5-Aminolevulinic acid-mediated sonosensitization of rat RG2 glioma cells in vitro.

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.

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells.

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/cm(2) to 8 W/cm(2) (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 24h 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/cm(2).

Influence of the ultrasound transducer bandwidth on selection of the complementary Golay bit code length.

In contrast to previously published papers [A. Nowicki, Z. Klimonda, M. Lewandowski, J. Litniewski, P.A. Lewin, I. Trots, Comparison of sound fields generated by different coded excitations - Experimental results, Ultrasonics 44 (1) (2006) 121-129; J. Litniewski, A. Nowicki, Z. Klimonda, M. Lewandowski, Sound fields for coded excitations in water and tissue: experimental approach, Ultrasound Med. Biol. 33 (4) (2007) 601-607], which examined the factors influencing the spatial resolution of coded complementary Golay sequences (CGS), this paper investigates the effect of ultrasound imaging transducer's fractional bandwidth on the gain of the compressed echo signal for different spectral widths of the CGS. Two different bit lengths were considered, specifically one and two cycles. Three transducers having fractional bandwidth of 25%, 58% and 80% and operating at frequencies 6, 4.4 and 6 MHz, respectively were examined (one of the 6 MHz sources was focused and made of composite material). The experimental results have shown that by increasing the code length, i.e. decreasing the bandwidth, the compressed echo amplitude could be enhanced. The smaller the bandwidth was the larger was the gain; the pulse-echo sensitivity of the echo amplitude increased by 1.88, 1.62 and 1.47, for 25%, 58% and 80% bandwidths, respectively. These results indicate that two cycles bit length excitation is more suitable for use with bandwidth limited commercially available imaging transducers. Further, the time resolution is retained for transducers with two cycles excitation providing the fractional bandwidth is lower than approximately 90%. The results of this work also show that adjusting the code length allows signal-to-noise-ratio (SNR) to be enhanced while using limited (less that 80%) bandwidth imaging transducers. Also, for such bandwidth limited transducers two cycles excitation would not decrease the time resolution, obtained with "conventional" spike excitation. Hence, CGS excitation could be successfully implemented with the existing, relatively narrow band imaging transducers without the need to use usually more expensive wideband, composite ones.

Estimation of ultrasonic attenuation in a bone using coded excitation.

This paper describes a novel approach to estimate broadband ultrasound attenuation (BUA) in a bone structure in human in vivo using coded excitation. BUA is an accepted indicator for assessment of osteoporosis. In the tested approach a coded acoustic signal is emitted and then the received echoes are compressed into brief, high amplitude pulses making use of matched filters and correlation receivers. In this way the acoustic peak pressure amplitude probing the tissue can be markedly decreased whereas the average transmitted intensity increases proportionally to the length of the code. This paper examines the properties of three different transmission schemes, based on Barker code, chirp and Golay code. The system designed is capable of generating 16 bits complementary Golay code (CGC), linear frequency modulated (LFM) chirp and 13-bit Barker code (BC) at 0.5 and 1 MHz center frequencies. Both in vivo data acquired from healthy heel bones and in vitro data obtained from human calcaneus were examined and the comparison between the results using coded excitation and two cycles sine burst is presented. It is shown that CGC system allows the effective range of frequencies employed in the measurement of broadband acoustic energy attenuation in the trabecular bone to be doubled in comparison to the standard 0.5 MHz pulse transmission. The algorithm used to calculate the pairs of Golay sequences of the different length, which provide the temporal side-lobe cancellation is also presented. Current efforts are focused on adapting the system developed for operation in pulse-echo mode; this would allow examination and diagnosis of bones with limited access such as hip bone.

Skin imaging with high frequency ultrasound - preliminary results.

This study presents the detailed construction and the principle of performance of high frequency (HF) ultrasound scanner for skin examination. The aim of this study was to show a difference between diseased and healthy skin and to evaluate the usefulness of the scanner in monitoring of therapeutic efficacy of morphea and lichen sclerosus et atrophicus (LSA). We examined 48 patients aged between 15 and 64 years; 25 patients with plaque-type morphea, nine patients with linear morphea and 14 patients with LSA. In the course of 18 months all patients were examined before, during and after treatment. In 29 patients ultrasonographic evidence of regression (decreasing of the skin thickness) was observed, in eight patients ultrasound examination showed progression and in 12 patients no difference in the ultrasound scan before and after treatment could be shown. Our study shows that HF ultrasound scanner is suitable to differentiate between the healthy and diseased skin in morphea and LSA as well as to evaluate the treatment efficacy of these diseases.

Estimation of acoustical streaming: theoretical model, Doppler measurements and optical visualisation.

An approximate solution for the streaming velocity generated by flat and weakly focused transducers was derived by directly solving the Dirichlet boundary conditions for the Poisson equation, the solution of the Navier-Stokes equation for the axial components of the streaming velocity. The theoretical model was verified experimentally using a 32 MHz pulsed Doppler unit. The experimental acoustical fields were produced by three different 4 mm diameter flat and focused transducers driven by the transmitter generating the average acoustic power within the range from 1 microW to 6 mW. The streaming velocity was measured along the ultrasonic beam from 0 to 2 cm. Streaming was induced in a solution of water and corn starch. The experimental results showed that for a given acoustic power the streaming velocity was independent of the starch density in water, changed from 0.3 to 40 grams of starch in 1 l of distilled water. For applied acoustic powers, the streaming velocity changed linearly from 0.2 to 40 mm/s. Both, the theoretical solutions for plane and focused waves and the experimental results were in good agreement. The streaming velocity field was also visualised using the particle image velocimetry (PIV) and two different evaluation methods. The first based on the FFT-based cross-correlation analysis between small sections for each pair of images and the second employing the algorithm of searching for local displacements between several images.

Acoustic streaming: comparison of low-amplitude linear model with streaming velocities measured by 32-MHz Doppler.

The pressure gradient along the ultrasonic beam results in medium streaming. Following Nyborg's analysis of the Navier-Stokes equation, Wu and Du developed an approximate solution for the streaming velocity generated by flat and weakly focused transducers. We have modified their solution of the Poisson equation by directly deriving the Dirichlet boundary conditions to be applied for this type of equation. Our numerical results (for the linear case) were about one half smaller for flat and weakly focused on Gaussian beam transducers compared to the results by Wu and Du. The theoretical calculations were verified using a purpose-designed 32-MHz pulsed Doppler unit. The applied average acoustic power was changed from 1 microW to 6 mW, the burst width was 0.5 microseconds and the pulse repetition frequency was 32 kHz. The experiments were done on 4-mm-diameter flat and focused (focal distance = 8 and 12 mm) transducers. The streaming was measured along the ultrasonic beam from 0-20 mm; at all positions, the maximum Doppler frequency was estimated from the recorded spectra. Streaming was induced in a solution of water and corn starch. The experimental results showed that, for a given acoustic power, the streaming velocity was independent of the starch density in water changed from 0.3-40 g of starch in 1 l of distilled water. For applied acoustic powers, the streaming velocity changed linearly from 0.2-40 mm/s. Both the theoretical solutions for plane and focused waves and the experimental results were in good agreement.

[Angio-ultrasonography--a method of examining the extracranial arteries]. / Angioultrasonografia--metoda badania tetnic pozaczaszkowych.

The authors describe a method of investigations of the extracranial cerebral arteries--angioultrasonography based on the Doppler effect. The study was done with a Polish apparatus designed and produced by the Experimental Laboratory of the Institute of Basic Technical Problems, Polish Academy of Sciences. The study confirmed the usefulness of this method in the diagnosis of arterial diseases involving the extracranial arteries supplying the brain. A high agreement was demonstrated between the results of angioultrasonography and classical angiography. Owing to the use of a grey scale it was possible to localize accurately the site of stenosis or occlusion of the studied artery.

Method for the measurement of the maximum Doppler frequency.

This paper describes a method for detection of the maximum Doppler frequency from a histogram of Doppler signals (HDS). The performance of the system is described, and its behavior in analysis of noise signals that have known lower and upper cut off frequencies is discussed. Experimental investigations show that the device correctly measures the maximum frequency envelope and can be useful in evaluating the maximum flow velocities in the blood circulation system.