Biology subjects

Lessard, M. D.

Publications and source records attributed to Lessard, M. D..

4 recordsLinked to original sources

bioRxiv2025

Pan-ASLM: a high-resolution and large-field-of-view light sheet microscope for Expansion Microscopy

Expansion microscopy, a super-resolution fluorescence microscopy technique in which samples are expanded up to [~]8,000 times (after 20-fold expansion) their original volume, places high demands on the microscopes used to image the expanded samples. To reveal nanoscale cellular ultrastructure in meaningful sample volumes, the instruments need to feature a large field of view and working distance. Simultaneously, they need to offer a high three-dimensional resolution to avoid counteracting the resolution improvement achieved by the expansion process. Here, we present pan-ASLM, a high resolution, large field-of-view light-sheet microscope developed for expanded samples, based on the Axially Swept Light Sheet Microscopy (ASLM) technique. pan-ASLM allows imaging over a 640 {micro}m x 640 {micro}m field of view with lateral and axial resolutions of 566 nm and 457 nm, respectively, and features an image acquisition speed of up to 20 fps (183 Mvoxels/sec). It offers [~]1200x higher imaging speed, a [~]7x larger field of view, and [~]2x better axial resolution than the standard confocal microscopes typically used for expanded samples. We validate the new microscope design through imaging of pan-expanded HeLa cells as well as mouse kidney and brain tissue.

biophysics↗

bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗

bioRxiv2020

3D Adaptive Optical Nanoscopy for Thick Specimen Imaging at sub-50 nm Resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here, we have developed a nanoscope that, by utilizing an advanced adaptive optics strategy, achieves sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue.

biophysics↗

bioRxiv2019

Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.\n\nOne Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

biophysics↗