Moreth, T. | bioRxiv Science

bioRxiv2020

Long-term live imaging of epithelial organoids and corresponding multiscale analysis reveal high heterogeneity and identifies core regulatory principles

Organoids are morphologically heterogeneous three-dimensional cell culture systems. To understand the cell organisation principles of their morphogenesis, we imaged hundreds of pancreas and liver organoids in parallel using light sheet and bright field microscopy for up to seven days. We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation and degeneration, and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright field macroscale analyses of entire cultures. Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying core regulatory principles of organoid morphogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=182 HEIGHT=200 SRC="FIGDIR/small/199463v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1df3118org.highwire.dtl.DTLVardef@104dc7org.highwire.dtl.DTLVardef@62512forg.highwire.dtl.DTLVardef@bff752_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

cell biology↗

bioRxiv2019

Ultra-thin fluorocarbon foils optimize multiscale imaging of three-dimensional native and optically cleared specimens

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive index along an optical path. Many light sheet fluorescence microscopes operate with a large chamber that contains an aqueous immersion medium and an inner specimen holder that contains the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimize the impact of the specimen holder on the optical quality, we use multi-facetted cuvettes fabricated with vacuum-formed ultra-thin fluorocarbon (FEP) foils The ultra-thin FEP-foil cuvettes have a wall thickness of about 12 {micro}m. They are resilient to fluidic exchanges, durable, mechanically stable and yet flexible. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs, thick tissue sections and dense organoid clusters. The cuvettes outperform many other sample-mounting techniques in terms of full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding, as well as their applicability for multiple-view imaging and automated segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The ultra-thin cuvettes allow the fixation and staining of the specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

cell biology↗