Biology subjects

Grieve, K.

Publications and source records attributed to Grieve, K..

4 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↗

bioRxiv2023

Foveal drusen increase fixation instability: fixational eye movements as biomarkers for pre-symptomatic AMD

PurposeThis study aims at linking subtle changes of fixational eye movements (FEM) in controls and in patients with foveal drusen using adaptive optics retinal imaging in order to find anatomo-functional markers for pre-symptomatic Age-related Macular Degeneration (AMD). MethodsWe recruited 7 young controls, 4 older controls and 16 presymptomatic AMD patients with foveal drusen from the Silversight Cohort. A high speed research-grade adaptive optics flood illumination ophthalmoscope (AO-FIO) was used for monocular retinal tracking of fixational eye movements. The system allows for sub-arcminute resolution, high-speed and distortion-free imaging of the foveal area. Foveal drusen position and size were documented using gaze-dependent imaging on a clinical-grade AO-FIO. ResultsFEM were measured with high precision (RMS-S2S=0.0015{degrees} on human eyes) and small foveal drusen (median=60{micro}m) were detected with high contrast imaging. Microsaccade amplitude, drift diffusion coefficient and ISOline Area (ISOA) were significantly larger for patients with foveal drusen compared with controls. Among the drusen participants, microsaccade amplitude was correlated to drusen eccentricity from the center of the fovea. ConclusionsA novel high-speed high-precision retinal tracking technique allowed for the characterization of FEM at the microscopic level. Foveal drusen altered fixation stability, resulting in compensatory FEM changes. Particularly, drusen at the foveolar level seemed to have a stronger impact on microsaccade amplitudes and ISOA. The unexpected anatomo-functional link between small foveal drusen and fixation stability opens up a new perspective of detecting oculomotor signatures of eye diseases at the presymptomatic stage.

neuroscience↗

bioRxiv2022

Imaging the structure and dynamic activity of retinal microglia and macrophage-like cells in the living human eye

PurposeWe recently showed how a refined sequential detection pattern and image processing pipeline for multi-offset adaptive optics scanning light ophthalmoscopy (AOSLO) can increase the contrast of weakly scattering inner retinal structures, including microglia. However, sequential detection was still time-consuming, preventing dynamics from being monitored over short intervals (< 3 mins). Here we show that simultaneous fiber-bundle (FB) detection can overcomes this limitation to reveal the structure and dynamic activity of microglia and macrophage-like cells in healthy and diseased retinae. MethodsWe designed and implemented a custom 7-fiber optical FB with one central confocal fiber and six larger fibers for multi-offset detection in AOSLO at a single focal plane. We imaged the ganglion cell layer at several locations at multiple timepoints (from minutes to weeks) in 8 healthy participants and in 4 patients with ocular infections or inflammation, including ocular syphilis and posterior uveitis. Microglia and immune cells were manually segmented to quantify cell morphometry and motility. ResultsFiber-bundle detection reduced single acquisition time to 20-30 seconds, enabling imaging over larger areas and monitoring of dynamics over shorter intervals. Presumed microglia in healthy retinas had an average diameter of 12.8 m and with a spectrum of morphologies including circular cells and elongated cells with visible processes. We also detected the somas of putative macroglia, potentially astrocytes, near the optic nerve head. Microglia moved slowly in normal eyes (0.02m/sec, on average) but speed increased in patients with active infections or inflammation (up to 2.37m/sec). Microglia activity was absent in a patient with chronic uveitis that was quiescent but apparent over short intervals in an active uveitis retina. In a patient with ocular syphilis imaged at multiple timepoints during treatment, macrophage-like cells containing granular internal structures were seen. Decreases in the quantity and motility of these immune cells were correlated with improvements to vision and other structural and systemic biomarkers. ConclusionsFB-AOSLO enable simplified optical setup with easy alignment and implementation. We imaged the fine-scale structure and dynamics of microglia and macrophage-like cells during active infection and inflammation in the living eye for the first time. In healthy eyes we also detected putative glia cell near the optic nerve head. FB-AOSLO offers promise as a powerful tool for detecting and monitoring retinal inflammation and infection in the living eye over short response to treatment.

bioengineering↗

bioRxiv2022

Retinal axial motion analysis and implications for real-time correction in human retinal imaging

High-resolution ophthalmic imaging devices including spectral-domain and full-field optical coherence tomography (SDOCT and FFOCT) are adversely affected by the presence of continuous involuntary retinal axial motion. Here, we thoroughly quantify and characterize retinal axial motion with both high temporal resolution (200,000 A-scans/s) and high axial resolution (4.5 {micro}m), recorded over a typical data acquisition duration of 3 s with an SDOCT device over 14 subjects. We demonstrate that although breath-holding can help decrease large-and-slow drifts, it increases small-and-fast fluctuations, which is not ideal when motion compensation is desired. Finally, by simulating the action of an axial motion stabilization control loop, we show that a loop rate of 1.2 kHz is ideal to achieve 100% robust clinical in-vivo retinal imaging.

bioengineering↗