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

Teleoperation of MRI-Compatible Robots with Hybrid Actuation and Haptic Feedback

Image guided surgery (IGS), which has been developing fast recently, benefits significantly from the superior accuracy of robots and magnetic resonance imaging (MRI) which is a great soft tissue imaging modality. Teleoperation is especially desired in the MRI because of the highly constrained space inside the closed-bore MRI and the lack of haptic feedback with the fully autonomous robotic systems. It also very well maintains the human in the loop that significantly enhances safety. This dissertation describes the development of teleoperation approaches and implementation on an example system for MRI with details of different key components. The dissertation firstly describes the general teleoperation architecture with modular software and hardware components. The MRI-compatible robot controller, driving technology as well as the robot navigation and control software are introduced. As a crucial step to determine the robot location inside the MRI, two methods of registration and tracking are discussed. The first method utilizes the existing Z shaped fiducial frame design but with a newly developed multi-image registration method which has higher accuracy with a smaller fiducial frame. The second method is a new fiducial design with a cylindrical shaped frame which is especially suitable for registration and tracking for needles. Alongside, a single-image based algorithm is developed to not only reach higher accuracy but also run faster. In addition, performance enhanced fiducial frame is also studied by integrating self-resonant coils. A surgical master-slave teleoperation system for the application of percutaneous interventional procedures under continuous MRI guidance is presented. The slave robot is a piezoelectric-actuated needle insertion robot with fiber optic force sensor integrated. The master robot is a pneumatic-driven haptic device which not only controls the position of the slave robot, but also renders the force associated with needle placement interventions to the surgeon. Both of master and slave robots mechanical design, kinematics, force sensing and feedback technologies are discussed. Force and position tracking results of the master-slave robot are demonstrated to validate the tracking performance of the integrated system. MRI compatibility is evaluated extensively. Teleoperated needle steering is also demonstrated under live MR imaging. A control system of a clinical grade MRI-compatible parallel 4-DOF surgical manipulator for minimally invasive in-bore prostate percutaneous interventions through the patient’s perineum is discussed in the end. The proposed manipulator takes advantage of four sliders actuated by piezoelectric motors and incremental rotary encoders, which are compatible with the MRI environment. Two generations of optical limit switches are designed to provide better safety features for real clinical use. The performance of both generations of the limit switch is tested. MRI guided accuracy and MRI-compatibility of whole robotic system is also evaluated. Two clinical prostate biopsy cases have been conducted with this assistive robot

Coupling nitrogen-vacancy centers in diamond to fiber-based Fabry-Pérot microcavities

This thesis investigates the coupling of the fluorescence of nitrogen-vacancy (NV) centers in diamond to tunable optical microresonators at ambient conditions, in particular in the regime of Purcell enhancement. We use fiber-based, open-access Fabry-Pérot cavities optimized for high finesse and ultra-small mode volume. Different regimes of cavity enhancement are studied that are complementary to each other: A first experiment relies on a high-finesse cavity with dielectric mirrors. The scaling laws of Purcell enhancement are explicitly demonstrated by a large-range variation of both the cavity mode volume (V = 16 − 600 µm^3 ) and the quality factor (Q = 6 · 10^3 − 2 · 10^6). We detect an enhancement of the emission spectral density by up to a factor of 300. The full potential of this resonator can be exploited with emitters having a linewidth which is narrower than the resonance linewidth of the cavity. This concept holds promise for the implementation of wavelength-tunable, narrow-band single-photon sources as well as the generation of indistinguishable single-photons at ambient conditions. However, for broad-band emitters like the NV center at room temperature, the emission lifetime is not affected noticeably in this configuration. In order to directly observe lifetime changes and Purcell-enhanced single-photon emission, we manufacture fiber-based cavities with silver-coated mirrors having ultra-small mode volumes, as small as V = 1.0 λ^3 = 0.34 µm^3. We demonstrate cavity-enhanced fluorescence imaging, which allows to locate and analyze several single NV centers with one cavity. The Purcell effect is evidenced by an enhanced fluorescence collection of up to 1.6 · 10^6 photons per second from single-NV centers and a tunable variation of the emission lifetime corresponding to an effective Purcell factor of up to 2. We furthermore investigate a benefcial regime of optical confinement where the Fabry-Pérot cavity mode is combined with additional mode confinement by the diamond nanocrystal itself, enabling sub-λ^3 mode volumes. We perform simulations that predict effective Purcell factors of up to 11 for NV centers and of up to 63 for silicon-vacancy centers, revealing a great potential for bright single-photon sources and effcient spin readout at ambient conditions.Diese Arbeit erforscht die Kopplung der Fluoreszenz von Stickstoff-Fehlstellen-Zentren (NV-Zentren) in Diamant mit durchstimmbaren optischen Mikroresonatoren bei Umgebungsbedingungen, insbesondere im Regime der Purcell Verstärkung. Hierzu benutzen wir faserbasierte, offen zugängliche Fabry-Pérot Resonatoren, die für hohe Finesse und ultrakleine Modenvolumen optimiert sind. Verschiedene, komplementäre Bereiche der Resonatorverstärkung werden untersucht. Ein erstes Experiment basiert auf einem Resonator mit hoher Finesse und dielektrischen Spiegeln. Das Skalierungsverhalten der Purcell Verstärkung wird ausführlich ausgewertet, indem man sowohl das Modenvolumen des Resonators (V = 16 − 600 µm^3 ) als auch dessen Güte (Q = 6 · 10^3 − 2 · 10^6) über einen weiten Bereich verändert. Die spektrale Leistungsdichte der Emission kann durch den Resonator um einen Faktor von bis zu 300 überhöht werden. Das gesamte Leistugsvermögen dieses Resonators kann mit schmalbandigen Emittern ausgenutzt werden, deren Emissionslinienbreite kleiner als die Linienbreite des Resonators ist. Dies ist ein vielversprechender Ansatz für die Umsetzung von schmalbandigen Einzelphotonenquellen mit durchstimmbarer Wellenlänge und für die Erzeugung ununterscheidbarer Einzelphotonen bei Umgebungsbedingungen. Jedoch bleibt die Lebenszeit der Emission für breitbandige Emitter, wie dem NV-Zentrum bei Raumtemperatur, in dieser Anordnung nahezu unbeeinflusst. Um eine Veränderung der Lebenszeit und durch den Purcell-Effekt verstärkte Einzelphotonenemission direkt zu beobachten, stellen wir Faserresonatoren mit silberbeschichteten Spiegeln und ultrakleinen Modenvolumen, bis hinab zu V = 1.0 λ^3 = 0.34 µm^3, her. Wir demonstrieren resonatorverstärkte Fluoreszenzbildgebung, die das Auffinden und Untersuchen von verschiedenen einzelnen NV-Zentren mit einem Resonator erlaubt. Der Purcell-Effekt wird über eine gesteigerte Aufsammlung der Fluoreszenz nachgewiesen, mit einer Rate von bis zu 1.6 · 10^6 Photonen pro Sekunde von einzelnen NV-Zentren und außerdem durch die abstimmbare Veränderung der Emissionslebenszeit, entsprechend einem effektiven Purcell Faktor von bis zu 2. Des Weiteren untersuchen wir ein vorteilhaftes Regime, in dem der Diamant Nanokristall selbst eine zusätzliche Einschränkung der optischen Mode bewirkt, die sich mit der Mode des Fabry-Pérot Resonators verbindet und Modenvolumen unter 1 λ^3 ermöglicht. Simulationen ergeben effektive Purcell Faktoren von bis zu 11 für NV-Zentren und von bis zu 63 für Silizium-Fehlstellen-Zentren, wodurch das große Potenzial für helle Einzelphotonenquellen und für effzientes Spin-Auslesen bei Umgebungsbedingungen aufgezeigt wird

Advances in High-Throughput Analysis: Automated Radiochemical Separations and Nanopillar based Separations and Field Enhanced Spectroscopy

Often the need to analyze a large number of samples coincide with critical time consternates. At such times, the implementation of high-throughput technologies is paramount. In this work we explore some viable pathways for high-throughput analysis and develop advancements in novel forms of detection of materials that are vital in the environmental, biological as well as national security arenas. Through the use of new protocols with high sensitivity and specificity as well as simplified chemical processing and sample preparation we aim to allow for improved throughput, fieldable detection, and rapid data acquisition of extensive sample sets. The methods developed in this work focus on unique platforms of the collection and analysis and combine them with automation and portability. Foremost, analytes of interest must be selectively isolated and concentrated by chemical and/or mechanical processes. Secondly, spectroscopic and physical properties are exploited and enhanced by employing viable detection platforms. Finally, automation and field portability are implemented through a combination of optimized robotics, minimized chemical preparation and/or unique lab on a chip type platforms. Presented are two sub areas of research. One focuses on the automation of a time consuming solid phase extraction process that is coupled to inductively coupled plasma mass spectrometry increasing sample throughput by orders of magnitude. The second focused on the fabrication and use of silicon nanopillars as a platform for separations and enhanced optical analysis. Each section of work focuses on the development of a practical, accessible, and deployable methods of analysis

Roadmap on structured light

Structured light refers to the generation and application of custom light fields. As the tools and technology to create and detect structured light have evolved, steadily the applications have begun to emerge. This roadmap touches on the key fields within structured light from the perspective of experts in those areas, providing insight into the current state and the challenges their respective fields face. Collectively the roadmap outlines the venerable nature of structured light research and the exciting prospects for the future that are yet to be realized.Peer ReviewedPostprint (published version

Fast fully automatic myocardial segmentation in 4D cine cardiac magnetic resonance datasets

Dissertação de mestrado integrado em Engenharia BiomédicaCardiovascular diseases (CVDs) are the leading cause of death in the world, representing 30% of all global deaths. Among others, assessment of the left ventricular (LV) morphology and global function using non-invasive cardiac imaging is an interesting technique for diagnosis and treatment follow-up of patients with CVDs. Nowadays, cardiac magnetic resonance (CMR) imaging is the gold-standard technique for the quantification of LV volumes, mass and ejection fraction, requiring the delineation of endocardial and epicardial contours of the left ventricle from cine MR images. In clinical practice, the physicians perform this segmentation manually, being a tedious, time consuming and unpractical task. Even though several (semi-)automated methods have been presented for LV CMR segmentation, fast, automatic and optimal boundaries assessment is still lacking, usually requiring the physician to manually correct the contours. In the present work, we propose a novel fast fully automatic 3D+time LV segmentation framework for CMR datasets. The proposed framework presents three conceptual blocks: 1) an automatic 2D mid-ventricular initialization and segmentation; 2) an automatic stack initialization followed by a 3D segmentation at the end-diastolic phase; and 3) a tracking procedure to delineate both endo and epicardial contours throughout the cardiac cycle. In each block, specific CMR-targeted algorithms are proposed for the different steps required. Hereto, we propose automatic and feasible initialization procedures. Moreover, we adapt the recent B-spline Explicit Active Surfaces (BEAS) framework to the properties of CMR image segmentation by integrating dedicated energy terms and making use of a cylindrical coordinate system that better fits the topology of CMR data. At last, two tracking methods are presented and compared. The proposed framework has been validated on 45 4D CMR datasets from a publicly available database and on a large database from an ongoing multi-center clinical trial with 318 4D datasets. In the technical validation, the framework showed competitive results against the state-of-the-art methods, presenting leading results in both accuracy and average computational time in the common database used for comparative purposes. Moreover, the results in the large scale clinical validation confirmed the high feasibility and robustness of the proposed framework for accurate LV morphology and global function assessment. In combination with the low computational burden of the method, the present methodology seems promising to be used in daily clinical practice.As doenças cardiovasculares (DCVs) são a principal causa de morte no mundo, representando 30% destas a nível global. Na prática clínica, uma técnica empregue no diagnóstico de pacientes com DCVs é a avaliação da morfologia e da função global do ventrículo esquerdo (VE), através de técnicas de imagiologia não-invasivas. Atualmente, a ressonância magnética cardíaca (RMC) é a modalidade de referência na quantificação dos volumes, massa e fração de ejeção do VE, exigindo a delimitação dos contornos do endocárdio e epicárdio a partir de imagens dinâmicas de RMC. Na prática clínica diária, o método preferencial é a segmentação manual. No entanto, esta é uma tarefa demorada, sujeita a erro humano e pouco prática. Apesar de até à data diversos métodos (semi)-automáticos terem sido apresentados para a segmentação do VE em imagens de RMC, ainda não existe um método capaz de avaliar idealmente os contornos de uma forma automática, rápida e precisa, levando a que geralmente o médico necessite de corrigir manualmente os contornos. No presente trabalho é proposta uma nova framework para a segmentação automática do VE em imagens 3D+tempo de RMC. O algoritmo apresenta três blocos principais: 1) uma inicialização e segmentação automática 2D num corte medial do ventrículo; 2) uma inicialização e segmentação tridimensional no volume correspondente ao final da diástole; e 3) um algoritmo de tracking para obter os contornos ao longo de todo o ciclo cardíaco. Neste sentido, são propostos procedimentos de inicialização automática com elevada robustez. Mais ainda, é proposta uma adaptação da recente framework “B-spline Explicit Active Surfaces” (BEAS) com a integração de uma energia específica para as imagens de RMC e utilizando uma formulação cilíndrica para tirar partido da topologia destas imagens. Por último, são apresentados e comparados dois algoritmos de tracking para a obtenção dos contornos ao longo do tempo. A framework proposta foi validada em 45 datasets de RMC provenientes de uma base de dados disponível ao público, bem como numa extensa base de dados com 318 datasets para uma validação clínica. Na avaliação técnica, a framework proposta obteve resultados competitivos quando comparada com outros métodos do estado da arte, tendo alcançado resultados de precisão e tempo computacional superiores a estes. Na validação clínica em larga escala, a framework provou apresentar elevada viabilidade e robustez na avaliação da morfologia e função global do VE. Em combinação com o baixo custo computacional do algoritmo, a presente metodologia apresenta uma perspetiva promissora para a sua aplicação na prática clínica diária