Stethoscope acoustics

Audible-frequency sounds from the human body are an invaluable diagnostic tool. For over 200 years, stethoscopes have been used to listen to these sounds. Despite this, the physics of how stethoscopes work remain poorly understood. While the stethoscope itself is a simple device, its performance depends on how it is coupled to both the patient and the clinician. Existing models do not adequately address these interactions, forcing design choices to be made based on simple heuristics. The aims of this thesis are to provide a theoretical framework for understanding the acoustics of stethoscopes, propose a low order model to simulate the response, and develop an experimental methodology to validate the model. When a stethoscope is pressed against the chest, body sounds induce small perturbations around the equilibrium position of the nonlinear chest-stethoscope system. In this thesis, a lumped-element approach is used to model these perturbations. The resulting models are validated using experiments conducted on a phantom (a laboratory model representing the human chest). Impedance measurements on the phantom and on the human chest allow differences between these systems to be accounted for. The models presented in this thesis capture the trends associated with each of the key design parameters. Minimising the cavity volume maximises the response, while tubing significantly attenuates low frequencies and introduces distorting standing-wave resonances. Using a diaphragm attenuates the response and shifts the resonances to higher frequencies, but also allows smaller air cavities to be used. Holding a stethoscope against the chest sets the equilibrium position of the coupled system and provides a damping-dominated impedance load on the chestpiece. The strong dependence of a stethoscope’s performance on external factors, such as the properties of the chest and the way it is held, makes it difficult to compare sensors objectively. The work presented in this thesis dispels various misconceptions about how stethoscopes work and can be used to inform design choices, ultimately improving the diagnostic capabilities of future stethoscopes.Magdalene College, Cambridge Philosophical Societ

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

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

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

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

A Three – tier bio-implantable sensor monitoring and communications platform

One major hindrance to the advent of novel bio-implantable sensor technologies is the need for a reliable power source and data communications platform capable of continuously, remotely, and wirelessly monitoring deeply implantable biomedical devices. This research proposes the feasibility and potential of combining well established, ‘human-friendly' inductive and ultrasonic technologies to produce a proof-of-concept, generic, multi-tier power transfer and data communication platform suitable for low-power, periodically-activated implantable analogue bio-sensors. In the inductive sub-system presented, 5 W of power is transferred across a 10 mm gap between a single pair of 39 mm (primary) and 33 mm (secondary) circular printed spiral coils (PSCs). These are printed using an 8000 dpi resolution photoplotter and fabricated on PCB by wet-etching, to the maximum permissible density. Our ultrasonic sub-system, consisting of a single pair of Pz21 (transmitter) and Pz26 (receiver) piezoelectric PZT ceramic discs driven by low-frequency, radial/planar excitation (-31 mode), without acoustic matching layers, is also reported here for the first time. The discs are characterised by propagation tank test and directly driven by the inductively coupled power to deliver 29 μW to a receiver (implant) employing a low voltage start-up IC positioned 70 mm deep within a homogeneous liquid phantom. No batteries are used. The deep implant is thus intermittently powered every 800 ms to charge a capacitor which enables its microcontroller, operating with a 500 kHz clock, to transmit a single nibble (4 bits) of digitized sensed data over a period of ~18 ms from deep within the phantom, to the outside world. A power transfer efficiency of 83% using our prototype CMOS logic-gate IC driver is reported for the inductively coupled part of the system. Overall prototype system power consumption is 2.3 W with a total power transfer efficiency of 1% achieved across the tiers

Ultrafast Ultrasound Imaging

Among medical imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), ultrasound imaging stands out due to its temporal resolution. Owing to the nature of medical ultrasound imaging, it has been used for not only observation of the morphology of living organs but also functional imaging, such as blood flow imaging and evaluation of the cardiac function. Ultrafast ultrasound imaging, which has recently become widely available, significantly increases the opportunities for medical functional imaging. Ultrafast ultrasound imaging typically enables imaging frame-rates of up to ten thousand frames per second (fps). Due to the extremely high temporal resolution, this enables visualization of rapid dynamic responses of biological tissues, which cannot be observed and analyzed by conventional ultrasound imaging. This Special Issue includes various studies of improvements to the performance of ultrafast ultrasoun

Novel Techniques for Tissue Imaging and Characterization Using Biomedical Ultrasound

The use of ultrasound technology in the biomedical field has been widely increased in recent decades. Ultrasound modalities are considered more safe and cost effective than others that use ionizing radiation. Moreover, the use of high-frequency ultrasound provides means of high-resolution and precise tissue assessment. Consequently, ultrasound elastic waves have been widely used to develop non-invasive techniques for tissue assessment. In this work, ultrasound waves have been used to develop non-invasive techniques for tissue imaging and characterization in three different applications.;Currently, there is a lack of imaging modalities to accurately predict minute structures and defects in the jawbone. In particular, the inability of 2D radiographic images to detect bony periodontal defects resulted from infection of the periodontium. They also may carry known risks of cancer generation or may be limited in accurate diagnosis scope. Ultrasonic guided waves are sensitive to changes in microstructural properties, while high-frequency ultrasound has been used to reconstruct high-resolution images for tissue. The use of these ultrasound techniques may provide means for early diagnosis of marrow ischemic disorders via detecting focal osteoporotic marrow defect, chronic nonsuppurative osteomyelitis, and cavitations in the mandible (jawbone). The first part of this work investigates the feasibility of using guided waves and high frequency ultrasound for non-invasive human jawbone assessment. The experimental design and the signal/image processing procedures for each technique are developed, and multiple in vitro studies are carried out using dentate and non-dentate mandibles. Results from both the ultrasonic guided waves analysis and the high frequency 3D echodentographic imaging suggest that these techniques show great potential in providing non-invasive methods to characterize the jawbone and detect periodontal diseases at earlier stages.;The second part of this work describes indirect technique for characterization via reconstructing high-resolution microscopic images. The availability of well-defined genetic strains and the ability to create transgenic and knockout mice makes mouse models extremely significant tools in different kinds of research. For example, noninvasive measurement of cardiovascular function in mouse hearts has become a valuable need when studying the development or treatment of various diseases. This work describes the development and testing of a single-element ultrasound imaging system that can reconstruct high-resolution brightness mode (B-mode) images for mouse hearts and blood vessels that can be used for quantitative measurements in vitro. Signal processing algorithms are applied on the received ultrasound signals including filtering, focusing, and envelope detection prior to image reconstruction. Additionally, image enhancement techniques and speckle reduction are adopted to improve the image resolution and quality. The system performance is evaluated using both phantom and in vitro studies using isolated mouse hearts and blood vessels from APOE-KO and its wild type control. This imaging system shall provide a basis for early and accurate detection of different kinds of diseases such as atherosclerosis in mouse model.;The last part of this work is initialized by the increasing need for a non-invasive method to assess vascular wall mechanics. Endothelial dysfunction is considered a key factor in the development of atherosclerosis. Flow-mediated vasodilatation (FMD) measurement in brachial and other conduit arteries has become a common method to assess the endothelial function in vivo. In spite of the direct relationship that could be between the arterial wall multi-component strains and the FMD response, direct measurement of wall strain tensor due to FMD has not yet been reported in the literature. In this work, a noninvasive direct ultrasound-based strain tensor measuring (STM) technique is presented to assess changes in the mechanical parameters of the vascular wall during post-occlusion reactive hyperemia and/or FMD, including local velocities and displacements, diameter change, local strain tensor and strain rates. The STM technique utilizes sequences of B-mode ultrasound images as its input with no extra hardware requirement. The accuracy of the STM algorithm is assessed using phantom, and in vivo studies using human subjects during pre- and post-occlusion. Good correlations are found between the post-occlusion responses of diameter change and local wall strains. Results indicate the validity and versatility of the STM algorithm, and describe how parameters other than the diameter change are sensitive to reactive hyperemia following occlusion. This work suggests that parameters such as local strains and strain rates within the arterial wall are promising metrics for the assessment of endothelial function, which can then be used for accurate assessment of atherosclerosis

Testing procedures and acquisition systems for contact sensor¿based vocal monitoring devices

L'abstract è presente nell'allegato / the abstract is in the attachmen