Ultra-High Field Strength MR Image-Guided Robotic Needle Delivery Device for In-Bore Small Animal Interventions

Current methods of accurate soft tissue injections in small animals are prone to many sources of error. Although efforts have been made to improve the accuracy of needle deliveries, none of the efforts have provided accurate soft tissue references. An MR image-guided robot was designed to function inside the bore of a 9.4T MR scanner to accurately deliver needles to locations within the mouse brain. The robot was designed to have no noticeable negative effects on the image quality and was localized in the MR images through the use of an MR image visible fiducial. The robot was mechanically calibrated and subsequently validated in an image-guided phantom experiment, where the mean needle targeting accuracy and needle trajectory accuracy were calculated to be 178 ± 54µm and 0.27 ± 0.65º, respectively. Finally, the device successfully demonstrated an image-guided needle targeting procedure in situ

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

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

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

Medical robots for MRI guided diagnosis and therapy

Magnetic Resonance Imaging (MRI) provides the capability of imaging tissue with fine resolution and superior soft tissue contrast, when compared with conventional ultrasound and CT imaging, which makes it an important tool for clinicians to perform more accurate diagnosis and image guided therapy. Medical robotic devices combining the high resolution anatomical images with real-time navigation, are ideal for precise and repeatable interventions. Despite these advantages, the MR environment imposes constraints on mechatronic devices operating within it. This thesis presents a study on the design and development of robotic systems for particular MR interventions, in which the issue of testing the MR compatibility of mechatronic components, actuation control, kinematics and workspace analysis, and mechanical and electrical design of the robot have been investigated. Two types of robotic systems have therefore been developed and evaluated along the above aspects. (i) A device for MR guided transrectal prostate biopsy: The system was designed from components which are proven to be MR compatible, actuated by pneumatic motors and ultrasonic motors, and tracked by optical position sensors and ducial markers. Clinical trials have been performed with the device on three patients, and the results reported have demonstrated its capability to perform needle positioning under MR guidance, with a procedure time of around 40mins and with no compromised image quality, which achieved our system speci cations. (ii) Limb positioning devices to facilitate the magic angle effect for diagnosis of tendinous injuries: Two systems were designed particularly for lower and upper limb positioning, which are actuated and tracked by the similar methods as the first device. A group of volunteers were recruited to conduct tests to verify the functionality of the systems. The results demonstrate the clear enhancement of the image quality with an increase in signal intensity up to 24 times in the tendon tissue caused by the magic angle effect, showing the feasibility of the proposed devices to be applied in clinical diagnosis

Robotic System Development for Precision MRI-Guided Needle-Based Interventions

This dissertation describes the development of a methodology for implementing robotic systems for interventional procedures under intraoperative Magnetic Resonance Imaging (MRI) guidance. MRI is an ideal imaging modality for surgical guidance of diagnostic and therapeutic procedures, thanks to its ability to perform high resolution, real-time, and high soft tissue contrast imaging without ionizing radiation. However, the strong magnetic field and sensitivity to radio frequency signals, as well as tightly confined scanner bore render great challenges to developing robotic systems within MRI environment. Discussed are potential solutions to address engineering topics related to development of MRI-compatible electro-mechanical systems and modeling of steerable needle interventions. A robotic framework is developed based on a modular design approach, supporting varying MRI-guided interventional procedures, with stereotactic neurosurgery and prostate cancer therapy as two driving exemplary applications. A piezoelectrically actuated electro-mechanical system is designed to provide precise needle placement in the bore of the scanner under interactive MRI-guidance, while overcoming the challenges inherent to MRI-guided procedures. This work presents the development of the robotic system in the aspects of requirements definition, clinical work flow development, mechanism optimization, control system design and experimental evaluation. A steerable needle is beneficial for interventional procedures with its capability to produce curved path, avoiding anatomical obstacles or compensating for needle placement errors. Two kinds of steerable needles are discussed, i.e. asymmetric-tip needle and concentric-tube cannula. A novel Gaussian-based ContinUous Rotation and Variable-curvature (CURV) model is proposed to steer asymmetric-tip needle, which enables variable curvature of the needle trajectory with independent control of needle rotation and insertion. While concentric-tube cannula is suitable for clinical applications where a curved trajectory is needed without relying on tissue interaction force. This dissertation addresses fundamental challenges in developing and deploying MRI-compatible robotic systems, and enables the technologies for MRI-guided needle-based interventions. This study applied and evaluated these techniques to a system for prostate biopsy that is currently in clinical trials, developed a neurosurgery robot prototype for interstitial thermal therapy of brain cancer under MRI guidance, and demonstrated needle steering using both asymmetric tip and pre-bent concentric-tube cannula approaches on a testbed

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

MRI compatible mechatronic devices to aid medical diagnosis and intervention

Imperial Users onl