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Nano-fabrication of cellular force sensors and surface coatings via dendritic solidificationPaneru, Govind January 1900 (has links)
Doctor of Philosophy / Department of Physics / Bret N. Flanders / Directed electrochemical nanowire assembly (DENA) is a method for fabricating nano-structured materials via electrochemical dendritic solidification. This thesis presents two new applications of nano-structured materials that are fabricated via the DENA methodology: cellular force sensors to probe adhesive sites on living cells and single-crystalline metallic dendrites as surface coating materials.
Fast migrating cells like D. discoideum, leukocytes, and breast cancer cells migrate by attachment and detachment of discrete adhesive contacts, known as actin foci, to the substrate where the cell transmits traction forces. Despite their importance in migration, the physics by which actin foci bind and release substrates is poorly understood. This gap is largely due to the compositional complexity of actin foci in living cells and to a lack of technique for directly probing these sub-cellular structures. Recent theoretical work predicts these adhesive structures to depend on the density of adhesion receptors in the contact sites, the receptor-substrate potential, and cell-medium surface tension. This thesis describes the fabrication of sub-microscopic force sensors composed of poly(3,4-ethylene dioxythiophene) fibers that can interface directly with sub-cellular targets such as actin foci. The spring constants of these fibers are in the range of 0.07-430 nN m-1. These fibers were used to characterize the strength and lifetime of adhesion between the single adhesive contacts of D. discoideum cells and the fibers, finding an average force of 3.1 ± 2.7 nN and lifetime of 23.4 ± 18.5 s. This capability is significant because direct measurement of these properties will be necessary to measure the cell-medium surface tension and to characterize the receptor-substrate potential in the next (future) stage of this project.
The fabrication of smart materials that are capable of the high dynamic range structural reconfiguration would lead to their use to confer hydrophobic, lipophobic, and anti-corrosive character to substrates in a regenerative manner. As a step towards this goal, we have extended the DENA method to enable repetitive growth and dissolution of metallic dendrites to substrates. The experimental parameters that control this process are the frequency and duty cycle of the alternating voltage signal that initiates the dendritic growth.
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Dynamique des forces motiles et brisure de symétrie chez la cellule migrante / Dynamics of motile forces and symmetry breaking in the migrating cellHennig, Katharina 17 October 2018 (has links)
La motilité cellulaire directionnelle au cours du développement de l'organisme et des tissus, l'homéostasie et la maladie nécessite une rupture de symétrie. Ce processus repose sur la capacité des cellules individuelles à établir une polarité avant-arrière, et peut se produire en l'absence de signaux externes. L'initiation de la migration a été attribuée à la polarisation spontanée des composants du cytosquelette, tandis que l'évolution spatio-temporelle des forces du cytosquelette résultant de l'interaction mécanique cellule-substrat continue n'a pas encore été résolue. Ici, nous établissons un test de migration microfabriqué unidimensionnel qui imite un environnement fibrillaire complexe in vivo tout en étant compatible avec les mesures de force à haute résolution, la microscopie quantitative et l'optogénétique. La quantification des paramètres morphométriques et mécaniques révèle un comportement de stick-slip générique initié par un détachement stochastique des contacts adhésifs d'un côté de la cellule dépendant de la contractilité, qui est suffisant pour conduire la motilité cellulaire directionnelle en absence de polarité du cytosquelette préétablie ou de gradients morphogènes. Un modèle théorique valide le rôle crucial de la dynamique d'adhésion au cours de la rupture de symétrie spontanée, en proposant que le phénomène examiné puisse émerger indépendamment d'un système auto-polarisant complexe. / Directional cell motility during organism and tissue development, homeostasis and disease requires symmetry breaking. This process relies on the ability of single cells to establish a front-rear polarity, and can occur in absence of external cues. The initiation of migration has been attributed to the spontaneous polarization of cytoskeleton components, while the spatio- temporal evolution of cytoskeletal forces arising from continuous mechanical cell-substrate interaction has yet to be resolved. Here, we establish a one- dimensional microfabricated migration assay that mimics complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to drive directional cell motility in absence of pre-established cytoskeleton polarity or morphogen gradients. A theoretical model validates the crucial role of adhesion dynamics during spontaneous symmetry breaking, proposing that the examined phenomenon can emerge independently of a complex self-polarizing system.
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Microrobotic Manipulation and Characterization of Biological CellsLiu, Xinyu 01 March 2010 (has links)
Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies.
Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility.
Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos.
For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.
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Microrobotic Manipulation and Characterization of Biological CellsLiu, Xinyu 01 March 2010 (has links)
Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies.
Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility.
Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos.
For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.
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