Single-cell segmentation

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Table of contents

Acknowledgements
Abstract
Foreword
1 Introduction
1.1 The biofilm, a sessile bacterial colony
1.1.1 Context
1.1.2 Biofilm physiology
1.1.3 Biofilm formation
1.2 The bacterial cell cycle
1.2.1 Elongation
1.2.2 Septation
1.3 Bacterial adhesion
1.3.1 Usual adhesion assays
1.3.2 Surface proteins implied in adhesion
1.4 Growth mechanics of a bacterial colony
1.4.1 Evidences of mechanical stress generation
1.4.2 Our approach
2 Materials and methods
2.1 Microscopy
2.2 Image analysis
2.2.1 Colony segmentation
2.2.2 Single-cell segmentation
2.2.3 Tracking
2.2.4 Precise length measurement and fine detection of the division
2.2.5 Morphological parameters
2.3 Growth assays between glass and agarose
2.3.1 Asymmetric adhesion assays
2.3.2 Reorganization following the first division assays
2.3.3 Microcolony growth assays
2.4 Laser ablation
2.4.1 Set-up
2.4.2 Ablation at the 2-cells stage
2.4.3 Ablation at the 3-cells stage
2.5 Traction force microscopy
2.5.1 Adaptation of the technique to prokaryotic cells study .
2.5.2 Experimental procedure
2.5.3 Tracking of beads and calculation of forces
2.5.4 Calibration of the gel stability
3 Results and interpretation
3.1 One and two cells stage
3.1.1 Asymmetric adhesion
3.1.2 Adhesion dynamics of pre-existing poles
3.1.3 Buckling following the first division
3.2 Single-cell dynamics and colony organization
3.2.1 Centrifugal displacements driven by uniform growth within the colony
3.2.2 Organization of bacteria inside a microcolony
3.3 Cell-substrate adhesion
3.3.1 Spatio-temporal dynamics
3.3.2 Rupture of adhesive bonds
3.3.3 Evidence for polar adhesion asymmetry in growing colonies
3.3.4 Strength of adhesins
3.4 Adhesion contribution in the morphogenesis
3.4.1 Influence on morphology
3.4.2 Role of adhesion in the transition from planar to a terraced colony
3.4.3 Adaptation to the environmental challenge
Conclusion and perspectives
A Bacterial strains used in this project
B Experimental protocols
B.1 Agarose/glass sample preparation protocol
B.2 Force measurement protocol
C Growth rate and bacterial size quantification
C.1 Colonies growing between agarose and glass
C.2 Colonies growing between agarose and polyacrylamide
D Preliminary experiments
D.1 Cell length measurements during septation
D.2 Reflection Interference Contrast Microscopy
E French summary / Résumé en français
E.1 Résumé global
E.2 Avant-propos
E.3 Chapitre 1 : Introduction
E.4 Chapitre 2 : Matériels et méthodes
E.5 Chapitre 3 : Résultats et interprétations
E.6 Conclusion et perspectives