Persistent cells, representing a small fraction of a bacterial population, are refractory to antibiotic therapy. It is a phenotypic transient state. The underlying mechanisms of persistence remain largely unknown, but even divergent hypotheses agree on a fundamental component: persistence arises from phenotypic heterogeneity. Novel approaches are needed to elucidate which factors allow some bacteria to persist but not others. High-content screening (HCS), which uses microscopy as a screening tool, enables the visualization of single-cell phenotypes and is thus particularly suited for studying bacterial persistence. Traditionally performed with eukaryotic cells in micro-well plate format, executing HCS with prokaryotic cells required the development of a screening novel strategy due to their much smaller size. Microfluidics has proved to be a remarkable toolbox for bacterial studies. In combination with time-lapse microscopy, it permits the study of dynamic processes with single-cell resolution in a tightly controlled microenvironment. Though numerous microfluidic platforms for prokaryotes imaging have been developped, the current state of the art does not allow the imaging of thousands of genotypes in parallel, as is required for high-content screening. The focus of this thesis is to develop a high-throughput microfluidic screening platform enabling high-content imaging of prokaryotic cells. This platform allows 1152 bacterial strains to be imaged in parallel on both the single-cell and sub-cellular level with precise control over the growth environment. First, we demonstrate the ability of the platform to effectively screen for both altered cell division phenotypes through subcellular markers imaging in a static environment and for phenotypes emerging in complex medium profiles, such as bacterial persistence. Second, we employed our platform in a genetic screen, exposing a mutant library of Mycobacterium smegmatis to the antibiotic Isoniazid (INH), and identified a total of 31 mutants with altered persistence rates during antibiotic exposure as compared to the wild-type. Eight of these mutants had decreased persistence rates while 23 mutants displayed increased persistence. Third, using the same platform we performed in depth analysis of mutants with altered rates of persistence in order to identify the cell division and cell death dynamics underlying persistence. This single-cell analysis revealed unexpected dynamics where a mutant with decreased persistence displayed a lower killing rate compared to the wild-type. This high-throughput microfluidic screening platform is highly versatile and can be used in a wide range of bacterial studies, as it is capable of imaging not only single cells, but also subcellular markers, such as those based on the cell cycle or bacterial septa. This novel technology thus opens new doors for microbiologists.