Fatima TAIKI : Optimization of vitreous cryo-sectioning for cryo electron tomography imaging of eukaryotic chromosomes in situ
PhD thesis under the supervision of Amélie Leforestier (LPS, Orsay) and Mikhail Eltsov (IGBMC, Illkirch)
In my PhD project, I focused on optimizing vitreous cryo-sectioning for cryo-electron tomography (cryo-ET) to study chromatin structure directly in its native cellular environment. My goal was to overcome the main technical challenges that have long limited the use of cryosectioning for cryo-ET—particularly poor section adhesion, instability during imaging, and low
throughput. To address these issues, I developed a series of methodological solutions, including a two-step electrostatic charging procedure to enhance section attachment to EM grids, and identified optimal support films that combine mechanical stability with high imaging quality. These advances made it possible to routinely collect large numbers of high-quality tomograms from tissue samples.
As experimental systems, we chose Drosophila melanogaster embryos and larval salivary glands, for which I developed preparation procedures using high-pressure freezing. By combining optimized vitrification and cryo-sectioning workflows with Volta Phase Plate imaging and deep-learning–based computational denoising, I obtained in situ cryo-ET datasets
that enable direct identification of nucleosomes and linker DNA within native chromatin. This allowed me to quantify linker geometry, observe nucleosome conformational variability, and identify non-canonical nucleosome particles in situ.
Overall, this work shows that, when optimized, cryo-sectioning can be a robust alternative to cryo-FIB milling for structural studies of tissues and multicellular organisms. The first results on polytene chromosomes in Drosophila salivary glands provide a promising basis for future investigations of chromatin organization and its relationship to gene regulation at the molecular level.
Composition du Jury
Examinateur : Matthieu GERARD — DR, I2BC Paris-Saclay, CNRS
Rapporteur & Examinateur : Christopher RUSSO — Group Leader, MRC Laboratory of Molecular Biology, Cambridge
Rapporteur & Examinateur : Benoît ZUBER — PR, Institute of Anatomy, Université de Berne
Examinatrice: Aurélie BERTIN — DR, Institut Curie, CNRS
Examinatrice : Maria BARBI — PR, LPTMC, Sorbonne Université