Biology subjects

Thouvenin, O.

Publications and source records attributed to Thouvenin, O..

7 recordsLinked to original sources

bioRxiv2026

Validation of optical coherence tomography as a tool to identify differentiation key drivers in 3D in vitro conjunctival models

Conjunctival in vitro models present a valuable system to investigate conjunctival tissue homeostasis and pathologies. Combinations of collagen and fibroblasts as a stroma equivalent and the supplementation with serum have been reported to promote the differentiation of epithelial cells. However, how the individual factors affect differentiation of ocular surface cells is insufficiently understood. In this study, we analyzed the effect of serum concentration, a collagen matrix, and fibroblasts on conjunctival differentiation in a 3D in vitro model. For this purpose, we developed a computational analysis pipeline for the quantification of optical coherence tomography (OCT) data sets, allowing a time resolved, non-invasive assessment of conjunctiva epithelium differentiation, including goblet cell density. High-resolution dynamic full-field OCT (D-FFOCT) was employed to verify the identity of goblet cells. Conjunctival markers were further analyzed via histology, real-time quantitative PCR, and ELISA to confirm the data of the OCT analysis pipeline. We found that serum is required to induce epithelial differentiation while higher concentrations of 5 - 10% impaired epithelial development. The culture on a collagen matrix increased conjunctival markers upon stimulation with serum, while the co-culture with fibroblasts increased epithelial stratification. Increased serum concentration resulted in the increased occurrence of goblet cells of up to 20 cells/mm{superscript 2}. Altogether, the complementary analyses confirmed the quantified OCT data. Summarized, we identified the combination of serum (3%), collagen, and fibroblasts as a condition resulting in the highest physiological resemblance. Altogether, our study emphasizes the need for fine-tuning of culture conditions for 3D in vitro models. SIGNIFICANCE STATEMENTPhysiologically relevant in vitro conjunctiva models are essential for studying ocular surface homeostasis and disease. Our study refines 3D conjunctival culture conditions to more closely resemble native tissue by systematically identifying serum concentration, collagen scaffolds and fibroblasts as key drivers of epithelial differentiation. By applying non-invasive optical coherence tomography analysis, we enable longitudinal assessment of tissue maturation, addressing the need for non-destructive, repeatable tissue analysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/698626v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@13efafaorg.highwire.dtl.DTLVardef@1cf4529org.highwire.dtl.DTLVardef@c5c63forg.highwire.dtl.DTLVardef@5a85c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Adaptive Optics Rolling Slit Ophthalmoscope: Combining cellular-resolution, high-speed and large field-of-view in a multimodal retinal imager

Label-free optical imaging systems capable of monitoring dynamic biological processes over a large field-of-view (FOV) are essential for advancing our understanding of retinal and neurovascular health. However, existing imaging modalities often involve trade-offs between spatial resolution, contrast, FOV, and frame rate. In this study, we present the Adaptive Optics Rolling Slit Ophthalmoscope (AO-RSO), a novel imaging system that integrates the high-speed, wide-FOV capabilities of camera-based systems with the enhanced contrast and multimodal functionality of point-scanning techniques--without compromising resolution or speed. The AO-RSO utilizes line illumination synchronized with the rolling shutter of a high-speed sCMOS camera, enabling precise and dynamic spatial selection of detected photons: back-scattered photons for near-confocal bright-field imaging and forward-scattered photons for phase-contrast imaging of translucent retinal features. This system successfully visualizes diverse retinal structures, including cone and rod photoreceptors, nerve fiber bundles, red blood cells, vessel walls, and ganglion cells, across a wide retinal area (4.5{degrees}x 2.5{degrees}). With a large FOV and frame rates up to 200 Hz, the AO-RSO enables the quantification of blood flow, tracking of red blood cells within hundreds of capillaries, and evaluation of thousands of photoreceptors in the living human retina. This capability opens new opportunities for functional neuronal imaging, neurovascular coupling studies, and the early detection of retinal and neurodegenerative diseases.

bioengineering↗

bioRxiv2024

Measuring flicker induced vasodilation at a high spatial and temporal resolution in the human retina.

Neurovascular coupling (NVC) is a crucial process in which blood flow is dynamically adjusted to meet the metabolic demands of active neurons. In this study, we introduce an innovative method for in-vivo imaging of retinal blood vessel dilation in response to visible light stimulation, offering insights into the functional regulation of retinal blood flow. Using high-resolution, high-speed phase contrast imaging, we continuously monitored retinal vessel dynamics in eight healthy subjects, capturing precise temporal and spatial changes in vessel size under both stimulated and baseline conditions. We have measured a significant vessel dilation of 5.2% {+/-} 1.6% (4.4 {micro}m {+/-} 1.3 {micro}m) during flicker-stimulation compared to a dilation of 2.5% {+/-} 1% (2.1 {micro}m {+/-} 0.9 {micro}m) without stimulation. The flexibility of our method also allowed for the exploration of various acquisition and stimulation settings, broadening possibilities for investigating neurovascular coupling in the retina. This work not only enhances our understanding of neurovascular coupling but also has the potential to identify new biomarkers for vision-impairing conditions and neurodegenerative diseases. SignificanceNeurovascular coupling is a fundamental mechanism of the brain which locally regulates blood flow in response to changes in neuronal activity. Here, we present a new imaging technique that enables measuring vasodilation in response to visible light stimulation of the neurons at high spatial and temporal resolution in the living human retina. Owing to the unprecedented measurement precision of the presented technique, we provide new insights into the inner working of functional blood flow regulation in the retina. Clinically, this method holds promise for vascular dysfunction early detection, which can improve diagnosis of retinal and neurodegenerative diseases, as well as assist the development of new targeted therapies.

neuroscience↗

bioRxiv2022

Label-free virus-antibody interaction monitoring in real-time by common-path interferometry

Viruses can affect all life forms, raising concerns about virus detection and quantification of small nanoparticles. In this paper we use a label-free, full-field, incoherently illuminated common path interferometric method to detect, track, and quantify biotic nanoparticles. The detection consists of amplifying the light scattered by single nanoparticles in the sample solution. Then, the use of single-particle tracking analysis is used to monitor the change in particle diffusive mobility. With this approach, the recognition signature of T5 phages with purified antibodies targeting the major capsid protein is detected in a few minutes. We also tracked the interaction between SPP1 phages and physiological non-purified serum-containing multiples antibodies molecules. The first interactions occur after around one minute, and the recognition signature is detectable after minutes. In addition, we have been able to differentiate two populations of similar size of empty and full (encapsulating DNA) capsids of T5 in a heterogeneous solution demonstrating the robustness of this label-free detection approach. Furthermore, by combining the diffusion coefficient to the number of tracked particles, we were able to estimate the affinity of the virus-antibodies reaction.

biophysics↗

bioRxiv2019

Sensory neurons contacting the cerebrospinal fluid require the Reissner fiber to detect spinal curvature in vivo

Recent evidence indicate active roles for the cerebrospinal fluid (CSF) on body axis development and morphogenesis of the spine implying CSF-contacting neurons (CSF-cNs) in the spinal cord. CSF-cNs project a ciliated apical extension into the central canal that is enriched in the channel PKD2L1 and enables the detection of spinal curvature in a directional manner. Dorsolateral CSF-cNs ipsilaterally respond to lateral bending while ventral CSF-cNs respond to longitudinal bending. Historically, the implication of the Reissner fiber (RF), a long extracellular thread in the CSF, to CSF-cN sensory functions has remained a subject of debate. Here, we reveal using electron microscopy in zebrafish larvae that the RF is in close vicinity with cilia and microvilli of ventral and dorsolateral CSF-cNs. We investigate in vivo the role of cilia and the Reissner fiber in the mechanosensory functions of CSF-cNs by combining calcium imaging with patch-clamp recordings. We show that disruption of cilia motility affects CSF-cN sensory responses to passive and active curvature of the spinal cord without affecting the Pkd2l1 channel activity. Since ciliary defects alter the formation of the Reissner fiber, we investigated whether the Reissner fiber contributes to CSF-cN mechanosensitivity in vivo. Using a hypomorphic mutation in the scospondin gene that forbids the aggregation of SCO-spondin into a fiber, we demonstrate in vivo that the Reissner fiber per se is critical for CSF-cN mechanosensory function. Our study uncovers that neurons contacting the cerebrospinal fluid functionally interact with the Reissner fiber to detect spinal curvature in the vertebrate spinal cord. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/861344v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@3c7baaorg.highwire.dtl.DTLVardef@db094forg.highwire.dtl.DTLVardef@4041c6org.highwire.dtl.DTLVardef@9e3dae_HPS_FORMAT_FIGEXP M_FIG C_FIG eToCThe role of the Reissner fiber, a long extracellular thread running in the cerebrospinal fluid (CSF), has been since its discovery in 1860 a subject of debate. Orts-DelImmagine et al. report that the Reissner fiber plays a critical role in the detection of spinal curvature by sensory neurons contacting the CSF. HighlightsO_LISince its discovery, the role of the Reissner fiber has long been a subject of debate C_LIO_LIMechanoreception in CSF-contacting neurons (CSF-cNs) in vivo requires the Reissner fiber C_LIO_LICSF-cN apical extension is in close vicinity of the Reissner fiber C_LIO_LICSF-cNs and the Reissner fiber form in vivo a sensory organ detecting spinal curvature C_LI

neuroscience↗

bioRxiv2019

Origin of bidirectionality of cerebrospinal fluid flow and impact on long-range transport between brain and spinal cord

The circulation of cerebrospinal fluid (CSF) plays pivotal roles for body axis formation and brain development. During embryogenesis, CSF is rich in particles and proteins and flows bidirectionally in the central canal. The origins of bidirectional flow and its impact on development are unknown. Experiments combined with modeling and simulations demonstrate that the bidirectionality of CSF flow is generated locally by caudally-polarized motile cilia confined to the ventral wall of the central canal. Such active bidirectional flow of the CSF accelerates the long-range transport of particles propagating rostrally and caudally. In addition, spontaneous muscle contractions increase local CSF flow and consequently enhance long-range transport of extracellular lipidic particles. Focal ablation of the channel connecting brain ventricles to the central canal reduces embryo length, indicating that long-range transport contributes to embryonic growth. Our study also demonstrates that at this early stage, motile cilia ensure the proper formation of the central canal.

biophysics↗

bioRxiv2018

Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature

Flow of cerebrospinal fluid (CSF) may contribute to spine morphogenesis, as mutations affecting both cilia motility and CSF flow lead to scoliosis1. However, the mechanisms underlying detection of the CSF flow in the central canal of the spinal cord remain elusive. Here we used full-field optical coherence tomography (FF-OCT) and bead tracking to demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the zebrafish mutant cfap298tm304, previously known as kurly, reduction of cilia motility slows transport down the length of central canal. To investigate downstream mechanisms that could transduce CSF flow, we performed calcium imaging in sensory neurons contacting the CSF (CSF-cNs) and found that disruption in cilia motility impaired the activity of CSF-cNs. CSF-cNs across species express the transient receptor potential channel PKD2L1, also known as TRPP3, which contributes to CSF-cN chemosensory properties. Using calcium imaging and whole-cell patch clamp recordings, we found that the loss of the Pkd2l1 channel in pkd2l1 mutant embryos also abolished CSF-cN activity. Whole-cell recordings further demonstrated that opening of a single channel is sufficient to trigger action potentials in wild type CSF-cNs. Recording from isolated cells in vitro, we showed that CSF-cNs are mechanosensory cells that respond to pressure in a Pkd2l1-dependent manner. Interestingly, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. Our study indicates that CSF-cNs are mechanosensory cells whose spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to the maintenance of the natural curvature of the spine.

neuroscience↗