3D immuno‐confocal image reconstruction of fibroblast cytoskeleton and nucleus architecture

Abstract

Computational models of cellular structures generally rely on simplifying approximations and assumptions that limit biological accuracy. This study presents a comprehensive image processing pipeline for creating unified three-dimensional (3D) reconstructions of the cell cytoskeletal networks and nuclei. Confocal image stacks of these cellular structures were reconstructed to 3D isosurfaces (Imaris), then tessellations were simplified to reduce the number of elements in initial meshes by applying quadric edge collapse decimation with preserved topology boundaries (MeshLab). Geometries were remeshed to ensure uniformity (Instant Meshes) and the resulting 3D meshes exported (ABAQUS) for downstream application. The protocol has been applied successfully to fibroblast cytoskeletal reorganisation in the scleral connective tissue of the eye, under mechanical load that mimics internal eye pressure. While the method herein is specifically employed to reconstruct immunofluorescent confocal imaging data, it is also more widely applicable to other biological imaging modalities where accurate 3D cell structures are required.

Delayed reorganisation of F-actin cytoskeleton and reversible chromatin condensation in scleral fibroblasts under simulated pathological strain

“…Scleral fibroblasts were obtained, using an explant crawl-out approach, from 20 bovine eyes (mean age of 23.4 ± 4.4 months) within 12 h post-mortem (Maddock Kembrey Meats Lt abattoir, Maesteg, UK) as previously described [ 9 ]. Ethical approval was not required as the tissue was a by-product of the food industry.…”

“…Scleral fibroblasts were labelled for F-actin (Alexa-488™ phalloidin (1:40 dilution); Invitrogen, UK), β-tubulin microtubules (mouse anti-tubulin E7 antibody (1:500 dilution; Developmental Studies Hybridoma Bank, USA) and vimentin intermediate filaments (mouse anti-vimentin V9 antibody; 1:100 dilution) prior to mounting in Vectashield™ antifade medium containing 1.5 μg/ml DAPI (Vector Laboratories, USA) as previously described [ 9 , 16 ]. Membranes were divided into segments by cutting, sections were co-labelled for vimentin and F-actin and separately labelled for β-tubulin and representative cells taken from the same well for consistency.…”