Roadmap for optofluidics

Optofluidics, nominally the research area where optics and fluidics merge, is a relatively new research field and it is only in the last decade that there has been a large increase in the number of optofluidic. applications, as well as in the number of research groups, devoted to the topic. Nowadays optofluidics applications include, without being limited to, lab-on-a-chip devices, fluid-based and controlled lenses, optical sensors for fluids and for suspended particles, biosensors, imaging tools, etc. The long list of potential optofluidics applications, which have been recently demonstrated, suggests that optofluidic technologies will become more and more common in everyday life in the future, causing a significant impact on many aspects of our society. A characteristic of this research field, deriving from both its interdisciplinary origin and applications, is that in order to develop suitable solutions a. combination of a deep knowledge in different fields, ranging from materials science to photonics, from microfluidics to molecular biology and biophysics,. is often required. As a direct consequence, also being able to understand the long-term evolution of optofluidics research is not. easy. In this article, we report several expert contributions on different topics. so as to provide guidance for young scientists. At the same time, we hope that this document will also prove useful for funding institutions and stakeholders. to better understand the perspectives and opportunities offered by this research field

Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

Utilisation de nanoparticules magnétiques pour perturber la localisation spatiotemporelle de protéines de signalisation

An increasing number of studies highlight the importance of signaling localization. We developed methods to perturb this localization using magnetic nanoparticles. Proteins of interest are grafted on magnetic nanoparticles, allowing to magnetically localize them. We first propose a new method to engineer directly a spatial gradient of signaling protein concentration within in cell extract droplets using super-paramagnetic nanoparticles. We observed a link between a spatial asymmetry in biochemical cues and microtubules aster positional information. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. We then examined the possibility to magnetically perturb endosomes position in HeLa cell. We found the experimental conditions to achieve this goal. Finally, we used directly cytoskeleton elements as actin filament to trigger asymmetrically confined signaling proteins and trigger microtubule assembly, in cell extract droplets. More generally, these results show how symmetry breaking within cells can be induced and studied using magnetic nanoparticles and biophysical tools.De plus en plus d’études soulignent l’importance de la localisation intracellulaire des voies de signalisation. Nous avons développé des méthodes permettant de perturber cette localisation à l’aide de nanoparticules magnétiques. Ces dernières sont fonctionnalisées avec les protéines d’intérêts et deviennent ainsi un vecteur permettant de contrôler la localisation de la signalisation. Nous avons tout d’abord appliqué cette méthode dans un système modèle, des gouttes d’extrait cellulaire de Xénope, dans lesquelles nous avons créé artificiellement un gradient de protéines de signalisation à l’aide de nanoparticules magnétiques. Nous avons mis en évidence l’influence d’une asymétrie biochimique sur la localisation d’asters de microtubules. Dans un deuxième temps nous avons examiné la possibilité d’appliquer cette méthode dans des cellules HeLa adhérentes, pour perturber la localisation d’endosomes de signalisation rendus magnétiques. Nous avons cherché à optimiser les conditions expérimentales nécessaires pour contrôler la position d’endosomes de signalisation magnétiques Enfin, un troisième projet dont les résultats préliminaires sont présentés dans cette thèse, a consisté à utiliser un actuateur, non plus magnétique, mais biologique pour confiner une cascade de signalisation. Plus précisément la contraction d’un réseau d’actine confiné dans des gouttes d’extrait cellulaire est utilisée pour localiser des protéines de signalisation. Ces résultats démontrent l’intérêt de nanoparticules magnétiques pour induire et étudier des phénomènes de brisures de symétries dans des environnements biologique

Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy

Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits