Contrast Enhanced Ultrasound: An Overview

Application of ultrasound contrast medium to traditional medical sonography is known as Contrast-enhanced ultrasound (CEUS) that enhances contrast during ultrasnography by increasing ultrasound backscatter (reflection) of the ultrasound waves. Contrast-enhanced ultrasound can be used in diagnostic imaging, organ edge delineation, echocardiography, blood volume perfusion, lesion characterization, drug or gene delivery, molecular imaging etc. Different types of contrast media used in ultrasonography are agitated saline, microbubbles, nanobubbles etc. Adverse reactions of contrast agent include headache, hypersensitivity abdominal pain, diarrhoea, dyspepsia, hypertension, leg cramps etc. Although CEUS is popular now a days but with certain limitations such as more heat production with increase in frequency, short life of microbubbles, continuous monitoring, occasional microvasculature rupture and haemolysis. In conclusion, CEUS is an advanced technique for absolute quantification of tissue perfusion, drugs and genes delivery, differential diagnosis and monitoring therapy response.

Design and Application of Signal Modeling, Segmentation and Classification Methods for High-Frequency Ultrasound Backscatter Signals

In this study, we explore the possibility of monitoring program cell death (apoptosis) and classifying clusters of apoptotic cells based on the changes in high frequency ultrasound backscatter signals from these cells. One of the hallmarks of cancer is that the fail [sic] in the apoptosis mechanism in cells. Therefore this research carries the promise of designing more refined and more effective cancer therapies. The ultrasound signals are modeled through the Autoregressive (AR) modeling technique. The proper model order is calculated by tracking the error criteria derived from statistical properties of the original and modeled signal. In the next stage, five machine learning classifiers are developed to classify backscatter signals based on their AR coefficients. In clinical applications ultrasound backscatter signals from tissues and tumors are most likely to be non-stationary. Therefore analyzing such signals requires signal segmentation techniques. We developed recursive least square lattice filter for adaptive segmentation of ultrasound backscatter signals from multiple cell types into blocks of stationary segments, and model and classify the segments individually. In this thesis we demonstrate the accuracy of modeling, segmentation and classification techniques to detect signals from different cell pellets based on the signal processing and machine learning techniques.

Design and Application of Signal Modeling, Segmentation and Classification Methods for High-Frequency Ultrasound Backscatter Signals

In this study, we explore the possibility of monitoring program cell death (apoptosis) and classifying clusters of apoptotic cells based on the changes in high frequency ultrasound backscatter signals from these cells. One of the hallmarks of cancer is that the fail [sic] in the apoptosis mechanism in cells. Therefore this research carries the promise of designing more refined and more effective cancer therapies. The ultrasound signals are modeled through the Autoregressive (AR) modeling technique. The proper model order is calculated by tracking the error criteria derived from statistical properties of the original and modeled signal. In the next stage, five machine learning classifiers are developed to classify backscatter signals based on their AR coefficients. In clinical applications ultrasound backscatter signals from tissues and tumors are most likely to be non-stationary. Therefore analyzing such signals requires signal segmentation techniques. We developed recursive least square lattice filter for adaptive segmentation of ultrasound backscatter signals from multiple cell types into blocks of stationary segments, and model and classify the segments individually. In this thesis we demonstrate the accuracy of modeling, segmentation and classification techniques to detect signals from different cell pellets based on the signal processing and machine learning techniques.

Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

Quantitative Ultrasound Characterization of Tumor Cell Death: Ultrasound-Stimulated Microbubbles for Radiation Enhancement

The aim of this study was to assess the efficacy of quantitative ultrasound imaging in characterizing cancer cell death caused by enhanced radiation treatments. This investigation focused on developing this ultrasound modality as an imaging-based non-invasive method that can be used to monitor therapeutic ultrasound and radiation effects. High-frequency (25 MHz) ultrasound was used to image tumor responses caused by ultrasound-stimulated microbubbles in combination with radiation. Human prostate xenografts grown in severe combined immunodeficiency (SCID) mice were treated using 8, 80, or 1000 µL/kg of microbubbles stimulated with ultrasound at 250, 570, or 750 kPa, and exposed to 0, 2, or 8 Gy of radiation. Tumors were imaged prior to treatment and 24 hours after treatment. Spectral analysis of images acquired from treated tumors revealed overall increases in ultrasound backscatter intensity and the spectral intercept parameter. The increase in backscatter intensity compared to the control ranged from 1.9±1.6 dB for the clinical imaging dose of microbubbles (8 µL/kg, 250 kPa, 2 Gy) to 7.0±4.1 dB for the most extreme treatment condition (1000 µL/kg, 750 kPa, 8 Gy). In parallel, in situ end-labelling (ISEL) staining, ceramide, and cyclophilin A staining demonstrated increases in cell death due to DNA fragmentation, ceramide-mediated apoptosis, and release of cyclophilin A as a result of cell membrane permeabilization, respectively. Quantitative ultrasound results indicated changes that paralleled increases in cell death observed from histology analyses supporting its use for non-invasive monitoring of cancer treatment outcomes.

Development of microfluidic acoustic flow cytometry based on simultaneous ultrasound backscatter and photoacoustics for micron and sub-micron size objects

I developed a flow cytometer based on simultaneous detection of ultra-high frequency ultrasound backscatter and photoacoustic waves from individual micron scale objects, such as, cells, microparticles, and microbubbles owing in a microuidic channel. Individual micron scale objects are ow focused through a focal zone, where both ultrasound and laser pulses focus, in a microchannel of a polydimethylsiloxane (PDMS) based microuidic device. At the focal zone, the objects are simultaneously insonified by ultrasound (center frequency 375 MHz) and irradiated by nanosecond laser (532 nm wavelength) pulses. The interactions generate ultrasound backscatter and photoacoustic signals from the individual objects, which are strongly dependent on their size, morphology, and biomechanical properties, such as the Young's modulus, and optical absorption properties. These parameters can be extracted by analyzing the unique spectral features of the detected signals. At frequencies less than 100 MHz, the signals from the micron scale objects do not contain these unique spectral signatures, thus higher frequencies are required. Cell analysis is the main application of interest using the acoustic flow cytometer. Combining ultrasound backscatter and photoacoustics results in sufficient information about a single cell that can be used for single cell analysis and for diagnostics applications. However, the usage of this system is not limited to biological cells. This system can also be used for analyzing individual microbubbles, which are used as ultrasound contrast agents. During my research, a novel microuidic technique is developed to generate microbubbles of desired sizes by shrinking microbubbles from O(100) _m by applying a suitable vacuum pressure. These shrunken bubbles of different sizes can be used as samples to validate the acoustic ow system for microbubble analysis.

Measurement of viscoelastic properties of treated and untreated cancer cells using passive microrheology

Experiments have shown that there is an increase in ultrasound backscatter from cells during cell death. Since cell scattering depends on the mechanical property variations, one step towards a better understanding of the phenomenon involves measuring the cells' viscoelastic properties. Two promising techniques used for such studies are particle tracking microrheology (1P) and two-point microrheology (2P). The main aim of this work is to develop and test the ability of both to measure changes in viscous and elastic moduli of breast cancer cells during chemotherapeutic treatments. First, the viscosities of glycerol-water mixtures measured using microrheology were found to be within 5% of rheometer values. The viscous and elastic moduli of 4% and 6% poly(ethylene oxide) solutions were successfully measured at 30°C and 37°C. For MCF-7 cells, a 10-fold increase in the elastic modulus was observed using 1P, without a corresponding increase in the viscous modulus. Thus, it was shown that MCF-7 cells undergo an increase in stiffness during apoptosis.

Development of microfluidic acoustic flow cytometry based on simultaneous ultrasound backscatter and photoacoustics for micron and sub-micron size objects

I developed a flow cytometer based on simultaneous detection of ultra-high frequency ultrasound backscatter and photoacoustic waves from individual micron scale objects, such as, cells, microparticles, and microbubbles owing in a microuidic channel. Individual micron scale objects are ow focused through a focal zone, where both ultrasound and laser pulses focus, in a microchannel of a polydimethylsiloxane (PDMS) based microuidic device. At the focal zone, the objects are simultaneously insonified by ultrasound (center frequency 375 MHz) and irradiated by nanosecond laser (532 nm wavelength) pulses. The interactions generate ultrasound backscatter and photoacoustic signals from the individual objects, which are strongly dependent on their size, morphology, and biomechanical properties, such as the Young's modulus, and optical absorption properties. These parameters can be extracted by analyzing the unique spectral features of the detected signals. At frequencies less than 100 MHz, the signals from the micron scale objects do not contain these unique spectral signatures, thus higher frequencies are required. Cell analysis is the main application of interest using the acoustic flow cytometer. Combining ultrasound backscatter and photoacoustics results in sufficient information about a single cell that can be used for single cell analysis and for diagnostics applications. However, the usage of this system is not limited to biological cells. This system can also be used for analyzing individual microbubbles, which are used as ultrasound contrast agents. During my research, a novel microuidic technique is developed to generate microbubbles of desired sizes by shrinking microbubbles from O(100) _m by applying a suitable vacuum pressure. These shrunken bubbles of different sizes can be used as samples to validate the acoustic ow system for microbubble analysis.

Measurement of viscoelastic properties of treated and untreated cancer cells using passive microrheology

Experiments have shown that there is an increase in ultrasound backscatter from cells during cell death. Since cell scattering depends on the mechanical property variations, one step towards a better understanding of the phenomenon involves measuring the cells' viscoelastic properties. Two promising techniques used for such studies are particle tracking microrheology (1P) and two-point microrheology (2P). The main aim of this work is to develop and test the ability of both to measure changes in viscous and elastic moduli of breast cancer cells during chemotherapeutic treatments. First, the viscosities of glycerol-water mixtures measured using microrheology were found to be within 5% of rheometer values. The viscous and elastic moduli of 4% and 6% poly(ethylene oxide) solutions were successfully measured at 30°C and 37°C. For MCF-7 cells, a 10-fold increase in the elastic modulus was observed using 1P, without a corresponding increase in the viscous modulus. Thus, it was shown that MCF-7 cells undergo an increase in stiffness during apoptosis.

Towards Understanding the Nature of High Frequency Ultrasound Backscatter from Cells and Tissues: an Investigation of Backscatter Power Spectra from Different Concentrations of Cells of Different Sizes

Towards Understanding the Nature of High Frequency Ultrasound Backscatter from Cells and Tissues: an Investigation of Backscatter Power Spectra from Different Concentrations of Cells of Different Sizes