Highly Sensitive Shack-Hartmann Wavefront Sensor: Application to Non-Transparent Tissue Mimic Imaging with Adaptive Light-Sheet Fluorescence Microscopy.

High-quality in-depth imaging of three-dimensional samples remains a major challenge in modern microscopy. Selective plane illumination microscopy (SPIM) is a widely used technique that enables imaging of living tissues with subcellular resolution. However, scattering, absorption, and optical aberrations limit the depth at which useful imaging can be done. Adaptive optics (AOs) is a method capable of measuring and correcting aberrations in different kinds of fluorescence microscopes, thereby improving the performance of the optical system. We have incorporated a wavefront sensor adaptive optics scheme to SPIM (WAOSPIM) to correct aberrations induced by optically-thick samples, such as multi-cellular tumor spheroids (MCTS). Two-photon fluorescence provides us with a tool to produce a weak non-linear guide star (NGS) in any region of the field of view. The faintness of NGS; however, led us to develop a high-sensitivity Shack-Hartmann wavefront sensor (SHWS). This paper describes this newly developed SHWS and shows the correction capabilities of WAOSPIM using NGS in thick, inhomogeneous samples like MCTS. We report improvements of up to 79% for spatial frequencies corresponding to cellular and subcellular size features.

Publisher Correction: Membrane association of the bacterial riboregulator Hfq and functional perspectives.

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Membrane association of the bacterial riboregulator Hfq and functional perspectives.

Hfq is a bacterial RNA binding protein that carries out several roles in genetic expression regulation, mainly at the post-transcriptional level. Previous studies have shown its importance in growth and virulence of bacteria. Here, we provide the direct observation of its ability to interact with membranes. This was established by co-sedimentation assay, cryo-transmission electron (cryo-TEM) and atomic force (AFM) microscopies. Furthermore, our results suggest a role for its C-terminus amyloidogenic domain in membrane disruption. Precisely, AFM images of lipid bilayers in contact with Hfq C-terminus fibrils show the emergence of holes with a size dependent on the time of interaction. Cryo-TEM observations also show that liposomes are in contact with clusters of fibrils, with occasional deformation of the vesicles and afterward the apparition of a multitude of tiny vesicles in the proximity of the fibrils, suggesting peptide-induced breakage of the liposomes. Finally, circular dichroism spectroscopy demonstrated a change in the secondary structure of Hfq C-terminus upon interaction with liposomes. Altogether, these results show an unexpected property of Hfq and suggest a possible new role for the protein, exporting sRNA outside of the bacterial cell.