Biology subjects

Ricard, C.

Publications and source records attributed to Ricard, C..

3 recordsLinked to original sources

bioRxiv2026

Diagnosing Neurodegenerative Diseases by Label-Free 3-D Imaging of Intestinal Samples

Early diagnosis of Parkinsons disease (PD) remains challenging because motor symptoms appear only after extensive neurodegeneration, and a definitive diagnosis still relies on post-mortem neuropathology. Increasing evidence implicates the enteric nervous system (ENS) in prodromal disease stages, but routine ENS-based diagnosis is limited by the complexity of intestinal tissue organization and the need for specific labeling strategies. Here, we developed a label-free autofluorescence (AF) imaging workflow combined with unbiased morphometric analysis to identify neurodegenerative alterations in fixed human colonic tissue. Using a correlative multiscale imaging approach, we generated a database of almost 800 high-resolution confocal images from myenteric and submucosal plexuses of controls, PD, and Alzheimers disease (AD) patients. Blind evaluation by four expert histologists showed reliable identification of control tissue but lower sensitivity for pathological cases, reflecting the heterogeneous distribution of ENS lesions. Semi-quantitative and morphometric image analyses identified a distinct population of enlarged enteric neurons, termed large neural cells (LNCs), strongly enriched in PD and AD compared with controls. LNCs contained autofluorescent cytoplasmic inclusions and frequently prominent nucleoli, both features largely absent from control tissue independent of aging. Co-localization with the amyloid-binding probe Amytracker (AmyT) demonstrated that AF granules correspond to {beta}-sheet-rich protein aggregates rather than merely age-related lipofuscin granules. Similar alterations were detected in intact three-dimensional (3-D) colonic biopsies, demonstrating the feasibility of volumetric ENS imaging without tissue clearing. Together, our results establish label-free AF imaging as a rapid and clinically compatible strategy for detecting enteric neurodegenerative pathology. This approach provides a framework for the future development of ENS-based biomarkers and supports the use of volumetric intestinal imaging for early diagnosis of neurodegenerative diseases.

pathology↗

bioRxiv2022

Label-free, fast, 2-photon volume imaging of the structural organization of peripheral neurons and glia in the enteric ganglia

The enteric nervous system (ENS), sometimes considered as a second brain due to its large autonomy from central circuits is made of interconnected plexuses organized in a mesh-like network lining the gastrointestinal tract. Originally described as a leading actor of the regulation of digestion, bowel advance and intestinal secretion, its implication in various neuropathologies has recently been demonstrated. However, with few exceptions, its morphology and functions have been studied on thin sections of the intestinal wall or in dissected explants. Due to its intricate morphology, precious information on its three-dimensional (3-D) architecture and connectivity is often lost. In this context, we have developed a fast, label-free 3-D imaging method of the ENS, based on intrinsic signals of the tissue. We adapted a fast tissue-clearing protocol based on a high refractive-index aqueous solution, and then characterized the autofluorescence signals arising from the various cellular and sub-cellular components of the ENS. Immunofluorescence and spectral recordings complete this characterization. Finally, we demonstrate the fast acquisition of detailed 3-D image stacks of unlabeled mouse intestine, across the whole intestinal wall and including both the myenteric and submucosal enteric nervous plexuses using a new spinning-disk two-photon microscope. The combination of fast clearing (less than 15min for 73 % transparency), autofluorescence imaging and rapid volumetric imaging (less than a minute for the acquisition of s z-stack of 100 planes (150*150 {micro}m) at 300-nm spatial resolution) paves the way for new applications in fundamental and clinical research.

neuroscience↗

bioRxiv2018

Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that - in many aspects - recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that - unlike existing two- or three-lens designs - it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

biophysics↗