Suivi de culture cellulaire par imagerie sans lentille

par Srikanth Vinjimore Kesavan

Thèse de doctorat en Physique - Cergy

Sous la direction de Bernard Chalmond.

Soutenue le 15-12-2014

à Cergy-Pontoise , dans le cadre de École doctorale Sciences et ingénierie (Cergy-Pontoise, Val d'Oise) , en partenariat avec Laboratoire de Physique des Matériaux et des Surfaces (Cergy-Pontoise, Val d'Oise) (laboratoire) et de Laboratoire de Physique des Matériaux et des Surfaces / LPMS (laboratoire) .

Le président du jury était Cédric Allier.

Le jury était composé de Jean-Marc Dinten, Pierre Rocheteau, Xavier Gidrol, Spencer L. Shorte.

Les rapporteurs étaient Antoine Delon, Manuel Théry.


  • Résumé

    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.

  • Titre traduit

    Measurement of morphological modifications of cell population using lensless imaging.


  • Résumé

    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.


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