bottom-up-vitro-methods-to-assay-ultrastructural-organization

Bottom-Up <em>In Vitro</em> Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

Sorbonne Université

Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm. 

PSF engineering in multifocus microscopy for increased depth volumetric imaging.

Multifocus microscopy (MFM) allows sensitive and fast three-dimensional imaging. It relies on the efficient design of diffraction phase gratings yielding homogeneous intensities in desired diffraction orders. Such performances are however guaranteed only for a specific wavelength. Here, we discuss a novel approach for designing binary phase gratings with dual color properties and improved diffraction efficiency for MFM. We simulate binary diffraction gratings with tunable phase shifts to explore its best diffraction performances. We report the design and fabrication of a binary array generator of 3 × 3 equal-intensity diffraction orders with 74% efficiency, 95% uniformity and dual color capability. The multicolor properties of this new design are highlighted by two-color MFM imaging. Finally, we discuss the basics of extending this approach to a variety of diffraction pattern designs.

Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings.

Experimentally recorded point cloud data, such as those generated by single-molecule localization microscopy, are continuously increasing in size and dimension. Gaining an intuitive understanding and facilitating the analysis of such multidimensional data remains challenging. Here we report a new open-source software platform, Genuage, that enables the easy perception of, interaction with and analysis of multidimensional point clouds in virtual reality. Genuage is compatible with arbitrary multidimensional data extending beyond single-molecule localization microscopy.

Genuage: visualize and analyze multidimensional single-molecule point cloud data in virtual reality.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.

Article	Year
PSF engineering in multifocus microscopy for increased depth volumetric imaging. Biomedical optics express\| PubMed ID: 27231584	2016
Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings. Scientific reports\| PubMed ID: 28706216	2017
Genuage: visualize and analyze multidimensional single-molecule point cloud data in virtual reality. Nature methods\| PubMed ID: 32958921	2020
Molecular basis of CTCF binding polarity in genome folding. Nature communications\| PubMed ID: 33154377	2020

Bassam Hajj

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