Biology subjects

Hajj, B.

Publications and source records attributed to Hajj, B..

8 recordsLinked to original sources

bioRxiv2025

Polarization MultiFocus Microscopy for volumetric super-resolution and orientation imaging of biofilaments

Accessing molecular orientation in single molecule localization microscopy (SMLM) offers valuable insights into molecular ordering and organization in biological structures. Conventional single-molecule orientation-localization microscopy (SMOLM) methods typically rely on either engineering the microscopes point-spread function (PSF) to encode the orientation information or on polarization resolved detection. While PSF engineering enables detailed orientation analysis, it often requires complex computational analysis and suffers from reduced performance in dense cellular environments due to PSF spreading and overlap. In contrast, polarization-based approaches are easier to implement and are more fit when imaging dense samples but are unable to retrieve the axial information of single molecules. To overcome this limitation, we introduce the Polarization MultiFocus Microscope (PolMFM), a novel method for simultaneously retrieving the orientation and 3D position of single molecules. PolMFM combines the orientation measurement capabilities of a 4-polarization splitting scheme with a 3-planes multifocus microscope (MFM) enabling the reconstruction of molecular 2D orientation, wobble, and axial localization in a single acquisition. Through simulations, we demonstrate that PolMFM accurately recovers both orientation and 3D position, despite PSF defocusing. Experimental validation with reference samples shows that PolMFM matches the orientation precision of 4-Polar STORM, while uniquely adding axial information. We demonstrate the power of PolMFM by resolving the orientation and 3D positions of molecules in actin filaments in fixed cells, and by revealing that chromatin in crickets undergoes major reorganization and increased ordering during spermiogenesis. These findings highlight the potential of PolMFM for high-precision, multidimensional super-resolution imaging in complex and crowded biological environments.

biophysics↗

bioRxiv2025

Septin Architecture Dictates Size-Dependent Diffusion Barriers on biomimetic Membranes

Septins are cytoskeletal filaments bound to the inner leaflet of the plasma membrane, essential for cell division. They are involved in the formation of diffusion barriers for membrane-bound components. Whether septins directly function as barriers or as part of a broader regulatory cascade of events remains unclear. We addressed this using in vitro reconstituted assay of biomimetic synthetic membranes. We probed whether the diffusion of biomimetic fluorescent objects with tunable steric hindrance - mimicking membrane bound proteins - is constrained by septins. Our results indicate that (i) lipids and transmembrane proteins lacking cytosolic domains diffuse freely in the presence of septins (ii) membrane-bound objects with large cytosolic domains experience size-dependent diffusion constraints; and (iii) the ability of septins to act as diffusion barriers is finely tuned by their intrinsic filament organization. These findings demonstrate that septins can directly impose size-selective diffusion barriers and that their filament organization critically tunes this function.

biophysics↗

bioRxiv2022

A specific mesh-like organization of human septin octameric complex drives membrane reshaping and curvature sensitivity

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane, we have used a combination of methods to assay their ultrastructural organization, their curvature sensitivity as well as their role in membrane reshaping. In contrast to budding yeast septins, on membranes, human septins systematically organize into a two-layered mesh of orthogonal filaments instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is curvature sensitive and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarsegrained computed simulation to understand their mechanisms. Our results highlight the specificity of animal septins as opposed to fungal proteins.

biophysics↗

bioRxiv2022

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

In recent years, the development of new image analysis approaches has highlighted the possibility of recovering superresolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.

biophysics↗

bioRxiv2022

SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos,and background subtraction

While fluorescent microscopy imaging has become the spearhead of modern biology as it is able to generate long-term videos depicting 4D nanoscale cell behaviors, it is still limited by the optical aberrations and the photon budget available in the specimen and to some extend to photo-toxicity. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when pushing the illumination to the low levels in order to limit photo-damages. Moreover, as the processing of very large temporal series of images considerably slows down the analysis, special attention must be paid to the feasibility and scalability of the developed restoration algorithms. To address these specifications, we present a very flexible method designed to restore 2D-3D+Time fluorescent images and subtract undesirable out-of-focus background. We assume that the images are sparse and piece-wise smooth, and are corrupted by mixed Poisson-Gaussian noise. To recover the unknown image, we consider a novel convex and non-quadratic regularizer Sparse Hessian Variation) defined as the mixed norms which gathers image intensity and spatial second-order derivatives. This resulting restoration algorithm named SPITFIR(e) (SParse fIT for Fluorescence Image Restoration) utilizes the primal-dual optimization principle for energy minimization and can be used to process large images acquired with varied fluorescence microscopy modalities. It is nearly parameter-free as the practitioner needs only to specify the amount of desired sparsity (weak, moderate, high). Experimental results in lattice light sheet, stimulated emission depletion, multifocus microscopy, spinning disk confocal, and wide-field microscopy demonstrate the generic ability of the SPITFIR(e) algorithm to efficiently reduce noise and blur, and to subtract undesirable fluorescent background, while avoiding the emergence of deconvolution artifacts.

bioinformatics↗

bioRxiv2020

DIVA: natural navigation inside 3D images usingvirtual reality

As three-dimensional microscopy becomes commonplace in biological re-search, there is an increasing need for researchers to be able to view experimental image stacks in a natural three-dimensional viewing context. Through stereoscopy and motion tracking, commercial virtual reality headsets provide a solution to this important visualization challenge by allowing researchers to view volumetric objects in an entirely intuitive fashion. With this motivation, we present DIVA, a user-friendly software tool that automatically creates detailed three-dimensional reconstructions of raw experimental image stacks that are integrated in virtual reality. In DIVAs immersive virtual environment, users can view, manipulate and perform volumetric measurements on their microscopy images as they would to real physical objects. In contrast to similar solutions, our software provides high-quality volume rendering with native TIFF file compatibility. We benchmark the software with diverse image types including those generated by confocal, light-sheet and electron microscopy. DIVA is available at https://diva.pasteur.fr and will be regularly updated.

bioinformatics↗

bioRxiv2020

Genuage: a virtual reality platform for visualizing and analyzing multidimensional point cloud data

The quantity of experimentally recorded point cloud data, such generated in single-molecule experiments, is increasing continuously in both size and dimension. Gaining an intuitive understanding of the data and facilitating multi-dimensional data analysis remains a challenge. It is especially challenging when static distribution properties are not predictive of dynamical properties. Here, we report a new open-source software platform, Genuage, that enables the easy perception, interaction and analysis of complex multidimensional point cloud datasets by leveraging virtual reality. We illustrate the benefit of the Genuage with examples of three-dimensional static and dynamic localization microscopy datasets, as well as some synthetic datasets. Genuage has a large breadth of usage modes, due to its compatibility with arbitrary multidimensional data types extending beyond the single-molecule research community.

biophysics↗

bioRxiv2019

Molecular basis of CTCF binding polarity in genome folding

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 proteins (Merkenschlager & Nora, 2016; Fudenberg, Abdennur, Imakaev, Goloborodko, & Mirny, 2017). While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize DNA loops (de Wit et al., 2015; Guo et al., 2015; Rao et al., 2014; Vietri Rudan et al., 2015), the molecular basis of this polarity remains mysterious. Here we report that CTCF positions cohesin but does not control its overall binding or dynamics on chromatin by single molecule live imaging. Using an inducible complementation system, we found that CTCF mutants lacking the N-terminus cannot insulate TADs properly, despite normal binding. Cohesin remained at CTCF sites in this mutant, albeit with reduced enrichment. Given that the orientation of the CTCF motif presents the CTCF N-terminus towards cohesin as it translocates from the interior of TADs, these observations provide a molecular explanation for how the polarity of CTCF binding sites determines the genomic distribution of chromatin loops.

molecular biology↗