Impact of α-Synuclein Fibrillar Strains and β-Amyloid Assemblies on Mouse Cortical Neurons Endo-Lysosomal Logistics

Abstract

Visual Abstract

In-vivo fast non-linear microscopy reveals impairment of fast axonal transport induced by molecular motor imbalances in the brain of zebrafish larvae

“…Incidentally, the large variation of intensity of the vast majority of spots indicate that each endosome most probably contain a single nanoKTP, because the presence, within a vesicle, of multiple randomly oriented nanocrystals would yield a much lower intensity contrast. This observation confirms that in vivo , like in primary cultures, 22,37 neuron internalize at most one solid nanoparticle per vesicle. The nanoKTP still appear as single particles per transported vesicular compartment 24 hours after injection, despite the possibility for endolysosomes to merge.…”

“…Driving the mirror galvanometers of the microscope at their resonance frequency (8 kHz), we acquired continuous 512 × 352 pixels raster scans at a constant depth position ( ≈ 100 µ m) below the skin with a line average of 2, leading to a refreshing rate of 20 frames/s ( i.e. 50 ms per full frame scan) identical to the one we use in wide-field acquisition on cultured neurons, 18,29 that enhances the sensitivity to short pauses. In order to avoid degradation of the diffraction limited spatial resolution by sampling, we selected a pixel size of 173 nm, which fulfills the Shannon criteria as it is smaller than half the diffraction-limited spot radius of two-photon excited microscopy .…”

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae

“…Incidentally, the large variation of intensity of the vast majority of spots indicates that each endosome most probably contains a single nanoKTP, because the presence, within a vesicle, of multiple randomly oriented nanocrystals would yield a much lower intensity contrast. This observation confirms that in vivo , like in primary cultures, , neurons internalize at most one solid nanoparticle per vesicle. The nanoKTP still appear as single particles per transported vesicular compartment 24 h after injection, despite the possibility for endolysosomes to merge.…”

“…Driving the mirror galvanometers of the microscope at their resonance frequency (8 kHz), we acquired continuous 512 × 352 pixels raster scans at a constant depth position (≈100 μm) below the skin with a line average of 2, leading to a refreshing rate of 20 frames/s (i.e., 50 ms per full frame scan), identical to the one we use in wide-field acquisition on cultured neurons, , that enhances the sensitivity to short pauses. In order to avoid degradation of the diffraction-limited spatial resolution by sampling, we selected a pixel size of 173 nm, which fulfills the Shannon criteria, as it is smaller than half the diffraction-limited spot radius of two-photon-excited microscopy ρ Airy 2 ph /2.…”

3D Real-Time Two-Photon Microscopy Device for Single-Particle Holographic Tracking (3D-Red Shot)

“…We finally evaluated the ability of our setup to track single endosomes in living cells as a proof of principle of its compatibility with live biological sample imaging. We labeled endosomes of 2D-cultured mouse neuroblast (Neuro-2A) cells with NC via the spontaneous uptake by endocytosis of NC added to the culture medium, as established in previous work. , Figure a,b display two highly directional trajectories, acquired over 2 min, superimposed on bright-field microscopy images. The directional behavior proves that it is neither a diffusive motion in the medium nor on the cell membrane.…”