File:Pipeline for multiscale hyperdimensional analysis of organoids.webp

English: (a) Scheme showing the pipeline where cerebral organoids are grown from stem cells, fixed in 4% PFA, then post-fixed with SHIELD poly-epoxide crosslinker. Organoids are then delipidated and labeled with antibodies using eFLASH for rapid uniform staining. An axially sweeping light-sheet microscope was used for rapid (15 min per organoid) imaging of cleared intact organoids at 0.65 × 0.65 × 2.0 µm voxel resolution. Quantitative analysis applies automated algorithms and convolutional neural networks to measure multiscale features. We applied this pipeline for unbiased high-dimensional phenotyping of different experimental models. (b) SHIELD is compatible with many common markers for cerebral organoids. Data show antibody staining of SHIELD-cleared day 45 organoids using antibodies listed in Supplementary Table 1. (c) After delipidation and immersion in refractive index matching solution (dPROTOS), organoids are optically transparent and can be imaged with standard confocal microscopy. Grid = 1 mm. (d) SHIELD preserves endogenous fluorescence, mRNA and protein epitopes. Images show an organoid section containing GFP-labeled cells co-stained with anti-GFP antibody and a GFP mRNA FISH probe (e) A 3D render of day 35 cerebral organoid stained using Syto16 (nuclear dye, blue), β3-tubulin (neuronal marker, green) and vimentin (glial marker, red) or Syto16 (nuclear dye, blue); TBR1 (postmitotic neuron, green); SOX2 (progenitor cells, red). Images show a 3D render and a 2D view of the XZ plane to show whole-tissue labeling. Scale bars (yellow, 100 µm; white, 200 µm).