Ce travail de thèse se situe à l’interface de deux domaines : la culture cellulaire en trois dimensions et l’imagerie sans lentille.Fournissant un protocole de culture cellulaire plus réaliste sur le plan physiologique, le passage des cultures monocouches (2D) à des cultures tridimensionnelles (3D) - via l’utilisation de gels extracellulaires dans lesquels les cellules peuvent se développer dans les trois dimensions - permet de faire de grandes avancées dans de nombreux domaines en biologie tels que l’organogénèse, l’oncologie et la médecine régénérative. Ces nouveaux objets à étudier crée un besoin en matière d’imagerie 3D.De son côté, l’imagerie sans lentille 2D fournit un moyen robuste, peu cher, sans marquage et non toxique, d’étudier les cultures cellulaires en deux dimensions sur de grandes échelles et sur de longues périodes. Ce type de microscopie enregistre l’image des interférences produites par l’échantillon biologique traversé par une lumière cohérente. Connaissant la physique de la propagation de la lumière, ces hologrammes sont rétro-propagés numériquement pour reconstruire l’objet recherché. L’algorithme de reconstruction remplace les lentilles absentes dans le rôle de la formation de l’image.Le but de cette thèse est de montrer la possibilité d’adapter cette technologie sans lentille à l’imagerie des cultures cellulaires en 3D. De nouveaux prototypes de microscopes sans lentille sont conçus en parallèle du développement d’algorithmes de reconstructions tomographiques dédiés.Concernant les prototypes, plusieurs solutions sont testées pour converger vers un schéma alliant deux conditions. La première est le choix de la simplicité d’utilisation avec une culture cellulaire en boîte de Petri standard et ne nécessitant aucune préparation spécifique ou aucun changement de contenant. Cette condition entraînant de fortes contraintes géométriques sur l’architecture, la deuxième est de trouver la meilleure couverture angulaire possible des angles d’éclairage. Enfin, une version adaptée aux conditions en incubateur est développée et testée avec succès.Concernant les algorithmes, quatre types de solutions sont proposés, basées sur le théorème de diffraction de Fourier classiquement utilisé en tomographie diffractive optique. Toutes cherchent à corriger deux problèmes inhérents au microscope sans lentille : l’absence de l’information de phase, le capteur n’étant sensible qu’à l’intensité de l’onde reçue, et la couverture angulaire limitée. Le premier algorithme se limite à remplacer la phase inconnue par celle d’une onde incidente plane. Rapide, cette méthode est néanmoins source de nombreux artefacts. La deuxième solution, en approximant l’objet 3D inconnu par un plan moyen, utilise les outils de la microscopie sans lentille 2D pour retrouver cette phase manquante via une approche inverse. La troisième solution consiste à implémenter une approche inverse régularisée sur l’objet 3D à reconstruire. C’est la méthode la plus efficace pour compenser les deux problèmes mentionnés, mais elle est très lente. La quatrième et dernière solution est basée sur un algorithme de type Gerchberg-Saxton modifié avec une étape de régularisation sur l’objet.Toutes ces méthodes sont comparées et testées avec succès sur des simulations numériques et des données expérimentales. Des comparaisons avec des acquisitions au microscope classique montrent la validité des reconstructions en matière de tailles et de formes des objets reconstruits ainsi que la précision de leur positionnement tridimensionnel. Elles permettent de reconstruire des volumes de plusieurs dizaines de millimètres cubes de cultures cellulaires 3D, inaccessibles en microscopie standard.Par ailleurs, les données spatio-temporelles obtenues avec succès en incubateur montrent aussi la pertinence de ce type d’imagerie en mettant en évidence des interactions dynamiques sur de grandes échelles des cellules entres elles ainsi qu’avec leur environnement tridimensionnel. / This PhD work is at the interface of two fields: 3D cell culture and lens-free imaging.Providing a more realistic cell culture protocol on the physiological level, switching from single-layer (2D) cultures to three-dimensional (3D) cultures - via the use of extracellular gel in which cells can grow in three dimensions - is at the origin of several breakthroughs in several fields such as developmental biology, oncology and regenerative medicine. The study of these new 3D structures creates a need in terms of 3D imaging.On another side, 2D lens-free imaging provides a robust, inexpensive, non-labeling and non-toxic tool to study cell cultures in two dimensions over large scales and over long periods of time. This type of microscopy records the interferences produced by a coherent light scattered by the biological sample. Knowing the physics of the light propagation, these holograms are retro-propagated numerically to reconstruct the unknown object. The reconstruction algorithm replaces the absent lenses in the role of image formation.The aim of this PhD is to show the possibility of adapting this lens-free technology for imaging 3D cell culture. New lens-free microscopes are designed and built along with the development of dedicated tomographic reconstruction algorithms.Concerning the prototypes, several solutions are tested to finally converge to a scheme combining two conditions. The first requirement is the choice of simplicity of use with a cell culture in standard Petri dish and requiring no specific preparation or change of container. The second condition is to find the best possible angular coverage of lighting angles in regards of the geometric constraint imposed by the first requirement. Finally, an incubator-proof version is successfully built and tested.Regarding the algorithms, four major types of solutions are implemented, all based on the Fourier diffraction theorem, conventionally used in optical diffractive tomography. All methods aim to correct two inherent problems of a lens-free microscope: the absence of phase information, the sensor being sensitive only to the intensity of the incident wave, and the limited angular coverage. The first algorithm simply replaces the unknown phase with that of an incident plane wave. However, this method is fast but it is the source of many artifacts. The second solution tries to estimate the missing phase by approximating the unknown object by an average plane and uses the tools of the 2D lens-free microscopy to recover the missing phase in an inverse problem approach. The third solution consists in implementing a regularized inverse problem approach on the 3D object to reconstruct. This is the most effective method to deal with the two problems mentioned above but it is very slow. The fourth and last solution is based on a modified Gerchberg-Saxton algorithm with a regularization step on the object.All these methods are compared and tested successfully on numerical simulations and experimental data. Comparisons with conventional microscope acquisitions show the validity of the reconstructions in terms of shape and positioning of the retrieved objects as well as the accuracy of their three-dimensional positioning. Biological samples are reconstructed with volumes of several tens of cubic millimeters, inaccessible in standard microscopy.Moreover, 3D time-lapse data successfully obtained in incubators show the relevance of this type of imaging by highlighting large-scale interactions between cells or between cells and their three-dimensional environment.

Biological studies always start from curious observations. This is exemplified by description of cells for the first time by Robert Hooke in 1665, observed using his microscope. Since then the field of microscopy and cell biology grew hand in hand, with one field pushing the growth of the other and vice-versa. From basic description of cells in 1665, with parallel advancements in microscopy, we have travelled a long way to understand sub-cellular processes and molecular mechanisms. With each day, our understanding of cells increases and several questions are being posed and answered. Several high-resolution microscopic techniques are being introduced (PALM, STED, STORM, etc.) that push the resolution limit to few tens of nm, taking us to a new era where ‘seeing is believing'. Having said this, it is to be noted that the world of cells is vast, with information spread from nanometers to millimetres, and also over extended time-period, implying that not just one microscopic technique could acquire all the available information. The knowledge in the field of cell biology comes from a combination of imaging and quantifying techniques that complement one another.Majority of modern-day microscopic techniques focuses on increasing resolution which, is achieved at the expense of cost, compactness, simplicity, and field of view. The substantial decrease in the field of observation limits the visibility to a few single cells at best. Therefore, despite our ability to peer through the cells using increasingly powerful optical instruments, fundamental biology questions remain unanswered at mesoscopic scales. A global view of cell population with significant statistics both in terms of space and time is necessary to understand the dynamics of cell biology, taking in to account the heterogeneity of the population and the cell-cell variability. Mesoscopic information is as important as microscopic information. Although the latter gains access to sub-cellular functions, it is the former that leads to high-throughput, label-free measurements. By focussing on simplicity, cost, feasibility, field of view, and time-lapse in-incubator imaging, we developed ‘Lensfree Video Microscope' based on digital in-line holography that is capable of providing a new perspective to cell culture monitoring by being able to capture the kinetics of thousands of cells simultaneously. In this thesis, we present our lensfree video microscope and its applications in in-vitro cell culture monitoring and quantification.We validated the system by performing more than 20,000 hours of real-time imaging, in diverse conditions (e.g.: 37°C, 4°C, 0% O2, etc.) observing varied cell types and culture conditions (e.g.: primary cells, human stem cells, fibroblasts, endothelial cells, epithelial cells, 2D/3D cell culture, etc.). This permitted us to develop label-free cell based assays to study the major cellular events – cell adhesion and spreading, cell division, cell division orientation, cell migration, cell differentiation, network formation, and cell death. The results that we obtained respect the heterogeneity of the population, cell to cell variability (a raising concern in the biological community) and the massiveness of the population, whilst adhering to the standard cell culture practices - a rare combination that is seldom attained by existing real-time monitoring methods.We believe that our microscope and associated metrics would complement existing techniques by bridging the gap between mesoscopic and microscopic information. / Biological studies always start from curious observations. This is exemplified by description of cells for the first time by Robert Hooke in 1665, observed using his microscope. Since then the field of microscopy and cell biology grew hand in hand, with one field pushing the growth of the other and vice-versa. From basic description of cells in 1665, with parallel advancements in microscopy, we have travelled a long way to understand sub-cellular processes and molecular mechanisms. With each day, our understanding of cells increases and several questions are being posed and answered. Several high-resolution microscopic techniques are being introduced (PALM, STED, STORM, etc.) that push the resolution limit to few tens of nm, taking us to a new era where ‘seeing is believing'. Having said this, it is to be noted that the world of cells is vast, with information spread from nanometers to millimetres, and also over extended time-period, implying that not just one microscopic technique could acquire all the available information. The knowledge in the field of cell biology comes from a combination of imaging and quantifying techniques that complement one another.Majority of modern-day microscopic techniques focuses on increasing resolution which, is achieved at the expense of cost, compactness, simplicity, and field of view. The substantial decrease in the field of observation limits the visibility to a few single cells at best. Therefore, despite our ability to peer through the cells using increasingly powerful optical instruments, fundamental biology questions remain unanswered at mesoscopic scales. A global view of cell population with significant statistics both in terms of space and time is necessary to understand the dynamics of cell biology, taking in to account the heterogeneity of the population and the cell-cell variability. Mesoscopic information is as important as microscopic information. Although the latter gains access to sub-cellular functions, it is the former that leads to high-throughput, label-free measurements. By focussing on simplicity, cost, feasibility, field of view, and time-lapse in-incubator imaging, we developed ‘Lensfree Video Microscope' based on digital in-line holography that is capable of providing a new perspective to cell culture monitoring by being able to capture the kinetics of thousands of cells simultaneously. In this thesis, we present our lensfree video microscope and its applications in in-vitro cell culture monitoring and quantification.We validated the system by performing more than 20,000 hours of real-time imaging, in diverse conditions (e.g.: 37°C, 4°C, 0% O2, etc.) observing varied cell types and culture conditions (e.g.: primary cells, human stem cells, fibroblasts, endothelial cells, epithelial cells, 2D/3D cell culture, etc.). This permitted us to develop label-free cell based assays to study the major cellular events – cell adhesion and spreading, cell division, cell division orientation, cell migration, cell differentiation, network formation, and cell death. The results that we obtained respect the heterogeneity of the population, cell to cell variability (a raising concern in the biological community) and the massiveness of the population, whilst adhering to the standard cell culture practices - a rare combination that is seldom attained by existing real-time monitoring methods.We believe that our microscope and associated metrics would complement existing techniques by bridging the gap between mesoscopic and microscopic information.