Doctor of Philosophy

dissertationThis dissertation presents original research that improves the ability of magnetic resonance imaging (MRI) to measure temperature in aqueous tissue using the proton resonance frequency (PRF) shift and T1 measurements in fat tissue in order to monitor focused ultrasound (FUS) treatments. The inherent errors involved in measuring the longitudinal relaxation time T1 using the variable flip angle method with a two-dimensional (2D) acquisition are presented. The edges of the slice profile can contribute a significant amount of signal for large flip angles at steady state, which causes significant errors in the T1 estimate. Only a narrow range of flip angle combinations provided accurate T1 estimates. Respiration motion causes phase artifacts, which lead to errors when measuring temperature changes using the PRF method. A respiration correction method for 3D imaging temperature of the breast is presented. Free induction decay (FID) navigators were used to measure and correct phase offsets induced by respiration. The precision of PRF temperature measurements within the breast was improved by an average factor of 2.1 with final temperature precision of approximately 1 °C. Locating the position of the ultrasound focus in MR coordinates of an ultrasound transducer with multiple degrees of freedom can be difficult. A rapid method for predicting the position using 3 tracker coils with a special MRI pulse iv sequence is presented. The Euclidean transformation of the coil's current positions to their calibration positions was used to predict the current focus position. The focus position was predicted to within approximately 2.1 mm in less than 1 s. MRI typically has tradeoffs between imaging field of view and spatial and temporal resolution. A method for acquiring a large field of view with high spatial and temporal resolution is presented. This method used a multiecho pseudo-golden angle stack of stars imaging sequence to acquire the large field of view with high spatial resolution and k-space weighted image contrast (KWIC) to increase the temporal resolution. The pseudo-golden angle allowed for removal of artifacts introduced by the KWIC reconstruction algorithm. The multiple echoes allowed for high readout bandwidth to reduce blurring due to off resonance and chemical shift as well as provide separate water/fat images, estimates of the initial signal magnitude M(0), T2 * time constant, and combination of echo phases. The combined echo phases provided significant improvement to the PRF temperature precision, and ranged from ~0.3-1.0 °C within human breast. M(0) and T2 * values can possibly be used as a measure of temperature in fat

Ultrasound metrology and phantom materials for validation of photoacoustic thermometry

High intensity focused ultrasound is an emerging non-invasive cancer therapy during which a focused ultrasound beam is used to destroy cancer cells within a confined volume of tissue. In order to increase its successful implementation in practice, an imaging modality capable of accurately mapping the induced temperature rise in tissue is necessary. Photoacoustic thermometry, a rapidly emerging technique for non-invasive temperature monitoring, exploits the temperature dependence of the Grüneisen parameter of tissues, which leads to changes in the recorded photoacoustic signal amplitude with temperature. However, the implementation of photoacoustic thermometry approaches is hindered by a lack of rigorous validation. This includes both the equipment and methodology used. This work investigates the effect of temperature on ultrasound transducers used in photoacoustic thermometry imaging as well as characterisation of potential phantom materials for its validation. The variation in transducer sensitivity with temperature is investigated using two approaches. The first one utilises a reference transducer whose output power is known as a function of temperature to characterise the sensitivity of the hydrophone. As the knowledge of variability of transducer output with temperature is not readily available, two standard metrology techniques using radiation force balances and laser vibrometry are extended beyond room temperature to characterise the effect of temperature on the output of PZT tranducers. For the second approach to transducer sensitivity calibration, a novel method is developed utilising water as a laser-generated ultrasound source and validated using the self-reciprocity calibration method. The calibrated hydrophone is then used to characterise the relevant temperature-dependent properties of several phantom materials in a custom-built setup. The measurement results are used to determine the most suitable phantom for photoacoustic thermometry. Finally, the phantom is heated and imaged in a proof-of-concept photoacoustic thermometry setup using a linear array. These contributions are of vital importance for allowing the translation of photoacoustic thermometry into clinical practice

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

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Doctor of Philosophy

dissertationFocused ultrasound (FUS) is a promising noninvasive and radiation-free cancer therapy that selectively delivers high-intensity acoustic energy to a small target volume. This dissertation presents original research that improves the speed, safety, and efficacy of FUS therapies under magnetic resonance imaging (MRI) guidance. First, a new adaptive model-predictive controller is presented that leverages the ability of MRI to measure temperature inside the patient at near real-time speeds. The controller uses MR temperature feedback to dynamically derive and update a patient-specific thermal model, and optimizes the treatment based on the model's predictions. Treatment safety is a key element of the controller's design, and it can actively protect healthy tissue from unwanted damage. In vivo and simulation studies indicate the controller can safeguard healthy tissue and accelerate treatments by as much as 50%. Significant tradeoffs exist between treatment speed, and safety, which makes a real-time controller absolutely necessary for carrying out efficient, effective, and safe treatments while also highlighting the importance of continued research into optimal treatment planning. Next, two new methods for performing 3D MR acoustic radiation force imaging (MR-ARFI) are presented. Both techniques measure the tissue displacement induced by short bursts of focused ultrasound, and provide a safe way to visualize the ultrasound beam's location. In some scenarios, ARFI is a necessity for proper targeting since traditional MR thermometry cannot measure temperature in fat. The first technique for performing 3D ARFI introduces a novel unbalanced bipolar motion encoding gradient. The results demonstrate that this technique is safe, and that 3D displacement maps can be attained time-efficiently even in organs that contain fat, such as breast. The second technique measures 3D ARFI simultaneously with temperature monitoring. This method uses a multi-contrast gradient recalled echo sequence which makes multiple readings of the data without increasing scan time. This improves the signal to noise ratio and makes it possible to separate the effects of tissue heating vs displacement. Both of the 3D MR-ARFI techniques complement the presented controllersince proper positioning of the focal spot is critical to achieving fast and safe treatments

Towards Closed-loop, Robot Assisted Percutaneous Interventions under MRI Guidance

Image guided therapy procedures under MRI guidance has been a focused research area over past decade. Also, over the last decade, various MRI guided robotic devices have been developed and used clinically for percutaneous interventions, such as prostate biopsy, brachytherapy, and tissue ablation. Though MRI provides better soft tissue contrast compared to Computed Tomography and Ultrasound, it poses various challenges like constrained space, less ergonomic patient access and limited material choices due to its high magnetic field. Even after, advancements in MRI compatible actuation methods and robotic devices using them, most MRI guided interventions are still open-loop in nature and relies on preoperative or intraoperative images. In this thesis, an intraoperative MRI guided robotic system for prostate biopsy comprising of an MRI compatible 4-DOF robotic manipulator, robot controller and control application with Clinical User Interface (CUI) and surgical planning applications (3DSlicer and RadVision) is presented. This system utilizes intraoperative images acquired after each full or partial needle insertion for needle tip localization. Presented system was approved by Institutional Review Board at Brigham and Women\u27s Hospital(BWH) and has been used in 30 patient trials. Successful translation of such a system utilizing intraoperative MR images motivated towards the development of a system architecture for close-loop, real-time MRI guided percutaneous interventions. Robot assisted, close-loop intervention could help in accurate positioning and localization of the therapy delivery instrument, improve physician and patient comfort and allow real-time therapy monitoring. Also, utilizing real-time MR images could allow correction of surgical instrument trajectory and controlled therapy delivery. Two of the applications validating the presented architecture; closed-loop needle steering and MRI guided brain tumor ablation are demonstrated under real-time MRI guidance

Temperature Mapping using Mid-Field Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI) is a non-invasive imaging modality with excellent soft tissue contrast and sensitivity to tissue temperature. MRI use is growing in Canada with expectation that this is expected to continue in the medium term, with more wide adoption of MRI and in particular a renewed focus on MR systems which deviate from the most commonly used 1.5T field strength system. By implementing systems which do not use as strong magnets and instead operate Generally, as the field strength of an MR system decreases, the signal received when imaging also decreases, which makes it difficult to implement some applications which are standard at higher field. One such application is temperature mapping on a these \u3c1T \u3esystems, which can be used to monitor thermal therapies interventionally. This thesis addresses the potentials for implementing temperature mapping at 0.5T, both in the creation of a tissue mimicking phantom which can be used to compare temperature mapping methods and implementing temperature maps both in vivo and in the custom phantom. As well, motivated by the sensitivity that thermal mapping has to external disturbances, the challenges that these accessible MR systems face when being in non-specialized environments is addressed, as this can potentially limit the efficacy of temperature mapping. This work ultimately demonstrates the acceptable capabilities of a 0.5T system to map temperatures with an adequate temporal resolution, along with presenting practical solutions to operating a system in non-traditional locations