Institute of Fundamental Technological Research

The HIFU ablation technique is limited by the long duration of the procedure, which results from the large difference between the size of the HIFU beam’s focus and the tumor size. Ablation of large tumors requires treating them with a sequence of single HIFU beams, with a specific time interval in-between. The aim of this study was to evaluate the biological effects induced in a malignant solid tumor of the rat mammary gland, implanted in adult Wistar rats, during HIFU treatment according to a new ablation plan which allowed researchers to significantly shorten the duration of the procedure. We used a custom, automated, ultrasound imaging-guided HIFU ablation device. Tumors with a 1 mm thickness margin of healthy tissue were subjected to HIFU. Three days later, the animals were sacrificed, and the HIFU-treated tissues were harvested. The biological effects were studied, employing morphological, histological, immunohistochemical, and ultrastructural techniques. Massive cell death, hemorrhages, tissue loss, influx of immune cells, and induction of pro-inflammatory cytokines were observed in the HIFU-treated tumors. No damage to healthy tissues was observed in the area surrounding the safety margin. These results confirmed the efficacy of the proposed shortened duration of the HIFU ablation procedure and its potential for the treatment of solid tumors.

HIFU thermal ablation, breast cancer model, treatment plan, morphology, histology, ultrastructure, immune response, cell death, apoptosis, necrosis

High Intensity Focused Ultrasound (HIFU) is used in clinical practice for thermal ablation of malignant and benign solid tumors located in various organs. One of the reason limiting the wider use of this technology is the long treatment time resulting from i.a. the large difference between the size of the focal volume of the heating beam and the size of the tumor. Therefore, the treatment of large tumors requires scanning their volume with a sequence of single heating beams, the focus of which is moved in the focal plane along a specific trajectory with specific time and distance interval between sonications. To avoid an undesirable increase in the temperature of healthy tissues surrounding the tumor during scanning, the acoustic power and exposure time of each HIFU beam as well as the time intervals between sonications should be selected in such a way as to cover the entire volume of the tumor with necrosis as quickly as possible. This would reduce the costs of treatment. The aim of this study was to quantitatively evaluate the hypothesis that selecting the average acoustic power and exposure time for each individual heating beam, as well as the temporal intervals between sonications, can significantly shorten treatment time. Using 3D numerical simulations, the dependence of the duration of treatment of a tumor with a diameter of 5 mm or 9 mm (requiring multiple exposure to the HIFU beam) on the sonication parameters (acoustic power, exposure time) of each single beam capable of delivering the threshold thermal dose (CEM43 = 240 min) to the treated tissue volume was examined. The treatment duration was determined as the sum of exposure times to individual beams and time intervals between sonications. The tumor was located inside the ex vivo tissue sample at a depth of 12.6 mm. The thickness of the water layer between the HIFU transducer and the tissue was 50 mm. The sonication and scanning parameters selected using the developed algorithm shortened the duration of the ablation procedure by almost 14 times for a 5-mm tumor and 20 times for a 9-mm tumor compared to the duration of the same ablation plan when a HIFU beam was used of a constant acoustic power, constant exposure time (3 s) and constant long time intervals (120 s) between sonications. Results of calculations of the location and size of the necrotic lesion formed were experimentally verified on ex vivo pork loin samples, showing good agreement between them. In this way, it was proven that the proper selection of sonication and scanning parameters for each HIFU beam allows to significantly shorten the time of HIFU therapy.

HIFU ablation planning,HIFU therapy duration shortening,Tissue ex vivo,k-wave model,Experimental verification of therapy accuracy,Numerical simulation

Uveal melanoma is the most common intraocular cancer. The current eye-sparing treatment options include mostly plaque brachytherapy. However, the effectiveness of these methods is still unsatisfactory. In this article, we review several possible new treatment options. These methods may be based on the physical destruction of the cancerous cells by applying ultrasounds. Another approach may be based on improving the penetration of the anti-cancer agents. It seems that the most promising technologies from this group are based on enhancing drug delivery by applying electric current. Finally, new advanced nanoparticles are developed to combine diagnostic imaging and therapy (i.e., theranostics). However, these methods are mostly at an early stage of development. More advanced studies on experimental animals and clinical trials would be needed to introduce some of these techniques to routine clinical practice.

uveal melanoma, HIFU, iontophoresis, electrotherapy, nanoparticles, theranostics

We have designed and built low-cost compact ultrasound imaging-guided robotic HIFU (High-Intensity Focused Ultrasound) ablation device for thermal damage of solid tumors in small animals. Before this device is used to treat animals, experimental studies on ex vivo tissues were necessary to assess the accuracy of its targeting, ensuring the safety of therapy. The objective of this study was to assess the targeting accuracy of our device in the focal and axial plane of the HIFU beam using ex vivo tissue embedded in a reference cylindrical chamber inside which a coaxial cylindrical volume with a smaller diameter was ablated. HIFU beams with selected acoustic parameters, generated by a singe-element bowl-shaped 64-mm HIFU transducer operating at 1.08 MHz or 3.21 MHz frequency, were propagated in two-layer media: water-tissue (50 mm-40 mm) and focused at 12.6-mm depth below the tissue surface. Cylindrical necrotic lesions of various size were created by moving the chamber using a computercontrolled precise positioning unit. Lesions formed were compared with those intended for treatment using various visualization methods and displacement between their centers were determined. The targeting accuracy in the focal and axial planes was found to be respectively about 98% and 86% when determined from photos and about 88% and 76% when determined from MR images. The displacement between the centers of the necrotic lesion formed and planned for treatment was about 1 mm in the focal plane and about 2 mm in the axial plane. Our ablation device can be used as an effective and safe tool to plan, monitor and treat solid tumors in small animals and to test new anti-cancer drugs in preclinical studies.

We have designed and built ultrasound imaging-guided HIFU ablative device for preclinical studies on small animals. Before this device is used to treat animals, ex vivo tissue studies were necessary to determine the location and extent of necrotic lesions created inside tissue samples by HIFU beams depending on their acoustic properties. This will allow to plan the beam movement trajectory and the distance and time intervals between exposures leading to necrosis covering the entire treated volume without damaging the surrounding tissues. This is crucial for therapy safety. The objective of this study was to assess the impact of sonication parameters on the size of necrotic lesions formed by HIFU beams generated by 64-mm bowl-shaped transducer used, operating at 1.08 MHz or 3.21 MHz. Multiple necrotic lesions were created in pork loin samples at 12.6-mm depth below tissue surface during 3-s exposure to HIFU beams with fixed duty-cycle and varied pulse-duration or fixed pulse-duration and varied duty-cycle, propagated in two-layer media: water-tissue. After exposures, the necrotic lesions were visualized using magnetic resonance imaging and optical imaging (photos) after sectioning the samples. Quantitative analysis of the obtained results allowed to select the optimal sonication and beam movement parameters to suport planning of effective therapy.

automated ultrasound imaging-guided HIFU ablation system, ex vivo tissue, ultrasonic exposure parameters, extent of necrotic lesions

Malignant glial tumors (gliomas) are the second (after cerebral stroke) cause of death from diseases of the central nervous system. The current routine therapy, involving a combination of tumor resection, radio-, and chemo-therapy, only modestly improves survival. Sonodynamic therapy (SDT) has been broadly defined as a synergistic effect of sonication applied in combination with substances referred to as "sonosensitizers". The current review focuses on the possibility of the use of tumor-seeking sonosensitizers, in particular 5-aminolevulinic acid, to control recurring gliomas. In this application, SDT employs a principle similar to that of the more widely-known photodynamic therapy of superficially located cancers, the difference being the use of ultrasound instead of light to deliver the energy necessary to eliminate the sensitized malignant cells. The ability of ultrasound to penetrate brain tissues makes it possible to reach deeply localized intracranial tumors such as gliomas. The major potential advantage of this variant of SDT is its relative non-invasiveness and possibility of repeated application. Until now, there have been no clinical data regarding the efficacy and safety of such treatment for malignant gliomas, but the preclinical data are encouraging.

glioma, ultrasound, sonodynamic therapy, ALA

Image-guided High Intensity Focused Ultrasound (HIFU) technique is dynamically developing technology for treating solid tumors due to its non-invasive nature. Before a HIFU ablation system is ready for use, the exposure parameters of the HIFU beam capable of destroying the treated tissue without damaging the surrounding tissues should be selected to ensure the safety of therapy. The purpose of this work was to select the threshold acoustic power as well as the step and rate of movement of the HIFU beam, generated by a transducer intended to be used in the HIFU ablation system being developed, by using an array of thermocouples and numerical simulations. For experiments a bowl-shaped 64-mm, 1.05 MHz HIFU transducer with a 62.6 mm focal length (f-number 0.98) generated pulsed waves propagating in two-layer media: water/ex vivo pork loin tissue (50 mm/40 mm) was used. To determine a threshold power of the HIFU beam capable of creating the necrotic lesion in a small volume within the tested tissue during less than 3 s each tissue sample was sonicated by multiple parallel HIFU beams of different acoustic power focused at a depth of 12.6 mm below the tissue surface. Location of the maximum heating as well as the relaxation time of the tested tissue were determined from temperature variations recorded during and after sonication by five thermo-couples placed along the acoustic axis of each HIFU beam as well as from numerical simulations. The obtained results enabled to assess the location of each necrotic lesion as well as to determine the step and rate of the HIFU beam movement. The location and extent of the necrotic lesions created was verified using ultrasound images of tissue after sonication and visual inspection after cutting the samples. The threshold acoustic power of the HIFU beam capable of creating the local necrotic lesion in the tested tissue within 3 s without damaging of surrounding tissues was found to be 24 W, and the pause between sonications was found to be more than 40 s.

automated HIFU ablation system, threshold acoustic power of HIFU beam, ex vivo tissue, necrotic lesion, thermocouple array

A technique using pulsed High Intensity Focused Ultrasound (HIFU) to destroy deep-seated solid tumors is a promising noninvasive therapeutic approach. A main purpose of this study was to design and test a HIFU transducer suitable for preclinical studies of efficacy of tested, anti-cancer drugs, activated by HIFU beams, in the treatment of a variety of solid tumors implanted to various organs of small animals at the depth of the order of 1–2 cm under the skin. To allow focusing of the beam, generated by such transducer, within treated tissue at different depths, a spherical, 2-MHz, 29-mm diameter annular phased array transducer was designed and built. To prove its potential for preclinical studies on small animals, multiple thermal lesions were induced in a pork loin ex vivo by heating beams of the same: 6 W, or 12 W, or 18 W acoustic power and 25 mm, 30 mm, and 35 mm focal lengths. Time delay for each annulus was controlled electronically to provide beam focusing within tissue at the depths of 10 mm, 15 mm, and 20 mm. The exposure time required to induce local necrosis was determined at different depths using thermocouples. Location and extent of thermal lesions determined from numerical simulations were compared with those measured using ultrasound and magnetic resonance imaging techniques and verified by a digital caliper after cutting the tested tissue samples. Quantitative analysis of the results showed that the location and extent of necrotic lesions on the magnetic resonance images are consistent with those predicted numerically and measured by caliper. The edges of lesions were clearly outlined although on ultrasound images they were fuzzy. This allows to conclude that the use of the transducer designed offers an effective noninvasive tool not only to induce local necrotic lesions within treated tissue without damaging the surrounding tissue structures but also to test various chemotherapeutics activated by the HIFU beams in preclinical studies on small animals.

spherical annular phased array transducer, pulsed HIFU beam, electronically adjustable focal length, local tissue heating, thermal ablation, necrotic lesion

The purpose of this work was to determine the influence of standing waves and possible multiple reflections under the conditions often encountered in examining the effects of ultrasound exposure on the cell cultures in vitro. More specifically, the goal was to quantitatively ascertain the influence of ultrasound exposure under free field (FF) and standing waves (SW) and multiple reflections (MR) conditions (SWMR) on the biological endpoint (50% cell necrosis). Such information would help in designing the experiments, in which the geometry of the container with biological tissue may prevent FF conditions to be established and in which the ultrasound generated temperature elevation is undesirable. This goal was accomplished by performing systematic, side-by-side experiments in vitro with C6 rat glioma cancer cells using 12 well and 96 well plates. It was determined that to obtain 50% of cell viability using the 12 well plates, the spatial average, temporal average (ISATA) intensities of 0.32 W/cm2 and 5.89 W/cm2 were needed under SWMR and FF conditions, respectively. For 96 well plates the results were 0.80 W/cm2 and 2.86 W/cm2 respectively. The corresponding, hydrophone measured pRMS maximum pressure amplitude values, were 0.71 MPa, 0.75 MPa, 0.75 MPa and 0.73 MPa, respectively. These results suggest that pRMS pressure amplitude was independent of the measurement set-up geometry and hence could be used to predict the cells' mortality threshold under any in vitro experimental conditions or even as a starting point for (pre-clinical) in vivo tests. The described procedure of the hydrophone measurements of the pRMS maximum pressure amplitude at the k/2 distance (here 0.75 mm) from the cell's level at the bottom of the dish or plate provides the guideline allowing the difference between the FF and SWMR conditions to be determined in any experimental setup. The outcome of the measurements also indicates that SWMR exposure might be useful at any ultrasound assisted therapy experiments as it permits to reduce thermal effects. Although the results presented are valid for the experimental conditions used in this study they can be generalized. The analysis developed provides methodology facilitating independent laboratories to determine their specific ultrasound exposure parameters for a given biological end-point under standing waves and multiple reflections conditions. The analysis also permits verification of the outcome of the experiments mimicking pre- and clinical environment between different, unaffiliated teams of researchers.

Standing wave, Ultrasound pressure, Ultrasound intensity, C6 glioma, Anticancer therapy, Sonodynamic therapy, Ultrasound bio-effects

In Poland cancer is the second cause of death overall, and the first before 65. Demand for new anticancer therapies is increasing every year. The main objective of studies on medical and technical aspects of new anticancer methods is to reduce unwanted side effects and costs associated with conventional methods of treatment. Percutaneous (noninvasive) HIFU (High Intensity Focused Ultrasound) technique gives the chance to radically reduce both of these factors. The main goal of this work is automation of HIFU technology for producing thermal damage to the entire volume of a solid breast tumor implanted into a rat mammary gland using the proposed bi-modal ultrasound equipment, enabling the ultrasonic heating of a small volume within the tumor under the ultrasonic imaging control, as well as 3D scanning of the heating beam focus throughout the entire tumor volume. Design of the proposed equipment includes the heating probe of low frequency (about 1MHz), allowing penetration of pulsed focused waves into tissues, and the linear phased array probe of high frequency (from 4 MHz to 10 MHz), allowing visualization of the locally heated area inside the tumor in real time. Automatic 3D scanning of the heating beam focus provides the thermal damage to its entire volume.

High Intensity Focused Ultrasound beam, focal volume, tissue damage

In therapeutic applications of High Intensity Focused Ultrasound (HIFU) the guidance of the HIFU beam and especially its focal plane is of crucial importance. This guidance is needed to appropriately target the focal plane and hence the whole focal volume inside the tumor tissue prior to thermo-ablative treatment and beginning of tissue necrosis. This is currently done using Magnetic Resonance Imaging that is relatively expensive. In this study an ultrasound method, which calculates the variations of speed of sound in the locally heated tissue volume by analyzing the phase shifts of echo-signals received by an ultrasound scanner from this very volume is presented. To improve spatial resolution of B-mode imaging and minimize the uncertainty of temperature estimation the acoustic signals were transmitted and received by 8 MHz linear phased array employing Synthetic Transmit Aperture (STA) technique. Initially, the validity of the algorithm developed was verified experimentally in a tissue-mimicking phantom heated from 20.6 to 48.6°C. Subsequently, the method was tested using a pork loin sample heated locally by a 2 MHz pulsed HIFU beam with focal intensity ISATA of 129 W/cm2. The temperature calibration of 2D maps of changes in the sound velocity induced by heating was performed by comparison of the algorithm-determined changes in the sound velocity with the temperatures measured by thermocouples located in the heated tissue volume. The method developed enabled ultrasound temperature imaging of the heated tissue volume from the very inception of heating with the contrast-to-noise ratio of 3.5–12 dB in the temperature range 21–56°C. Concurrently performed, conventional B-mode imaging revealed CNR close to zero dB until the temperature reached 50°C causing necrosis. The data presented suggest that the proposed method could offer an alternative to MRI-guided temperature imaging for prediction of the location and extent of the thermal lesion prior to applying the final HIFU treatment.

Ultrasonic temperature imaging, HIFU, Echo phase shift, Velocity image contrast

Sonodynamic therapy (SDT) is a promising technique based on the ability of certain substances, called sonosensitizers, to sensitize cancer cells to non-thermal effects of low-energy ultrasound waves, allowing their destruction. Sonosensitization is thought to induce cell death by direct physical effects such as cavitation and acoustical streaming as well as by complementary chemical reactions generating oxygen free radicals. One of the promising sonosensitizers is 5-aminolevulinic acid (ALA) which upon selective uptake by cancer cells is metabolized and accumulated as protoporphyrin IX. The objective of the study was to describe ALA-mediated sonodynamic effects in vitro on a rat RG2 glioma cell line. Glioma cells, seeded at the bottom of 96-well plates and incubated with ALA (10 μg/ml) for 6 h, were exposed to the sinusoidal US pulses with a resonance frequency of 1 MHz, 1000 μs duration, 0.4 duty-cycle, and average acoustic power varying from 2 W to 6 W. Ultrasound waves were generated by a flat circular piezoelectric transducer with a diameter of 25 mm. Cell viability was determined by MTT assay. Structural cellular changes were visualized with a fluorescence microscope. Signs of cytotoxicity such as a decrease in cell viability, chromatin condensation and apoptosis were found. ALA-mediated SDT evokes cytotoxic effects of low intensity US on rat RG2 glioma cells in vitro. This cell line is indicated for further preclinical assessment of SDT in in vivo conditions.

5-aminolevulinic acid, sonodynamic therapy, rat RG2 glioma cells, cell viability

Therapeutic and surgical applications of High Intensity Focused Ultrasound (HIFU) require monitoring of local temperature rises induced inside tissues. It is needed to appropriately target the focal plane, and hence the whole focal volume inside the tumor tissue, prior to thermo-ablative treatment, and the beginning of tissue necrosis. In this study we present an ultrasound method, which calculates the variations of the speed of sound in the locally heated tissue. Changes in velocity correspond to temperature change. The method calculates a 2D distribution of changes in the sound velocity, by estimation of the local phase shifts of RF echo-signals backscattered from the heated tissue volume (the focal volume of the HIFU beam), and received by an ultrasound scanner (23). The technique enabled temperature imaging of the heated tissue volume from the very inception of heating. The results indicated that the contrast sensitivity for imaging of relative changes in the sound speed was on the order of 0.06%; corresponding to an increase in the tissue temperature by about 2 °C.

HIFU, echo phase shift, parametric imaging, velocity/brightness CNR

A tissue thermal conductivity (Ks) is an important parameter which knowledge is essential whenever thermal fields induced in selected organs are predicted. The main objective of this study was to develop an alternative ultrasonic method for determining Ks of tissues in vitro suitable for living tissues. First, the method involves measuring of temperature-time T(t) rises induced in a tested tissue sample by a pulsed focused ultrasound with measured acoustic properties using thermocouples located on the acoustic beam axis. Measurements were performed for 20-cycle tone bursts with a 2 MHz frequency, 0.2 duty-cycle and 3 different initial pressures corresponding to average acoustic powers equal to 0.7 W, 1.4 W and 2.1 W generated from a circular focused transducer with a diameter of 15 mm and f-number of 1.7 in a two-layer system of media: water/beef liver. Measurement results allowed to determine position of maximum heating located inside the beef liver. It was found that this position is at the same axial distance from the source as the maximum peak-peak pressure calculated for each nonlinear beam produced in the two-layer system of media. Then, the method involves modeling of T(t) at the point of maximum heating and fitting it to the experimental data by adjusting Ks. The averaged value of Ks determined by the proposed method was found to be 0.5±0.02 W/(m·°C) being in good agreement with values determined by other methods. The proposed method is suitable for determining Ks of some animal tissues in vivo (for example a rat liver).

Acoustics, Sound pressure, Beef, Thermal conductivity, Thermocouples, Nonlinear systems, Sound waves, Bioacoustics

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43°C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity ISATA (spatial averaged, temporal averaged) varied from 0.33 W/cm2 to 8 W/cm2 (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43°C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm2.

Cancer cells, Photo-sensitizers, Sonodynamic therapy, Thermal effects, Ultrasonic beam properties

Therapeutic and surgical applications of focused ultrasound require monitoring of local temperature rises induced inside tissues. From an economic and practical point of view ultrasonic imaging techniques seem to be the most suitable for the temperature control. This paper presents an implementation of the ultrasonic echoes displacement estimation technique for monitoring of local temperature rise in tissue during its heating by focused ultrasound The results of the estimation were compared to the temperature measured with thermocouple. The obtained results enable to evaluate the temperature fields induced in tissues by pulsed focused ultrasonic beams using non-invasive imaging ultrasound technique

HIFU, therapeutic ultrasound, ultrasonic imaging, echo strain estimation

Sonodynamic therapy is the ultrasound dependent enhancement of the cytotoxic activities of certain drugs called sonosensitizers. The study of therapeutic efficacy of ultrasound is always preceded by in-vitro tests. In this work, two in-vitro sonication procedures were compared. One with the transducer positioned bellow the cell colony, radiating upward, with standing wave reflected from the water-air surface, the second, in the free field conditions. Efficiency of the cancer cells necrosis caused by ultrasound was compared with acoustical field intensity ISPTA measured by a hydrophone. The standing wave conditions effectively increased the intensity of the ultrasonic wave at the level of cells. To achieve 50% of cell viability, the intensity ISATA, decreased from 5.8 W/cm2 to 0.3 W/cm2. In summary, sonication in the standing wave conditions can effectively and reproducibly destroy cells by ensuring the sterility and without the risk of overheating.

ultrasound, sonodynamic therapy, cancer cells, necrosis

In many therapeutic applications of a pulsed focused ultrasound with various intensities the finite-amplitude acoustic waves propagate in water before penetrating into tissues and their local heating. Water is used as the matching, cooling and harmonics generating medium. In order to design ultrasonic probes for various therapeutic applications based on the local tissue heating induced in selected organs as well as to plan ultrasonic regimes of treatment a knowledge of pressure variations in pulsed focused nonlinear acoustic beams produced in layered media is necessary. The main objective of this work was to verify experimentally the applicability of the recently developed numerical model based on the Time- Averaged Wave Envelope (TAWE) approach (Wójcik et al., 2006) as an effective research tool for predicting the pulsed focused nonlinear fields produced in two-layer media comprising of water and testedmaterials (with attenuation arbitrarily dependent on frequency) by clinically relevant axially-symmetric therapeutic sources. First, the model was verified in water as a reference medium with known linear and nonlinear acoustic properties. The measurements in water were carried out at a 25◦C temperature using a 2.25 MHz circular focused (f/3.0) transducer with an effective diameter of 29 mm. The measurement results obtained for 8-cycle tone bursts with three different initial pressure amplitudes varied between 37 kPa and 113 kPa were compared with the numerical predictions obtained for the source boundary condition parameters determined experimentally. The comparison of the experimental results with those simulated numerically has shown that the model based on the TAWE approach predicts well both the spatial-peak and spatial-spectral pressure variations in the pulsed focused nonlinear beams produced by the transducer used in water for all excitation levels complying with the condition corresponding to weak or moderate source-pressure levels. Quantitative analysis of the simulated nonlinear beams from circular transducers with ka ≫ 1 allowed to show that the axial distance at which sudden accretion of the 2nd or higher harmonics amplitude appears is specific for this transducer regardless of the excitation level providing weak to moderate nonlinear fields. For the transducer used, the axial distance at which the 2nd harmonics amplitude suddenly begins to grow was found to be equal to 60 mm. Then, the model was verified experimentally for two-layer parallel media comprising of a 60-mm water layer and a 60-mm layer of 1.3-butanediol (99%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). This medium was selected because of its tissue-mimicking acoustic properties and known nonlinearity parameter B/A. The measurements of both, the peak- and harmonic-pressure variations in the pulsed nonlinear acoustic beams produced in two-layer media (water/1.3-butanediol) were performed for the same source boundary conditions as in water. The measurement results were compared with those simulated numerically. The good agreement between the measured data and numerical calculations has shown that the model based on the TAWE approach is well suited to predict both the peak and harmonic pressure variations in the pulsed focused nonlinear sound beams produced in layered media by clinically relevant therapeutic sources. Finally, the pulsed focused nonlinear fields from the transducer used in two-layer media: water/castor oil, water/silicone oil (Dow Corning Ltd., Coventry, UK), water/human brain and water/pig liver were predicted for various values of the nonlinearity parameter of tested media.

therapeutic ultrasound, circular focused transducers, pulsed nonlinear acoustic pressure beams, layered media, numerical modeling and experiments

This work addresses the difficulties in the measurements of the nonlinear medium parameter B/A and presents a modification of the finite amplitude method (FAM), one of the accepted procedures to determine this parameter. The modification is based on iterative, hybrid approach and entails the use of the versatile and comprehensive model to predict distortion of the pressure–time waveform and its subsequent comparison with the one experimentally determined. The measured p–t waveform contained at least 18 harmonics generated by 2.25 MHz, 29 mm effective diameter, single element, focused PZT source (f-number 3.5) and was recorded by Sonora membrane hydrophone calibrated in the frequency range 1– 40 MHz. The hydrophone was positioned coaxially at the distal end of the specially designed, two-section assembly comprising of one, fixed length (60 mm), water-filled cylindrical container and the second, variable length (60–120 mm) container that was filled with unknown medium. The details of the measurement chamber are described and the reasons for this specific design are analyzed. The data were collected with the variable length chamber filled with 1.3-butanediol, which was used as a close approximation of tissue mimicking phantom. The results obtained provide evidence that a novel combination of the FAM with the semi-empirical nonlinear propagation model based on the hyperbolic operator is capable of reducing the overall uncertainty of the B/A measurements as compared to those reported in the literature. The overall uncertainty of the method reported here was determined to be ±2%, which enhances the confidence in the numerical values of B/A measured for different, clinically relevant media. Optimization of the approach is also discussed and it is shown that it involves an iterative procedure that entails a careful selection of the acoustic source and its geometry and the axial distance over which the measurements need to be performed. The optimization also depends critically on the experimental determination of the source surface pressure amplitude.

pulsed finite-amplitude acoustic waves, nonlinear propagation, nonlinearity parameter B/A

The aim of this work is mathematical modeling and numerical calculation in space and time of temperature fields induced by low power focused ultrasound beams in soft tissue in vivo after few minutes exposure time. These numerical predictions are indispensable for planning of various ultrasound therapeutic applications. Both, the acoustic pressure distribution and power density of heat sources induced in tissue, were calculated using the numerical solution to the second order nonlinear differential wave equation describing propagation of the high intensity acoustic wave in three-layer structure of nonlinear attenuating media. The problem of the heat transfer in living tissues is modelled by the Pennes equation, which accounts for the effects of heat diffusion, blood perfusion losses and metabolism rate. Boundary conditions and geometry are chosen according to the anatomical dimensions of a rat liver. The obtained results are compared with those calculated previously and verified experimentally for temperature elevations induced by ultrasound in liver samples in vitro. The analysis of the results emphasizes the value of the blood perfusion and the values of heat conductivity on the temperature growth rate. The numerical calculations of temperature fields were performed using the ABAQUS FEM software package. The thermal and acoustic properties of the liver and water being the input parameters to the numerical model were taken from the published data in cited references. The range of thermal conductivity coefficient of living tissue is obtained from the model of two-phase composite medium with given microstructure. The first component is a “solid” tissue and the second one corresponds to blood vessels area. The circular focused ultrasonic transducer with a diameter of 15 mm, focal length of 25 mm and resonance frequency of 2 MHz has been used to generate the pulsed ultrasonic beam in a very introductory experiment in vivo, which has been performed. Numerical prediction confirms qualitatively its results.

focused ultrasound, soft tissues, local thermal fields, numerical modelling

Many therapeutic applications of pulsed focused ultrasound are based on heating of detected lesions which may be localized in tissues at different depths under the skin. In order to concentrate the acoustic energy inside tissues at desired depths a new approach using a planar multi-element annular array transducer with an electronically adjusted time-delay of excitation of its elements, was proposed. The 7-elements annular array transducer with 2.4 MHz center operating frequency and 20 mm outer diameter was produced. All its elements (central disc and 6 rings) had the same radiating area. The main purpose of this study was to investigate thermal fields induced in bovine liver in vitro by pulsed focused ultrasonic beams with various acoustic properties and electronically steered focal plane generated from the annular array transducer used. The measurements were performed for the radiating beams with the 20 mm focal depth. In order to maximize nonlinear effects introducing the important local temperature rise, the measurements have been performed in two-layer media comprising of a water layer, whose thickness was specific for the transducer used and equal to 13 mm, and the second layer of a bovine liver with a thickness of 20 mm. The thickness of the water layer was determined numerically as the axial distance where the amplitude of the second harmonics started to increase rapidly. The measurements of the temperature rise versus time were performed using a thermocouple placed inside the liver at the focus of the beam. The temperature rise induced in the bovine liver in vitro by beams with the average acoustic power of 1W, 2W, and 3W and duty cycle of 1/5, 1/15 and 1/30, respectively, have been measured. For each beam used the exposure time needed for the local tissue heating to the temperature of 43◦C (used in therapies based on ultrasonic enhancement of drug delivery or in therapies involving stimulation of immune system by enhancement of the heat shock proteins expression) and to the temperature of 56◦C (used in HIFU therapies) was determined. Two sets of measurements were done for each beam considered. First, the thermocouple measurement of the temperature rise was done and next, the real-time monitoring of dynamics of growth of the necrosis area by using ultrasonic imaging technique, while the sample was exposed to the same acoustic beam. It was found that the necrosis area becomes visible in the ultrasonic image only for beams with the average acoustic power of 3 W, although after cutting the sample the thermally ablated area was visible with the naked eye even for the beams with lower acoustic power. The quantitative analysis of the obtained results allowed to determine the exposure time needed to get the necrosis area visible in the ultrasonic image.

annular array transducer, pulsed focused nonlinear ultrasound, electronically moved focus, tissue heating, biological effects, tissue necrosis

Beneficial biological effects in soft tissues can be induced by focused ultrasound of low intensity (LIFU). For example, increasing of cells immunity to stress can be accomplished through the enhanced heat shock proteins (Hsp) expression induced by the low intensity focused ultrasound. The possibility to control the Hsp expression enhancement in soft tissues in vivo can be the potential new therapeutic approach to neurodegenerative diseases that utilizes the known feature of cells to increase their immunity to stresses through the Hsp expression enhancement. The controlling of the Hsp expression enhancement by adjusting the level of exposure to ultrasound energy would allow evaluating of ultrasound-mediated treatment efficiency. Our objective was to develop the numerical model capable of predicting in space and time temperature fields induced in multilayer nonlinear attenuating media by a circular focused transducer generating pulsed acoustic waves and to compare the results calculated for two-layer configuration of media: water - fresh rat liver with the experimental data. The measurements of temperature variations versus time at 5 points on the acoustic beam axis within the tissue sample were performed using 0.2-mm diameter thermocouples. Temperature fields were induced by the transducer with 15-mm diameter, 25-mm focal length and 2-MHz centre frequency generating tone bursts with the intensity ISPTA varied between 0.45 W/cm2 and 1.7 W/cm2 and duration varied between 20 and 500 cycles at the same 20-% duty cycle and 20-min exposure time. Quantitative analysis of the obtained results allowed to show that, for example, for the acoustic beam with intensity ISPTA = 1.13 W/cm2 exposure time to ultrasound should not be longer than 10 min to avoid cells necrosis following the 43-oC temperature threshold exceeding.

low intensity focused ultrasound, soft tissues, temperature fields, ultrasonic regimes, therapy efficiency

In this paper a new method utilizing nonlinear properties of tissues to improve contrast-to-noise ratio is presented. In our novel method the focused circular transducer is excited with two-tone bursts (including the 2.2 MHz fundamental and 4.4 MHz second harmonic frequencies) with specially coded polarization of each tone. This new approach was named Multitone Nonlinear Coding (MNC) because the choice of both tones polarization and amplitude law, allowing optimization of the probe receiving properties, depends on nonlinear properties of tissue. The numerical simulations of nonlinear fields in water and in tissue-like medium with absorption coefficient of 7 Np/(m•MHz) are performed. The comparison between the proposed method and the Pulse Inverse (PI) method is presented. The concept of the virtual fields was introduced to explain properties of both the Pulse Inversion and MNC methods and to compare their abilities. It was shown that for the same on-source pressure an application of the MNC method allows to decrease the mechanical index about 40 %, to improve lateral resolution from 10 to 30 % and to gain the signal-to-noise ratio up to 8 times with respect to the PI method.

harmonic imaging, ultrasonography, nonlinear propagation

The aim of this work is twofold. Firstly, to verify a theoretical model which is capable of predicting temperature fields appearing in soft tissues during their ultrasound treatment. Secondly, to analyze some aspects of the dynamics of Heat Shock Response induced by the heating process in the context of therapeutic treatment. The theoretical investigations and quantitive analysis of temperature increments at any field point versus time of heating process, depending on the heat source power, spatial distribution and duration as well as on the tissue thermal properties, has been carried out by Finite Element Method (FEM). The validation of the numerical model has been performed by comparison of the calculation results with the experimental data obtained by measuring in vitro of the 3D temperature increments induced in samples of the turkey and veal liver by the circular focused transducer with the diameter of 15 mm, focal length of 25 mm and resonance frequency of 2 MHz. Various ultrasonic regimes were considered. They were controlled by adjusting ultrasound power and exposure time. The heat shock proteins (HSP) and misfolded proteins (MFP) levels during the proposed cyclic sonification are presented.

heat-responsive gene therapy, temperature field, low-power focused ultrasound, soft tissues, ultrasonic regime control, heat sources distribution, heat shock proteins

Ultrasound is safe, convinient and inexpensive modality which may be useful for soft tissues treatment. A range of beneficial biological effects induced by ultrasound depends on the exposure level used during treatment. At high intensities instantaneous tissue necrosis is desired, whereas at lower intensities remedial reversible cellular effects may be produced. For example, increasing of cell immunity against stress can be obtained through the heat shock proteins (Hsp) expression enhancement. The possibility of the Hsp expression enhancement in soft tissues in vivo by means of controlled exposure to ultrasound would allow to evaluate the treatment efficiency. Ultrasonic regimes can be controlled by adjusting the ultrasound intensity, frequency, pulse duration, duty-cycle and exposure time. The goal of this work was to develop the numerical model capable of predicting in space and time the temperature fields induced by circular focused transducer generating tone bursts in multilayer nonlinear attenuating media, which is intended for the Hsp expression enhancement therapeutic applications. The acoustic pulsed pressure field generated by the transducer was calculated using our original 3D numerical solver [1]. For prediction of the temperature distributions in multilayer biological media the Pennes bio-heat transfer equation was solved numerically. The 3D thermal fields induced in a rat liver in vitro by a 2 MHz transducer of 15-mm diameter and 25-mm focal length during ultrasonic Hsp expression enhancement treatment using various acoustic beam intensities and exposure time was predicted.

multilayer biological media, ultrasound exposure parameters, local thermal fields, numerical prediction

The primary goal of this work was to verify experimentally the applicability of the recently introduced time-averaged wave envelope (TAWE) method as a tool for fast prediction of four dimensional (4D) pulsed nonlinear pressure fields from arbitrarily shaped acoustic sources in attenuating media. The experiments were performed in water at the fundamental frequency of 2.8 MHz for spherically focused (focal length F = 80 mm) square (20mm x 20 mm) and rectangular (10mm x 25 mm) sources similar to those used in the design of 1D linear arrays operating with ultrasonic imaging systems. The experimental results obtained with 10-cycle tone bursts at three different excitation levels corresponding to linear, moderately nonlinear and highly nonlinear propagation conditions (0.045, 0.225 and 0.45 MPa on-source pressure amplitude, respectively) were compared with those yielded using the TAWE approach. The comparison of the experimental results and numerical simulations has shown that the TAWE approach is well suited to predict (to within ± 1 dB) both, the spatial–temporal and spatial–spectral pressure variations in the pulsed nonlinear acoustic beams. The obtained results indicated that implementation of the TAWE approach enabled shortening of computation time in comparison with the time needed for prediction of the full 4D pulsed nonlinear acoustic fields using a conventional (Fourier-series) approach. The reduction in computation time depends on several parameters, including the source geometry, dimensions, fundamental resonance frequency, excitation level as well as the strength of the medium nonlinearity. For the non-axisymmetric focused transducers mentioned above and excited by a tone burst corresponding to moderately nonlinear and highly nonlinear conditions the execution time of computations was 3 and 12h, respectively, when using a 1.5 GHz clock frequency, 32-bit processor PC laptop with 2 GB RAM memory, only. Such prediction of the full 4D pulsed field is not possible when using conventional, Fourier-series scheme as it would require increasing the RAM memory by at least 2 orders of magnitude.

rectangular focused apertures, pulsed acoustic fields, nonlinear distortion, numerical modelling and experiments

The goal of this work was to verify experimentally the applicability of the recently developed Time-Averaged Wave Envelope (TAWE) method [1] as a tool for fast prediction of pulsed nonlinear pressure fields from focused nonaxisymmetric acoustic sources in attenuating media. The experiments were performed in water at the fundamental frequency of 2.8 MHz for spherically focused (focal length F = 80 mm) square (20 x 20 mm) and rectangular (10 x 25 mm) sources similar to those used in the design of 1D linear arrays operating with ultrasonic imaging systems. The experimental results obtained with 10-cycle tone bursts at three different excitation levels corresponding to linear, moderately nonlinear and highly nonlinear propagation conditions (0.045, 0.225 and 0.45 MPa on-source pressure amplitude, respectively) were compared with those yielded using the TAWE approach. Comparison of the experimental and numerical calculations results has shown that the TAWE approach is well suited to predict (to within ± 1 dB) both the spatial-temporal and spatial-spectral pressure variations in the pulsed nonlinear acoustic beams.

rectangular focused apertures, pulsed acoustic fields, nonlinear distortion, numerical modelling

The acoustic nonlinearity parameter, B/A, is a fundamental material constant characterizing nonlinear properties of biological media. Knowledge of the B/A of biological fluids or soft tissues through which pulsed acoustic waves generated from clinically relevant probes are propagating is necessary whenever high intensity pressure fields are produced. The numerical model recently developed in our lab, capable of predicting the pulsed sound fields generated from axisymmetric sources in nonlinear attenuating media, was a powerful instrument for investigating nonlinear acoustic fields produced from circular plane or focused sources in attenuating media in dependence on boundary condition parameters. Quantitative analysis of the obtained results enabled developing the alternative method for determination of the B/A parameter of biological media. First, the method involves measuring in the near field of a piezoelectric transducer the nonlinear waveform distortion of the pulsed acoustic wave propagating through the two-layer system of media: water-tested material. Then, the method involves numerical modeling, in frequency domain and under experimental boundary conditions, the nonlinear waveform distortion of the propagating wave by using the Time-Averaged Wave Envelope (TAWE) approach [1]. The obtained numerical simulation results were fitted to the experimental data by adjusting the B/A parameter of the tested material. The determined values of the B/A for standard media considered (corn oil, glycerol, pig blood, homogenized pig liver), whose density, sound velocity and attenuation law have been preliminary determined experimentally, are in a good agreement with those published. The proposed method ensure the decimal degree of accuracy, is relatively simple to use and requires small volume of tested materials that is important because of difficulty of their availability.

biological media, pulsed acoustic waves, nonlinear propagation, nonlinearity parameter

A novel, free from paraxial approximation and computationally efficient numerical algorithm capable of predicting 4D acoustic fields in lossy and nonlinear media from arbitrary shaped sources (relevant to probes used in medical ultrasonic imaging and therapeutic systems) is described. The new WE (wave envelopes) approach to nonlinear propagation modeling is based on the solution of the second order nonlinear differential wave equation reported in [J. Wojcik, J. Acoust. Soc. Am. 104 (1998) 2654-2663; V.P. Kuznetsov, Akust. Zh. 16 (1970) 548-553]. An incremental stepping scheme allows for forward wave propagation. The operator-splitting method accounts independently for the effects of full diffraction, absorption and nonlinear interactions of harmonics. The WE method represents the propagating pulsed acoustic wave as a superposition of wavelet-like sinusoidal pulses with carrier frequencies being the harmonics of the boundary tone burst disturbance. The model is valid for lossy media, arbitrarily shaped plane and focused sources, accounts for the effects of diffraction and can be applied to continuous as well as to pulsed waves. Depending on the source geometry, level of nonlinearity and frequency bandwidth, in comparison with the conventional approach the Time-Averaged Wave Envelopes (TAWE) method shortens computational time of the full 4D nonlinear field calculation by at least an order of magnitude; thus, predictions of nonlinear beam propagation from complex sources (such as phased arrays) can be available within 30-60 min using only a standard PC. The approximateratio between the computational time costs obtained by using the TAWE method and the conventional approach in calculations of the nonlinear interactions is proportional to (1/N)**2, and in memory consumption to 1/N where N is the average bandwidth of the individual wavelets. Numerical computations comparing the spatial field distributions obtained by using both the TAWE method and the conventional approach (based on a Fourier series representation of the propagating wave) are given for circular source geometry, which represents the most challenging case from the computational time point of view. For two cases, short (2 cycle) and long (8 cycle) 2 MHz bursts, the computational times were 10 min and 15 min versus 2 h and 8 h for the TAWE method versus the conventional method, respectively.

Nonliear propagation, Envelope waves, Fast calculations

The nonlinear pulsed acoustic pressure field from a focused square aperture is considered. Experimental measurements in water of a 4D sound field radiated from a 2.8 MHz focused square transducer of a 20 mm side and a 80 mm focal distance for excitation level producing an average acoustic pressure P0 = 0.14 MPa at its surface are presented. The obtained results are compared with the numerical calculation results for the same boundary conditions. The novel, free from paraxial approximation and computationally efficient numerical algorithm was used to simulate the 4D nonlinear pulsed pressure field from the nonaxisymmetric acoustic source. Our theoretical model was based on the Time-Averaged Pressure Envelope (TAPE) method recently developed that enable to represent the propagated pulsed disturbance as a superposition of sinusoidal wavelets with carrier frequencies being the harmonics of the initial tone burst and with envelopes determined by the TAPE method. The novel approach to the solution of the nonlinear wave equation enabled to simulate full 4D nonlinear field for given boundary conditions in a dozen or so minutes utilizing the computational power of the standard PC.

square spherically focused transducer, pulsed waves, nonlinear propagation in water, sound fields, numerical modelling

Imaging of living cells or tissues at a microscopic resolution, where GHz frequencies are used, provides a foundation for many new biological applications. The possible temperature increase causing a destructive influence on the living cells should be then avoided. However, there is no information on possible local temperature increases at these very high frequencies where, due to strongly focused ultrasonic beams, nonlinear propagation effects occur. Acoustic parameters of living cells were assumed to be close to those of water; therefore, the power density of heat sources in a water medium was determined as a basic quantity. Hence, the numerical solution of temperature distributions at the frequency of 1 GHz was computed for high and low powers generated by the transducer equal to 0.32 W and 0.002 W. In the first case, typical nonlinear propagation effects were demonstrated and, in the second one, propagation was almost linear. The focal temperature increase obtained in water equaled 14°C for the highest possible theoretical repetition frequency of fr = 10 MHz and for the thermal insulation at the sapphire lens-water boundary. Simultaneously, the scanning velocity of the tested object was assumed to be incomparably low in respect to the acoustic beam velocity. The maximum temperature increase in water occurred exactly at this boundary, being equal there to 20°C. It was shown that, first of all, the very high absorption of water was significant for the temperature distribution in the investigated region, suppressing the focal temperature peaks. Because the temperature increases are proportional to the repetition frequency, so for example, at its practical value of fr = 0.1 MHz, all temperature increases will be 100 times lower than listed above. For the low transducer power of 0.002 W, the corresponding temperature increases were about 140 times lower than those for the high power of 0.32 W. The presented solutions are devoted mainly to the reflection pulse mode; however, they can be also applied for the transmitting (continuous-wave) mode, as shown in an example. Pressure distributions were computed for the acoustic field of the microscope for the first and higher harmonics. Hence, at the frequency of 1 GHz, the effective focal radius in water measured as the −6-dB amplitude pressure drop was found to be 1,1 μm, and 0.7 μm for the second harmonic, independently of the assumed transducer power. So the width of the beam, scanning the living cells in the focal region, was equal to 2.2 μm at the fundamental frequency of 1 GHz.

Temperature, Acoustic microscopy, Living cells, Temperature increase, Pressure

Nonlinear propagation effects produced by focused pulses in blood were measured over a 20-cm range, being inspired by diagnostic applications in cardiology. The initial and maximum pressures applied during measurements in blood were equal to 0.40 MPapp and 0.76 MPapp, while the pressure estimated at the patient body surface equalled 0.70 MPapp. Measurements of the frequency characteristic and the linearity of the ultrasonic probe used in experiments were performed in water. A numerical procedure developed previously was applied in blood to calculate the pressure distribution of its first and second harmonics along the beam axis. The comparison of numerical and measured distributions in blood at a temperature of 37°C showed rather good agreement. Using numerical methods, a proportional growth of the second harmonic with the increased applied initial pressure was first observed, and finally the maximum limiting effect was found. In this way, much higher level of harmonics could be obtained. However, there arise the questions of the transmitting system construction and of the nonuniform resolution in the case of harmonic imaging when increasing the applied initial pressure.

Ultrasound, Pulses, Nonlinear propagation, Blood, Cardiology

Measurements in the very near field of piezoelectric transducers are fundamental for many ultrasonic problems. In such cases also the transducer vibrations should be known to perform mathematical models of radiated beams. Acoustic pressure measurements near to the transducer surface can give the necessary information. The pressure of the radiated wave at the transducer surface corresponds to its normal vibration velocity multiplied by the [ampersand]rho;c value of the medium. However, this is valid only for the central wave, when the edge wave of the transducer can be ignored. On the other hand, pressure measurements on and very near to the transducer surface are not possible because of the voltage leakage between the electronic transmitter and the PVDF hydrophone used in such measurements. By means of a numerical model, central and edge waves were found for a plane PZT transducer 7.5mm in radius, with the applied 2.7MHz voltage pulse composed of 3 cycles. Two types of boundary conditions of Dirichlet and Neumann were considered showing a negligible difference in the case of short pulses. Basing on numerical and experimental results, practical conditions were determined which make it possible to carry out pressure measurements in the very near field of the transducer, and hence to determine the transducer vibrations which are important for modeling ultrasonic pulse beams.

The effect of nonlinear propagation in fluid followed by soft tissue was studied both theoretically and experimentally for a most crucial case in obstetrical ultrasonography. For this purpose, short pressure pulses, with the duration time of 1.3 μs and a carrier frequency of 3 MHz, radiated by a concave transducer into water, with maximum intensities up to the value of 18 W/cm2, were computed and measured. The ultrasonic beam had the physical focus at the distance of 6.5 cm, where the highest focal intensity of ISPPA= 242 W/cm2 was obtained. In front of the transducer, at a distance of 7 cm, artificial tissue samples prepared on the basis of ground porcine kidney, with a thickness of 0.5, 1.5 and 3 cm, were placed in water. Pressure pulses and their spectral components were produced numerically and measured by means of a PVDF hydrophone in water before and after penetrating the tissue samples. The theoretical analysis and measurements were carried out, in every case, for two signal levels: for a high level assuring nonlinear propagation and for a low one where conditions of linear propagation were fulfilled. In this way, it was possible to compare directly the effects of nonlinear and linear propagation, in every case showing a good conformity of theoretical values with measured ones. A method of determination of the effective frequency response of the hydrophone was elaborated to enable quantitative comparisons of numerical and experimental results. The theoretical part of our study was based on a paper of Wójcik (1998), enabling us to compute the characteristic function of nonlinear increase of absorption. An agreement of up to 10% was obtained when comparing theoretical and measured values of these functions in the investigated beam in water and behind tissue samples. The results obtained showed that the recently given theory of nonlinear absorption, based on the spectral analysis and the elaborated numerical procedures, may be useful in various practical ultrasonic medical problems and also in technological applications.

Ultrasound, Pulses, Nonlinear propagation, Diagnostics

The theory of wave reflection from spherical obstacles was applied for determination of the cause of the shadow created by plane wave pulses incident on rigid, steel, gaseous spheres and on spheres made of kidney stones. The spheres were immersed in water which was assumed to be a tissuelike medium. Acoustic pressure distributions behind the spheres with the radii of 1 mm, 2.5 mm and 3.5 mm were determined at the frequency of 5 MHz. The use of the exact wave theory enabled us to take into account the diffraction effects. The computed pressure distributions were verified experimentally at the frequency of 5 MHz for a steel sphere with a 2.5-mm radius. The experimental and theoretical pulses were composed of about three ultrasonic frequency periods. Acoustic pressure distributions in the shadow zone of all spheres were shown in the amplitude axonometric projection, in the grey scale and also as acoustic isobar patterns. Our analysis confirmed existing simpler descriptions of the shadow from the point of view of reflection and refraction effects; however, our approach is more general, also including diffraction effects and assuming the pulse mode. The analysis has shown that gaseous spherical inclusions caused shadows with very high dynamics of acoustic pressures that were about 15 dB higher in relation to all the other spheres. The shadow length, determined as the length at which one observes a 6-dB drop of the acoustic pressure, followed the relation r−6dB = 3.7a2λ with the accuracy of about 20% independent of the sphere type. λ denotes the wavelength and a the sphere radius. Thus, a theoretical possibility of differentiating between gaseous and other inclusions and of estimation of the inclusion size in the millimeter range from the shadow was shown. The influence of the frequency-dependent attenuation on the shadow will be considered in the next study.

Shadow, Pulses, Spheres, Ultrasonography

Transient solution of the thermal conductivity equation for the three-dimensional case of the Gaussian ultrasonic focused beam was derived and applied for cases relevant to medical ultrasonography. Quantitative results for the case of a homogeneous medium with constant values of thermal coefficients and constant absorption as well as for the two-layer tissue model used in obstetrics were presented for various diagnostic probes used in ultrasonography. The possible effects of perfusion and nonlinear propagation were neglected. The results obtained are in agreement with results of other authors when considering the steady-state and the infinitely short insonation time. The computations show the influence of the insonation time on the temperature elevation, thus making it possible to introduce its value as a factor in limiting the possible harmful effects in ultrasonography. This has been shown in diagrams presenting the temperature distribution along the beam axis of 6 different diagnostic probes for various insonation times and demonstrating the corresponding temperature decrease when limiting the insonation time to 5 and 1 min. For instance, the highest temperature elevation (for probe number 1, see Table 1) decreases 2.6 and 5 times with respect to the steady-state temperature when the insonation time equals 5 and 1 min, respectively.

Temperature, Ultrasonography, Time, Hazard

Local ultrasonic tissue ablation is induced by a rapid (<3s) rise in temperature in a small ellipsoidal volume (about 13mm3) inside the tissue to a cytotoxic level when exposed to a high-intensity focused ultrasound (HIFU) beam. The aim of this study was to develop a numerical tool to predict the location and extent of a necrotic lesion formed locally inside the ex vivo tissue as a result of exposure to a single or multiple HIFU beam, ensuring the efficacy and safety of destroying solid tumors. The proposed tool was based on modelling the non-linear propagation of acoustic waves and heat transfer in heterogeneous media using the k-wave toolbox. The wave propagation equations were solved for two-layer (water/tissue) media. The source of the acoustic waves was a spherical bowl-shaped transducer with a resonance frequency of 1.08 MHz. The distribution of heat sources was determined from the calculated acoustic pressure distribution in the HIFU beam. The obtained temperature distributions during heating and cooling allowed calculation of the thermal dose and prediction of the extent of the necrotic lesion. The obtained results of numerical simulations were compared with the experimental data from previous studies. The mean difference between the calculated and measured length or diameter of a single exposure induced necrotic lesion was approximately 1 mm. In the case of a necrotic lesion induced by multiple exposures, the mean difference between the measured and calculated cross-sectional area of the planned necrotic lesion covered with necrosis was approximately 11.2 %.

HIFU, modelling, numerical simulations, treatment planning, ex vivo experiments, necrotic lesions, thermal ablation, experimental verification

Terapeutyczne i chirurgiczne zastosowania ogniskowych ultradźwięków wymagają monitorowania lokalnych zmian temperatury w tkance. Najkorzystniejsze z punktu widzenia użytkowego i ekonomicznego byłoby zastosowanie do tych celów technik ultradźwiękowych.
Praca przedstawia próbę zastosowania metody estymacji przemieszczenia ech do monitorowania zmian temperatury podczas ultradźwiękowego nagrzewania tkanki in vitro. Dane uzyskane drogą pomiarów ultradźwiękowych zostały przetworzone w celu wyznaczenia mapy przemieszczeń ech i odniesione do pomiarów rozkładu temperatury przeprowadzonych za pomocą termopar. Uzyskane wyniki umożliwiają ocenę pola temperatury i pozytywnie rokują połączeniu ultradźwiękowych technik nagrzewania i szacowania lokalnej temperatury tkanki.

obrazowanie temperatury, prędkość akustyczna

Nową metodą terapii antynowotworowej glejaków jest sonoterapia polegająca na jednoczesnym podawaniu leku kumulującego się w tkankach nowotworowych i napromieniowanie falą akustyczną. Skuteczność metody została sprawdzona na komórkach glejaka C6, poddanych działaniu kwasu aminolewulinowego (ALA) i fali ultradźwiękowej 1MHz o natężeniu 0.94, 1.88 i 3.77W/cm2 przez 3 min. Jednoczesne działanie fali akustycznej i ALA było skuteczniejsze (pozostało 15%-13% żywych komórek) niż samej fali akustycznej (41%-27%) dla natężeń 0.94 i 1.88W/cm2. Natężenie 3.77W/cm2 było wystarczające do zniszczenia większości komórek niezależnie od obecności ALA. Podczas eksperymentu zaobserwowano powstawanie fali stojącej, zwiększającej natężenie 7x-10x. Otrzymane wyniki wskazują na przydatność nowej techniki sonodynamicznej do terapii nowotworów.

ultradźwięki, sonoterapia, terapia antynowotworowa, glejaki

Therapeutic and surgical applications of focused ultrasound require monitoring of local temperature rises induced inside tissues. From an economic and practical point of view ultrasonic imaging techniques seem to be the best for a temperature control. In this work an attempt to apply the method of the ultrasonic echoes displacement estimation for monitoring local temperature rises in tissues during their heating by focused ultrasound is presented. The estimated temperature rise was compared with this measured by a thermocouple. The obtained results enable to evaluate the temperature fields induced in tissues by pulsed focused ultrasonic beams using non-invasive imaging ultrasound technique.

HIFU, therapeutic ultrasound, ultrasonic imaging, echo strain estimation

Many therapeutic applications of focused ultrasound are based on heating of a detected lesion which may be located inside tissues at different depths under a skin. In order to raise the tumor temperature the focal spot of the ultrasonic beam is guided to it. The focusing of acoustic energy in tissues at the desired depth can be achieved using single-element circular concave piezoelectric transducers with the selected diameters and radii of curvature. This solution is inefficient because it requires as many transducers as there are depths at which tumors are localized. In order to solve this problem a new approach, allowing to concentrate the acoustic energy at different depths inside tissues using a planar multi-element phased annular array with electronically steered focus, was proposed in this work. The main purpose of this study was to investigate the thermal fields induced in bovine liver in vitro by the pulsed focused ultrasound beam with various acoustic properties generated from the planar multi-element phased annular array with electronically steered focal spot. The array with 20 mm outer diameter and 2.4 MHz centre frequency has 7 elements with the same radiating surface. The electronic steering of the time-delay of excitation of each array element allowed to obtain the focal spot of the beam at any distance from the transducer surface. This paper presents measurement results performed for the beam focused at 20 mm distance. In order to maximize nonlinear propagation effects being one of the reason of the local temperature rise induced in tissues by focused ultrasound the measurements have been performed in two-layer parallel media of propagation comprising of a water layer, whose thickness was specific for the transducer used and equal to 13 mm, and of a bovine liver in vitro layer with a thickness of 27 mm. It was determined numerically as the axial distance at which the second harmonics amplitude for the tone burst generated by the transducer used in water starts to increase rapidly. The measurements of temperature rise versus time were performed using a thermocouple placed inside the liver at the beam focus. The temperature rises induced in the bovine liver in vitro for beams with the average acoustic power of 1W, 2W and 3W and duty cycle of 1/5, 1/15 and 1/30 have been measured. For each beam the exposure time needed for local heating of the liver to a temperature of 43 °C (used in therapies based on ultrasonic enhancement of drug delivery or therapies involving stimulation of immune system by enhancement of the heat shock proteins expression) and to a temperature of 56 °C (used in HIFU therapies) was determined. In each sample of liver for each considered beam at first, the measurement of temperature rise (with thermocouple) was carried out, and then, exposing the sample (without thermocouple) to the same beam the real-time monitoring of dynamics of the thermo ablation area growth was performed using the ultrasonic imaging technique. The necrosis spot becomes visible in the ultrasonic image only when the beam has sufficient acoustic power. The quantitative analysis of the obtained results allowed to determine the beam acoustic power and exposure time that are sufficient to visualize necrosis spot in the ultrasonic image.

phased annular array, pulsed focused nonlinear ultrasound beams, electronically movable focus, tissue heating, biological effects, tissue necrosis

The main aim of this work was numerical modeling of temperature fields induced in soft tissues in vivo by pulsed focused ultrasound during neurodegenerative disease treatment and experimental verification of the proposed model for a rat liver. The new therapeutic approach to neurodegenerative diseases consists of stimulation of enhanced expression of the Heat Shock Proteins (HSP) which are responsible for immunity of cells to stress. During therapy the temperature rise in tissues in vivo should not exceed 6 oC above level of the thermal norm (37 oC). First, the 3D acoustic pressure field, and the rate of heat production per unit volume due to that field, were calculated using our 3D numerical solver capable of predicting nonlinear propagation of pulsed high intensity waves generated from circular focused acoustic sources in multilayer configuration of attenuating media. The two-layer configuration of media (water - rat liver) assumed in calculations fairly well approximated both the real anatomic dimensions of rat liver and the geometric scheme of our experimental set-up. A numerical solution of the Pennes bio-heat transfer equation which accounted for the effects of heat diffusion, blood perfusion and metabolism rates, was employed to calculate the temperature fields induced in the rat liver by the ultrasonic beam. The numerical simulation results were verified experimentally using a thermocouple inserted in the liver of a rat under anesthesia at the beam focus. The quantitative analysis of the obtained results enabled estimation of the effects of several acoustic and thermal parameters of the rat liver in vivo on the temperature rise, as well as determination of exposure time for ultrasonic beams with varied acoustic power generated by a 2-MHz circular transducer of 15-mm diameter and 25-mm focal length, in order to avoid the tissue overheating that leads to cells necrosis, which would be unacceptable in neurodegenerative disease treatment.

low power ultrasound, tissue heating, HSP expression enhancement, cell immunity, degenerative deceases treatment

Recent research has shown that beneficial therapeutic effects in soft tissues can be induced by the low power ultrasound (LPUS). For example, increasing of cells immunity to stress can be accomplished through the heat shock proteins (Hsp) expression enhancement induced by the low intensity ultrasound. The possibility to control the Hsp expression enhancement in soft tissues in vivo by pulsed focused low power ultrasound may be the potential new therapeutic approach to the neuro-degenerative diseases that utilizes the known feature of cells to increase their immunity to stresses through the Hsp expression enhancement. The controlling of the Hsp expression enhancement by adjusting the level and time of exposure to ultrasound energy would allow evaluating of the ultrasound-mediated treatment efficiency. Ultrasonic regimes are controlled by adjusting the pulsed ultrasound waves intensity, frequency, duration, duty cycle and exposure time. Our objective was to develop the numerical model capable of predicting in space and time temperature fields induced by a circular focused transducer generating tone bursts in multilayer nonlinear attenuating media and to compare the numerically calculated results with the experimental data obtained for soft tissues in vitro. The acoustic pressure field in multilayer biological media was calculated using our original numerical solver. For prediction of temperature fields the Pennes’ bio-heat transfer equation was employed. The measurements of temperature distributions along and across the acoustic beam axis were performed using the experimental facility containing the temperature-controlled 1% saline solution tank, the tested tissue sample immersed in the tank and the USB-TEMP module connected with 8 thermocouples positioned within the tissue sample along and across the acoustic axis. Temperature measurements in vitro were carried out in a fresh rat liver using the 15 mm diameter, 25 mm focal length and 2 MHz central frequency transducer generating tone bursts with the spatial peak temporal average acoustic intensity varied between 0.325 and 1.95 W/cm2, duration varied from 20 to 500 cycles at the same 20 % duty cycle and the exposure time varied up to 20 minutes. The measurement data were compared with numerical simulation results obtained under experimental boundary conditions. Good agreement between the theoretical and measurement results for all cases considered has verified the validity and accuracy of our numerical model. The accuracy of the proposed model most of all depends on the accuracy of the source pressure measurements, estimation of tissues attenuation coefficients and their frequency-dependence law as well as determination of the tissue thermal conductivity. The measurement results confirmed that for pulses considered the temperature rise induced in tissue depends on the duty cycle, not on the tone burst duration. Quantitative analysis of the obtained results allowed to show that, for example, for the acoustic intensity ISPTA = 1.1 W/cm2 (P0 = 0.184 MPa) exposure time of the fresh rat liver should not exceed 10 min to avoid cells necrosis (T > 43 oC).

low intensity focused ultrasound, soft tissues, temperature fields, ultrasonic regimes, therapy efficiency

The new therapeutic approach to neurodegenerative diseases consisting in stimulation of enhanced expression of the Heat Shock Proteins (HSPs) responsible for immunity of cells to stress is proposed. During such therapy local temperature rises in tissues should not exceed 6 oC above the thermal norm (37 oC). Tissue heating at temperatures higher than 43 oC may lead to an inability of cells to divide or to their death. The main aim of this work is numerical modeling of temperature rises induced locally in soft tissues in vivo by pulsed focused ultrasound during neurodegenerative diseases therapy and experimental verification of correctness and accuracy of the proposed model in a rat liver. Both the pulsed acoustic fields and power density of heat sources produced locally in tissues due to those fields were calculated using our 3D numerical solver capable of predicting nonlinear propagation of pulsed finite-amplitude acoustic waves generated from circular focused sources in multilayer configuration of attenuating media with arbitrary attenuation law. The two-layer configuration of media (water - rat liver) assumed in calculations fairly well approximated both the real anatomic dimensions of the rat liver and geometric scheme of our experimental set-up. The Pennes bio-heat transfer equation accounting for the effects of heat diffusion, blood perfusion and metabolism rates was employed to calculate the temperature fields. The numerical simulation results were verified experimentally using a thermocouple positioned in the rat liver at the acoustic beam focus. The quantitative analysis of the obtained results enabled estimation of the effects of the source acoustic power, blood perfusion and metabolism rates on the temperature rises in the rat liver as well as determination of the level and time of exposure to ultrasound in order to avoid the tissue overheating above 43 oC that leads to cells necrosis. The ultrasonic 20-cycle tone bursts with 20 % duty cycle and initial acoustic pressure amplitude varied from 0.131 to 0.226 MPa were generated from a 2-MHz circular transducer with a 15-mm diameter and 25-mm focal length during 10-min exposure time. It was shown that for the 0.226-MPa source pressure the exposure time should not exceed 5 minutes in order to avoid the rat liver overheating. The thermal conductivity coefficient for the rat liver in vivo was determined to be 0.47.

pulsed focused ultrasound, tissues heating, HSPs expression enhancement, cells immunity, neurodegenerative diseases treatment

The theoretical and experimental studies of the finite amplitude acoustic waves propagation in attenuating media from nonaxisymmetric sources rather rarely can be found in literature in spite of the fact that probes of the rectangular geometry (such as linear phased arrays) are commonly used in clinical practice for medical ultrasonic imaging purposes. The main reason of such situation is a lack in simpler theoretical models and in computationally efficient numerical algorithms that are able to predict accurately the nonlinear effects in 4D ultrasound fields from pulsed, arbitrarily shaped sources (plane and focused) in biological media with arbitrary frequency-dependent absorption. In recent years the only study describing the computationally efficient numerical model that is able to simulate accurately the 4D nonlinear ultrasound field in water and in biological tissues from pulsed nonaxisymmetric sources was developed by Zemp et al. [1]. Their model is based on the second order operator-splitting method, proposed by Tavakkoli et al., with the modified fractional step scheme whereby the combined effects of diffraction and absorption are accounted for over half-steps and the effects of nonlinear harmonic interactions over full incremental steps. The computation of diffraction and absorption sub-steps was based on the angular spectrum technique with modified sampling method (to obtain computational savings due to larger axial propagation steps) while the computation of nonlinear steps was based on the time-domain solution to Burgers' equation. There are not reports yet describing an experimental confirmation of an agreement between the simulated nonlinear acoustic pulsed fields in water or in soft tissues from nonaxisymmetric focused sources (obtained by using the numerical model proposed) and nonlinear field from realistic probes.

Apertures, Numerical simulation, Acoustic pulses, Absorption, Ultrasonic imaging, Computational modeling, Acoustic propagation, Focusing, Phased arrays, Nonlinear acoustics

Ultradźwięki są stosowane w medycynie zarówno w diagnostyce - ultrasonografii jak i w terapii. Działanie terapeutyczne sprowadza się do efektów termicznych, uśmiercania komórek przez apoptozę oraz efektów sonomechanicznych. Terapie in-vivo są poprzedzane testami in-vitro komórek umieszczonych na szalce Petriego. W tej pracy zostaną przedstawione dwa efekty oddziaływania fali akustycznej na komórki: wspomaganie rozpuszczania skrzepliny oraz apoptoza komórek nowotworowych.
Do badań przygotowano zbiornik wypełniony odgazowaną wodą destylowaną z termostatem i mieszadłem elektromagnetycznym. W zbiorniku umieszczono płaski przetwornik ultradźwiękowy, promieniujący do góry, w kierunku zanurzonej w wodzie szalki Petriego ze skrzepliną lub komórkami.
W pierwszej części oddziaływano falą ultradźwiękową o częstotliwości 1.0 MHz i natężeniu Isata = 0.2 W/cm2 na skrzeplinę uzyskaną z kropli krwi 30 min po pobraniu. Skrzeplinę umieszczono w roztworze soli fizjologicznej z dodatkiem leku rozpuszczającego skrzeplinę tPA typu Actilyse 50 w stężeniu 10μg/ml i nadźwiękawiano przez 20 min. Eksperyment powtórzono z kolejnymi skrzeplinami stosując same ultradźwięki oraz sam lek Actilyse. Współdziałanie leku i fali ultradźwiękowej w ciągu 20 min spowodowało całkowite rozpuszczenie skrzepliny. W pozostałych przypadkach skrzeplina pozostała zmniejszając swoją objętość o 10-20%.
W drugiej części badano wpływ fali ultradźwiękowej 1.0 MHz na komórki nowotworowe szczurzego glejaka C6. Komórki wysiano na dnie szalki i hodowano w pożywce DMEM w temperaturze 37ºC. Następnie do pożywki dodano 1mM kwasu 5-aminolewulinowego ALA. Po 24h inkubacji, komórki poddano działaniu ultradźwięków o natężeniu Isata = 0.4, 0.8 oraz 1.2 W/cm2 przez 3 min. Po kolejnych 24h, żywotność komórek zbadano spektrofotometrem po dodaniu do pożywki odczynnika MTT assay. Badania powtórzono dla komórek bez ALA oraz bez ultradźwięków. Dla natężenia Isata = 1.2 W/cm2, żywotność komórek wynosiła 39% dla współdziałania ALA i ultradźwięków, 78% dla ALA oraz 99% dla ultradźwięków.
Przygotowane przez autorów stanowisko pomiarowe umożliwia prowadzenie badań in-vitro nad terapeutycznym oddziaływaniu ultradźwięków na komórki. Uzyskano pozytywne rezultaty w wykorzystaniu ultradźwięków zarówno do rozpuszczania skrzeplin – sonotrombolizy jak i uśmiercania komórek nowotworowych – terapii sonodynamicznej. Stosowano ultradźwięki małej mocy, niepowodujące zniszczenia komórek przez przegrzanie lub kawitację.

ultradźwięki terapeutyczne, sonotromboliza, tromboliza, leczenie nowotworów

To target a focal spot of an ultrasound beam on a tumor located deep inside tissues during thermo-ablative treatment by HIFU technique, beams with different focal distances are required. To be able to control a depth of local thermal fields induced in tissues by a single beam, both, the planar and concave 7-element annular phased array transducers with a 2 MHz frequency and 29 mm diameter generating beams with electronically controlled focal length were designed and produced. The radius of curvature (ROC) for the concave transducer was equal to 60 mm. Elements of each transducer had the same area to provide uniform pressure distribution on the radiating surface due to the same impedance and were excited by pulses with time delays providing the beam focusing in water at three different depths (25 mm, 30 mm, and 35 mm). To select sets of time delays for each focal depth the measurements of pressure waveforms on the axis of each beam generated in water were performed using a needle hydrophone. For these measurements 10-cycle tone bursts with 1 kHz PRF were used. In order to induce local thermo-ablative necrosis inside a tissue at three different depths (10mm, 15mm, and 20mm) a two-layer media of propagation comprising of 15-mm layer of water and 25-mm layer of pork loin was used. To heat the pork loin locally 20-cycle tone bursts with 0.2 duty-cycle and average acoustic power varied between12W and 18W (initial intensity ISATA varied between 2W/cm2 and 3W/cm2) was applied. In order to determine the exposure time required to induce necrosis (rise in temperature to 56 °C) inside the pork loin sample at the selected depth the thermocouples placed on the acoustic beam axis were used. After exposure to focused ultrasound three necrotic lesions were observed after cutting the tested tissue sample along the axes of the beams used. The obtained results proved the feasibility of controlling the depth of local tissue necrosis using pulsed focused ultrasound beams with electronically movable focal spot generated by the annular phased array transducer designed.

annular phased array transducer, pulsed High Intensity Focused Ultrasound, electronically movable focus, local tissue heating, thermal ablation, tissue necrosis