Understanding the details of how chromatin folds and the physical parameters governing chromatin behavior are among the most intriguing intellectual challenges in modern cell biology. Recent insights on chromosome conformation in yeast nuclei were gained owing to high-throughput molecular biology techniques, but the physical parameters governing chromosomes dynamics remain completely misunderstood. During my thesis, we have developed an original high speed 3D imaging technology for studying chromatin in living yeast, which allows accessing 3D dynamics with temporal resolutions of 15 ms and nanometer resolution by fluorescence microscopy. Our technology is based on the fabrication of V-shaped micromirrors by standard photolithography, and relies on stereovision methods for 3D reconstruction. These micromirrors were integrated into a lab on a chip, and several versions of the technology have been proposed to improve their properties. We demonstrated that this technology is suitable for tracking the movement of chromosomes in living cells, and we achieved among the best acquisition rates in the literature. Our technological developments were eventually used to explore the physical mechanisms behind the spatial fluctuations of chromosomes, and we observed that standard models of polymer physics can be used to extract quantitative information describing the organization and the spatial dynamics of genome.