose. This finding prompted us to assess whether we can image a radiation-induced stiffness distribution with quantitative ultrasound elastography and whether the stiffness distribution is correlated with the dose distribution. A methacrylic-acid-based gel was loaded with scatterers to create an ultrasound echoic signal. It was irradiated to create a rod-like region of increased stiffness with a 10 x 10 mm(2) cross-section. The gel block was compressed in a frame that restricted the movement of the gel to planes orthogonal to the long axis of the irradiated region and ultrasonic echo data were acquired in the central plane during compression. This simplified irradiation pattern and experimental set-up were designed to approximate plane-strain conditions and was chosen for proof of concept. The movement of the gel was tracked from ultrasound images of a different compressional state using cross-correlation, enabling a displacement map to be created. The shear modulus was reconstructed using an inverse algorithm. The role of the magnitude of the regularization parameter in the inverse problem and the boundary conditions in influencing the spatial distribution of stiffness and, thus, final dose contrast was investigated through parametric studies. These parameters were adjusted using prior knowledge about the stiffness in parts of the material, e.g., the background was not irradiated and therefore its stiffness was homogeneous. It was observed that a suitable choice for these reconstruction parameters was essential for a quantitative application of stiffness measurement such as dosimetry. The dose contrast and distribution found with the optimal parameters were close to those obtained with MRI. Initial results reported in this article are encouraging and indicate that with ongoing refinement of ultrasound elastography techniques and accompanying inverse algorithms, this approach could play an important role in gel dosimetry.

Targeted retroviral gene delivery using ultrasound.

BACKGROUND: Achieving specificity of delivery represents a major problem limiting the clinical application of retroviral vectors for gene therapy, whilst lack of efficiency and longevity of gene expression limit non-viral techniques. Ultrasound and microbubble contrast agents can be used to effect plasmid DNA delivery. We therefore sought to evaluate the potential for ultrasound/microbubble-mediated retroviral gene delivery. METHODS: An envelope-deficient retroviral vector, inherently incapable of target cell entry, was combined with cationic microbubbles and added to target cells. The cells were exposed to pulsed 1 MHz ultrasound for 5 s and subsequently analysed for marker gene expression. The acoustic pressure profile of the ultrasound field, to which transduction efficiency was related, was determined using a needle hydrophone. RESULTS: Ultrasound-targeted gene delivery to a restricted area of cells was achieved using virus-loaded microbubbles. Gene delivery efficiency was up to 2% near the beam focus. Significant transduction was restricted to areas exposed to > or = 0.4 MPa peak-negative acoustic pressure, despite uniform application of the vector. An acoustic pressure-dependence was demonstrated that can be exploited for targeted retroviral transduction. The mechanism of entry likely involves membrane perturbation in the vicinity of oscillating microbubbles, facilitating fusion of the viral and cell membranes. CONCLUSIONS: We have established the basis of a novel retroviral vector technology incorporating favourable aspects of existing viral and non-viral gene delivery vectors. In particular, transduction can be controlled by means of ultrasound exposure. The technology is ideally suited to targeted delivery following systemic vector administration.

Quantitative discrimination of pigmented lesions using three-dimensional high-resolution ultrasound reflex transmission imaging.

High-resolution ultrasound-reflex transmission imaging is a non-invasive method that can be performed in vivo. We have adapted and refined this technique for skin imaging. Scans can be analyzed to produce objective parameters. Previous work has highlighted sonographic differences between benign and malignant lesions. The aim of this study was to produce and test numerical parameters from ultrasound skin images that would quantify the acoustic differences between common pigmented lesions, which may aid their discrimination from melanoma. We report our findings for randomly selected patients referred from primary care with suspected melanoma. Those subsequently classified as malignant melanoma (MM), seborrheic keratosis (SK), and benign nevi by a consultant dermatologist (n=87) were imaged by high-resolution ultrasound-reflex transmission imaging. Using surrounding normal skin as a control, numerical sonographic parameters were derived for each lesion giving a relative measure of surface sound reflectance, intra-lesional sound reflection, total sound attenuation, and the relative uniformity of each parameter across the tumor. Significant quantitative differences existed between benign and malignant pigmented lesions studied. Sufficient discrimination was produced between MM (n=25), SKs (n=24) and other benign-pigmented lesions (n=38) to potentially reduce the referral of benign tumors by 65% without missing melanoma.

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound.

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.

Dual-frequency ultrasound examination of skin and subcutis thickness in breast cancer-related lymphedema.

Breast cancer-related lymphedema (BCRL) is a chronic swelling of the arm that sometimes follows breast cancer treatment. Clinically, both skin and subcutis are swollen. Edema is considered to be predominantly subcutaneous and of an even distribution. The purpose of this study was to quantify the degree and uniformity of skin and subcutis swelling around the forearms of women with BCRL. Ten women with BCRL were recruited. Both forearms were examined using 20 MHz ultrasound to visualize the skin and 7 MHz ultrasound to visualize the subcutis. Skin thickness was between the bottom of the entry-echo and the skin-subcutis boundary. Subcutis thickness was measured between the skin-subcutis boundary and the subcutis-muscle boundary. Both average skin thickness (1.97 +/- 1.00 mm) and average subcutis thickness (10.32 +/- 5.63 mm) were greater in the ipsilateral arm than in the contralateral arm (skin 1.12 +/- 0.14 mm, subcutis 5.58 +/- 2.04 mm, p < 0.01, t-test). The degree of increase in skin thickness did not vary around the arm (p > 0.05, ANOVA), while the degree of increase in subcutis thickness did vary (p < 0.05). Skin thickness correlated negatively with subcutis thickness in the contralateral arm, but correlated positively in the ipsilateral arm. The skin and subcutis are thickened in the ipsilateral arm of patients with BCRL. Skin thickness is increased uniformly around the arm and correlates strongly with the degree of swelling, while subcutis swelling varies. The measurement of skin thickness using ultrasound may form a useful clinical tool in the diagnosis of lymphedema and also aid further investigation of therapeutic techniques.