L'essor des microsystèmes contraint à développer des techniques qui permettent la production de pièces de plus en plus petites. Parmi ces techniques, l'injection de matériaux thermoplastiques dans des micro-moules, nommée microinjection, est un candidat de choix et commence à s'implanter dans le milieu industriel. Mais des verrous techniques et scientifiques empêchent son développement à plus grande échelle. Conscient de ces problèmes, le CEMEF de MinesParisTech, le PIMM de Arts et Métiers ParisTech et la société GETELEC ont déposé un projet ANR Mat&Pro intitulé Micronnect dont le but était à la fois de mettre au point un nouveau concept de machine de microinjection, d'étudier la rhéologie à haut taux de déformation, et pour nous au laboratoire PIMM, de comprendre et déterminer l'influence des hautes vitesses de déformation sur les microstructures et propriétés induites dans les polymères semi-cristallins qui représentent la grande majorité des polymères aujourd'hui utilisés en microinjection. Les études ont été menées sur un PEhD et un PA12 qui ont été injectés dans des plaques de faibles épaisseurs (200, 300 et 500 µm). Les analyses obtenues par la microscopie optique montrent que la morphologie " cœur-peau " n'est constituée que de deux couches visibles, une couche de peau transparente d'épaisseur constante et une couche de cœur assez uniforme. Les analyses combinées de diffusion et de diffraction des rayons X (SAXS et WAXS) avec un microfaisceau synchrotron nous ont permis de déterminer la microstructure induite dans l'épaisseur des pièces. Contrairement à l'injection en plus forte épaisseur, la couche de peau est constituée de shish-kebabs et s'avère la couche la plus orientée. L'épaisseur joue un rôle prépondérant avec des microstructures en shish-kebabs qui se prolongent jusqu'à cœur lorsque l'épaisseur diminue jusqu'à 0,3 ou 0,2 mm. La longueur d'écoulement, le temps d'injection et la température du moule ont également une influence significative. On recherchera des temps d'injection les plus rapides possibles, quelques centièmes de seconde, pour permettre à la fois un bon remplissage des cavités moulantes et une diminution des orientations cristallines, ce qui a pour effet une meilleure relaxation des chaînes de polymères après le remplissage et avant la cristallisation. On comprend alors que les technologies classiques de l'injection doivent évoluer.

This thesis investigates the cooling behaviour of polymers during the microinjection moulding process. The work included bespoke experimental mould design and manufacturing, material characterisation, infra-red temperature measurements, cooling analysis and cooling prediction using commercial simulation software. To measure surface temperature of the polymers, compounding of polypropylene and polystyrene with carbon black masterbatch was performed to make materials opaque for the IR camera. The effects of addition of carbon black masterbatch were analysed using differential scanning calorimetry and Fourier transform infrared spectroscopy. Sapphire windows formed part of the mould wall and allowed thermal measurements using an IR camera. They were laser machined on their inside surfaces to generate a range of finishes and structures. Their topographies were analysed using laser confocal microscope. The surface energy of sapphire windows was measured and compared to typical mould steel, employing a contact angle measurement technique and calculated using Owens-Wendt theory. A heating chamber was designed and manufactured to study spreading of polymer melts on sapphire and steel substrates. A design of experiments approach was taken to investigate the influence of surface finish and the main processing parameters on polymer cooling during microinjection moulding. Cooling curves were obtained over an area of 1.92 by 1.92 mm of the sapphire window. These experiments were conducted on the Battenfeld Microsystem 50 microinjection moulding machine. A simulation study of polymer cooling during the microinjection moulding process was performed using Moldflow software. Particular interest was paid to the effect of the values of the interfacial heat transfer coefficient (HTC) on the simulated cooling predictions. Predicted temperature curves were compared to experimentally obtained temperature distributions, to obtain HTC values valid for the material and processing parameters.

This thesis investigates the cooling behaviour of polymers during the microinjection moulding process. The work included bespoke experimental mould design and manufacturing, material characterisation, infra-red temperature measurements, cooling analysis and cooling prediction using commercial simulation software. To measure surface temperature of the polymers, compounding of polypropylene and polystyrene with carbon black masterbatch was performed to make materials opaque for the IR camera. The effects of addition of carbon black masterbatch were analysed using differential scanning calorimetry and Fourier transform infrared spectroscopy. Sapphire windows formed part of the mould wall and allowed thermal measurements using an IR camera. They were laser machined on their inside surfaces to generate a range of finishes and structures. Their topographies were analysed using laser confocal microscope. The surface energy of sapphire windows was measured and compared to typical mould steel, employing a contact angle measurement technique and calculated using Owens-Wendt theory. A heating chamber was designed and manufactured to study spreading of polymer melts on sapphire and steel substrates. A design of experiments approach was taken to investigate the influence of surface finish and the main processing parameters on polymer cooling during microinjection moulding. Cooling curves were obtained over an area of 1.92 by 1.92 mm of the sapphire window. These experiments were conducted on the Battenfeld Microsystem 50 microinjection moulding machine. A simulation study of polymer cooling during the microinjection moulding process was performed using Moldflow software. Particular interest was paid to the effect of the values of the interfacial heat transfer coefficient (HTC) on the simulated cooling predictions. Predicted temperature curves were compared to experimentally obtained temperature distributions, to obtain HTC values valid for the material and processing parameters.

<p>Microinjection is an established and reliable method to deliver transgenic constructs and other reagents to specific locations in the animal. Specifically, microinjection of a desired DNA construct into the distal gonad is the most widely used method to generate germ-line transformation of <em>C. elegans</em>. Although, current <em>C. elegans</em> microinjection method is an effective manner for creating transgenic worms, it requirements such as expensive multi DOF micromanipulator, detailed injection alignment procedure and skilled operator which makes the microinjection process slow and not suitable for scale to high throughput. Although many microfabricated microinjectors exist, none of them are capable of immobilizing a freely mobile animal such as <em>C.elegans</em> worm. In this research, a microfluidic microinjector was developed to simultaneously immobilize a freely mobile animal such as <em>C.elegans</em> and perform microinjection by using a simple and fast mechanism for needle actuation. The entire process of the microinjection takes ~30 seconds which includes 10s for worm loading and aligning, 5s needle penetration, 5s reagent injection and 5s worm unloading. The capability of the microinjector chip for creating transgenic <em>C. elegans</em> was illustrated (with success rate between 4% to 20%)</p> / Master of Science (MSc)

A series of experiments were used to evaluate three culture media and two incubation temperatures for their ability to support development of DNA microinjected porcine zygotes. Development in vitro was compared between embryos collected from postpubertal and prepubertal donors and between embryos injected with DNA into the pronucleus and the cytoplasm. Additionally, embryos were analyzed by the polymerase chain reaction (PCR) for the presence of the transgene. One-cell embryos (n=458) were recovered from 36 postpubertal gilts in Experiment 1. Injected and control embryos were cultured in modified media NCSU-23 (mNCSU-23), NCSU-37 (mNCSU-37), and CZB at 37°C and 38.8°C for 7 d. In Experiment 2, one-cell embryos (n=245) were collected from postpubertal (n=15) and prepubertal (n=14) gilts, microinjected with DNA, and cultured in medium mNCSU-23. Superovulated prepubertal gilts (n=22) were flushed in Experiment 3 to yield 343 one-cell embryos which had DNA injected into the cytoplasm or pronucleus. Whole embryos were assessed by PCR. Mean percentages of embryos developing to the expanded or hatched blastocyst stage in mNCSU-23 and mNCSU-37 did not differ from each other (p>.05), but both were greater than the development in CZB (p<.05). Development was greater at 38.8°C (p<.05) than at 37° C. Microinjection of DNA decreased the developmental percentage (p<.05) from that of non-injected controls. Embryos collected from postpubertal gilts had a higher percentage (68.0 ± 3.4) of expanded and hatched blastocysts than embryos from prepubertal donors (29.0 ± 4.6, p<.05). No difference was seen in development between embryos injected in the pronucleus or cytoplasm (p>. 05), but development for both was less than for control embryos (p<.05). Results of PCR analysis indicated that 40% of the embryos developing to the expanded blastocyst stage were positive for the transgene compared to a rate of 60% positive for degenerate embryos. These studies show that DNA microinjected porcine zygotes can be cultured to the expanded blastocyst stage in media mNCSU-23 and mNCSU-37 at 38.8°C. Microinjection of DNA decreases survival of embryos collected from both postpubertal and prepubertal sources, but postpubertal embryos exhibit a higher rate of development. Cytoplasmic injection does not improve embryo viability in vitro above that of pronuclear injection. Finally, whole embryo analysis by PCR is possible, but cross specificity of human Protein C and whey acidic protein (WAP) oligonucleotides for endogenous porcine DNA is strong and creates difficulty in applying PCR analysis to embryos microinjected with WAP-PC transgenes. / Master of Science

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.

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.