Remote refocusing light-sheet fluorescence microscopy for high-speed 2D and 3D imaging of calcium dynamics in cardiomyocytes

The high prevalence and poor prognosis of heart failure are two key drivers for research into cardiac electrophysiology and regeneration. Dyssynchrony in calcium release and loss of structural organization within individual cardiomyocytes (CM) has been linked to reduced contractile strength and arrhythmia. Correlating calcium dynamics and cell microstructure requires multidimensional imaging with high spatiotemporal resolution. In light-sheet fluorescence microscopy (LSFM), selective plane illumination enables fast optically sectioned imaging with lower phototoxicity, making it suitable for imaging subcellular dynamics. In this work, a custom remote refocusing LSFM system is applied to studying calcium dynamics in isolated CM, cardiac cell cultures and tissue slices. The spatial resolution of the LSFM system was modelled and experimentally characterized. Simulation of the illumination path in Zemax was used to estimate the light-sheet beam waist and confocal parameter. Automated MATLAB-based image analysis was used to quantify the optical sectioning and the 3D point spread function using Gaussian fitting of bead image intensity distributions. The results demonstrated improved and more uniform axial resolution and optical sectioning with the tighter focused beam used for axially swept light-sheet microscopy. High-speed dual-channel LSFM was used for 2D imaging of calcium dynamics in correlation with the t-tubule structure in left and right ventricle cardiomyocytes at 395 fps. The high spatio-temporal resolution enabled the characterization of calcium sparks. The use of para-nitro-blebbistatin (NBleb), a non-phototoxic, low fluorescence contraction uncoupler, allowed 2D-mapping of the spatial dyssynchrony of calcium transient development across the cell. Finally, aberration-free remote refocusing was used for high-speed volumetric imaging of calcium dynamics in human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM) and their co-culture with adult-CM. 3D-imaging at up to 8 Hz demonstrated the synchronization of calcium transients in co-culture, with increased coupling with longer co-culture duration, uninhibited by motion uncoupling with NBleb.Open Acces

Probing Cellular Uptake of Nanoparticles, One at a Time

Advanced fluorescence microscopy is the method of choice to study cellular uptake of nanoparticles with molecular specificity and nanoscale resolution; yet, direct visualization of nanoparticles entry into cells poses severe technical challenges. Here, we have combined super-resolution photoactivation localization microscopy (PALM) with single particle tracking (SPT) to visualize clathrin-mediated endocytosis (CME) of polystyrene nanoparticles at very high spatial and temporal resolution

Dynamic regulation of subcellular calcium handling in the atria:modifying effects of stretch and adrenergic stimulation

Atrial fibrillation is the fast and irregular heart rate that occurs when the upper chambers of the heart experience chaotic electrical activation. Three main factors contribute to the development of this disease: triggers, substrate and modifying factors. An arrhythmia is thus like a fire that needs a spark (Trigger) to ignite a pile of wood (Substrate) and depends on the humidity or accelerants (modifying factors) to burn faster or slower. This body of work takes a closer look at such modifying factors. The major finding of this thesis is that stretching atrial heart muscle cells releases Calcium ions from storage spaces within each cell. If these Calcium release events get frequent enough they can act as triggers for the arrhythmia. The thickness of the atrial muscle is heterogeneous, thus filling the atrium with blood distends thinner parts stronger than ticker portions. The varying degree of stretch might stimulate Calcium release predominantly from myocytes in thinner regions of the atria. This heterogeneity in spontaneous Calcium release can modify also the substrate. A comparable effect of stretch was previously described in the heart’s main chambers. However, it appears that the in the atria it depends on another mechanism, which could serve as a treatment target that mainly acts on the atria without negatively affecting the ventricle

Ono: an open platform for social robotics

In recent times, the focal point of research in robotics has shifted from industrial ro- bots toward robots that interact with humans in an intuitive and safe manner. This evolution has resulted in the subfield of social robotics, which pertains to robots that function in a human environment and that can communicate with humans in an int- uitive way, e.g. with facial expressions. Social robots have the potential to impact many different aspects of our lives, but one particularly promising application is the use of robots in therapy, such as the treatment of children with autism. Unfortunately, many of the existing social robots are neither suited for practical use in therapy nor for large scale studies, mainly because they are expensive, one-of-a-kind robots that are hard to modify to suit a specific need. We created Ono, a social robotics platform, to tackle these issues. Ono is composed entirely from off-the-shelf components and cheap materials, and can be built at a local FabLab at the fraction of the cost of other robots. Ono is also entirely open source and the modular design further encourages modification and reuse of parts of the platform

Fotoreceptores em campo elétrico

Orientadores: Sérgio Santos Mühlen, Richard Hans Wilhelm FunkTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de ComputaçãoResumo: Estudos anteriores e evidências experimentais sugerem um papel importante dos campos elétricos endógenos no direcionamento da migração celular, no desenvolvimento e na regeneração celular e na cicatrização de feridas. Em culturas de células, campos elétricos de corrente contínua influenciam a divisão celular, a polaridade, a forma e a motilidade das células. As distrofias das células fotoreceptoras são uma das principais causas de cegueira hereditária no mundo ocidental; a aplicação de campo elétrico poderia ser usada como um sinal direcional para o crescimento das células fotoreceptoras na direção do tecido danificado. Neste estudo, investigamos os efeitos do campo elétrico no estabelecimento da polaridade celular de fotoreceptores e na polarização de estruturas intracelulares, a fim de demonstrar que são regulados por um sinal elétrico externo. Utilizando um ensaio de migração bem estabelecido, células fotoreceptoras de retina de camundongos do tipo cone, 661W, foram estimuladas durante 5 horas com campo eléctrico de 5 V/cm. Através de técnicas de imunofluorescência, investigamos mudanças na posição de organelas importantes após a estimulação, como o núcleo e o complexo de Golgi (GA), e também de proteínas do citoesqueleto, tais como actina, microtúbulos e o centro organizador dos microtúbulos (MTOC). Além disso, investigamos também alterações no potencial da membrana plasmática e mitocondrial utilizando corantes específicos, na presença e ausência de campo elétrico aplicado. Em resposta ao estímulo direcional, as células 661W estenderam protuberâncias de membrana no sentido do catodo; elas se alongaram perpendicularmente ao campo elétrico e formaram uma borda frontal. Ocorreu migração significativa na direção do catodo. O centro organizador dos microtúbulos, o complexo de Golgi e actina foram reorientados na direcção da borda frontal das células (catodo), enquanto os microtúbulos se acumularam na borda traseira das células (anodo) e o núcleo foi translocado para a parte de trás das células, também na borda traseira. Após exposição ao campo elétrico, ambos os potenciais de membrana, plasmático e mitocondrial, se despolarizaram, especialmente no lado do catodo das células. Esse estudo contribuiu para uma melhor compreensão dos mecanismos de migração direcional devido ao campo elétrico das células 661W, que depende da despolarização das membranas plasmática e mitocondrial e da polarização induzida do citoequeleto de actina e microtúbulos, com subsequente polarização do núcleo, MTOC e GAAbstract: Previous studies and experimental evidences suggest an important role for endogenous electric fields in directing cell migration in wound healing, development and regeneration. In cultures, applied direct current electric field (dcEF) influences cell division, polarity, shape and motility. Photoreceptors dystrophies are one of the major causes of inherited blindness in the western world; application of EF could be used as a cue to direct photoreceptors cells to growth towards the damaged tissue. In this study we investigate the effects of dcEF in the establishment of photoreceptor cell polarity and polarization of intracellular structures, in order to demonstrate that they are regulated by an extracellular electrical cue. Using a well established migration assay, photoreceptors cone-like 661W mouse retinal cells were stimulated for 5 h with 5 V/cm electric field. Using immunofluorescence techniques we have investigated changes in position of important organelles after the stimulation, like Golgi Apparatus (GA) and nucleus, and also cytoskeletal proteins, such as the Microtubules Organizing Center (MTOC), actin and Microtubules (MT). Furthermore, we investigated changes in plasma and mitochondrial membrane potentials using ion reporter dyes in the presence and absence of an applied dcEF. In response to the directional stimulus, 661W cells have extended membrane protrusions towards cathode; they got elongated perpendicular to the dcEF and have formed a leading edge towards the direction of cues. Directional migration has occurred towards cathode. MTOC, GA and actin were reoriented in the direction of the leading edge of the cells (cathode), while the MT accumulated in the rear edge of the cells (anode) and the nucleus was translocated to the back of the cells, also in the rear edge. After dcEF exposure, both plasma and mitochondrial membranes were depolarized, especially in the cathode side of the cells. This study extended an understanding of the mechanism of the dcEF-directed 661W cell migration, which depends on plasma and mitochondrial membrane depolarization and an induced polarization of actin cytoskeleton and microtubules with subsequent polarization of nucleus, MTOC and GADoutoradoEngenharia BiomedicaDoutora em Engenharia Elétrica34548011846CAPE

The role of the femoral chordotonal organ in motor control, interleg coordination, and leg kinematics in Drosophila melanogaster

Legged locomotion in terrestrial animals is often essential for mating and survival, and locomotor behavior must be robust and adaptable in order to be successful. The behavioral plasticity demonstrated by animals’ ability to locomote across diverse types of terrains and to change their locomotion in a task-dependent manner highlights the flexible and modular nature of locomotor networks. The six legs of insects are under the multi-level control of local networks for each limb and limb joint in addition to over-arching central control of the local networks. These networks, consisting of pattern-generating groups of interneurons, motor neurons, and muscles, receive modifying and reinforcing feedback from sensory structures that encode motor output. Proprioceptors in the limbs monitoring their position and movement provide information to these networks that is essential for the adaptability and robustness of locomotor behavior. In insects, proprioceptors are highly diverse, and the exact role of each type in motor control has yet to be determined. Chordotonal organs, analogous to vertebrate muscle spindles, are proprioceptive stretch receptors that span joints and encode specific parameters of relative movement between body segments. In insects, when leg chordotonal organs are disabled or manipulated, interleg coordination and walking are affected, but the simple behavior of straight walking on a flat surface can still be performed. The femoral chordotonal organ (fCO) is the largest leg proprioceptor and monitors the position and movements of the tibia relative to the femur. It has long been studied for its importance in locomotor and postural control. In Drosophila melanogaster, an ideal model organism due its genetic tractability, investigations into the composition, connectivity, and function of the fCO are still in their infancy. The fCO in Drosophila contains anatomical subgroups, and the neurons within a subgroup demonstrate similar responses to movements about the femur-tibia joint. Collectively, the experiments laid out in this dissertation provide a multi-faceted analysis of the anatomy, connectivity, and functional importance of subgroups of fCO neurons in D. melanogaster. The dissertation is divided into four chapters, representing different aspects of this complex and intriguing system. First, I present a detailed analysis of the composition of the fCO and its connectivity within the peripheral and central nervous systems. I demonstrate that the fCO is made up of anatomically distinct groups of neurons, each with their own unique features in the legs and ventral nerve cord. Second, I investigated the neuropeptide profile of the fCO and demonstrate that some fCO neurons express a susbtance that is known to act as a neuromodulator. Third, I demonstrate the sufficiency of subsets of fCO neurons to elicit reflex responses, highlighting the role of the Drosophila fCO in postural control. Lastly, I take this a step further and look into the functional necessity of these neuronal subsets for intra- and interleg coordination during walking. The importance of the fCO in motor control in D. melanogaster has been considered rather minor, though research into the topic is very limited. In the work laid out herein, I highlight the complexity of the Drosophila fCO and its role in the determination of locomotor behavior

Overcoming conventional modeling limitations using image- driven lattice-boltzmann method simulations for biophysical applications

The challenges involved in modeling biological systems are significant and push the boundaries of conventional modeling. This is because biological systems are distinctly complex, and their emergent properties are results of the interplay of numerous components/processes. Unfortunately, conventional modeling approaches are often limited by their inability to capture all these complexities. By using in vivo data derived from biomedical imaging, image-based modeling is able to overcome this limitation. In this work, a combination of imaging data with the Lattice-Boltzmann Method for computational fluid dynamics (CFD) is applied to tissue engineering and thrombogenesis. Using this approach, some of the unanswered questions in both application areas are resolved. In the first application, numerical differences between two types of boundary conditions: “wall boundary condition” (WBC) and “periodic boundary condition” (PBC), which are commonly utilized for approximating shear stresses in tissue engineering scaffold simulations is investigated. Surface stresses in 3D scaffold reconstructions, obtained from high resolution microcomputed tomography images are calculated for both boundary condition types and compared with the actual whole scaffold values via image-based CFD simulations. It is found that, both boundary conditions follow the same spatial surface stress patterns as the whole scaffold simulations. However, they under-predict the absolute stress values approximately by a factor of two. Moreover, it is found that the error grows with higher scaffold porosity. Additionally, it is found that the PBC always resulted in a lower error than the WBC. In a second tissue engineering study, the dependence of culture time on the distribution and magnitude of fluid shear in tissue scaffolds cultured under flow perfusion is investigated. In the study, constructs are destructively evaluated with assays for cellularity and calcium deposition, imaged using µCT and reconstructed for CFD simulations. It is found that both the shear stress distributions within scaffolds consistently increase with culture time and correlate with increasing levels of mineralized tissues within the scaffold constructs as seen in calcium deposition data and µCT reconstructions. In the thrombogenesis application, detailed analysis of time lapse microscopy images showing yielding of thrombi in live mouse microvasculature is performed. Using these images, image-based CFD modeling is performed to calculate the fluid-induced shear stresses imposed on the thrombi’s surfaces by the surrounding blood flow. From the results, estimates of the yield stress (A critical parameter for quantifying the extent to which thrombi material can resist deformation and breakage) are obtained for different blood vessels. Further, it is shown that the yielding observed in thrombi occurs mostly in the outer shell region while the inner core remains intact. This suggests that the core material is different from the shell. To that end, we propose an alternative mechanism of thrombogenesis which could help explain this difference. Overall, the findings from this work reveal that image-based modeling is a versatile approach which can be applied to different biomedical application areas while overcoming the difficulties associated with conventional modeling