Computer assisted analysis of contrast enhanced ultrasound images for quantification in vascular diseases

Contrast enhanced ultrasound (CEUS) with microbubble contrast agents has shown great potential in imaging microvasculature, quantifying perfusion and hence detecting vascular diseases. However, most existing perfusion quantification methods based on image intensity, and are susceptible to confounding factors such as attenuation artefacts. Improving reproducibility is also a key challenge to clinical translation. Therefore, this thesis aims at developing attenuation correction and quantification techniques in CEUS with applications for detection and quantification of microvascular flow / perfusion. Firstly, a technique for automatic correction of attenuation effects in vascular imaging was developed and validated on a tissue mimicking phantom. The application of this technique to studying contrast enhancement of carotid adventitial vasa vasorum as a biomarker of radiation-induced atherosclerosis was demonstrated. The results showed great potential in reducing attenuation artefact and improve quantification in CEUS of carotid arteries. Furthermore, contrast intensity was shown to significantly increase in irradiated carotid arteries and could be a useful imaging biomarker for radiation-induced atherosclerosis. Secondly, a robust and automated tool for quantification of microbubble identification in CEUS image sequences using a temporal and spatial analysis was developed and validated on a flow phantom. The application of this technique to evaluate human musculoskeletal microcirculation with contrast enhanced ultrasound was demonstrated. The results showed an excellent accuracy and repeatability in quantifying active vascular density. It has great potential for clinical translation in the assessment of lower limb perfusion. Finally, a new bubble activity identification and quantification technique based on differential intensity projection in CEUS was developed and demonstrated with an in-vivo study, and applied to the quantification of intraplaque neovascularisation in an irradiated carotid artery of patients who were previously treated for head and neck cancer. The results showed a significantly more specific identification of bubble signals and had good agreement between the differential intensity-based technique and clinical visual assessment. This technique has potential to assist clinicians to diagnose and monitor intraplque neovascularisation.Open Acces

Respiratory Motion Compensation in Coronary Magnetic Resonance Angiography: Analysis and Optimization of Self-Navigation

Coronary Magnetic Resonance Imaging requires prolonged acquisition times; for this reason, respiratory movements of the heart have a great impact on the final image quality. The aim of this thesis was to provide possible optimization of the "self-navigation" approach to compensate this type of motion. Two developed methods were tested in 11 volunteer, thus providing statistically significant results. The purposed solutions provided optimal image quality in individal cases

Contrast echocardiography for cardiac quantifications

The indicator-dilution-theory for cardiac quantifications has always been limited in practice by the invasiveness of the available techniques. However, the recent introduction of stable ultrasound contrast agents opens new possibilities for indicator dilution measurements. This study describes a new and successful approach to overcome this invasiveness issue. We show a novel approach for minimally invasive quantification of several cardiac parameters based on the dilution of ultrasound contrast agents. A single peripheral injection of an ultrasound contrast agent bolus can result in the simultaneous assessment of cardiac output, pulmonary blood volume, and left and right ventricular ejection fraction. The bolus passage in different sites of the central circulation is detected by an ultrasound transducer. The detected acoustic (or video) intensities are processed and several indicator dilution curves are measured simultaneously. To this end, we exploit that for low concentrations the relation between contrast concentration and acoustic backscatter is approximately linear. The Local Density Random Walk Model is used to fit and interpret the indicator dilution curves for cardiac output, pulmonary blood volume, and ejection fraction measurements. Two fitting algorithms based either on a multiple linear regression in the logarithmic domain or on the solution of the moment equations are developed. The indicator dilution system can be also interpreted as a linear system and, therefore, characterized by an impulse response function. An adaptive Wiener deconvolution filter is implemented for robust dilution system identification. For ejection fraction measurements, the atrial and ventricular indicator dilution curves are measured and processed by the deconvolution filter, resulting in the estimate of the left ventricle dilution-system impulse response. This curve can be fitted and interpreted by a mono-compartment exponential model for the ejection fraction assessment. The proposed deconvolution filter is also used for the identification of the dilution system between right ventricle and left atrium. The Local Density Random Walk Model fit of the estimated impulse response allows the pulmonary blood volume assessment. Both cardiac output and pulmonary blood volume measurements are validated in vitro with accurate results (correlation coefficients larger than 0.99). The Pulmonary blood volume measurement feasibility is also tested in humans with promising results. The ejection fraction measurement is validated in-vivo. The impulse response approach allows accurate left ventricle ejection fraction estimates. Comparison with echocardiographic bi-plane measurements shows a correlation coefficient equal to 0.93. A dedicated image segmentation algorithm for videodensitometry has also been developed for automating the determination of regions of interest. The resulting algorithm has been integrated with the indicator dilution analysis system. The automatic determination of the measurement region results in improved dilution-curve signal-to-noise ratios. In conclusion, this study proves that quantification of cardiac output, pulmonary blood volume, and left and right ventricular ejection fraction by dilution of ultrasound contrast agents is feasible and accurate. Moreover, the proposed methods are applicable in different contexts (e.g., magnetic resonance imaging) and for different types of measurements, leading to a broad range of applications

Advancements and Breakthroughs in Ultrasound Imaging

Ultrasonic imaging is a powerful diagnostic tool available to medical practitioners, engineers and researchers today. Due to the relative safety, and the non-invasive nature, ultrasonic imaging has become one of the most rapidly advancing technologies. These rapid advances are directly related to the parallel advancements in electronics, computing, and transducer technology together with sophisticated signal processing techniques. This book focuses on state of the art developments in ultrasonic imaging applications and underlying technologies presented by leading practitioners and researchers from many parts of the world

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin