Extended Data Fig. 8: Reproducibility and absence of analysis spatial bias.
From: Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification
a:left: representative image of a neuroectoderm organoid after 8 days of differentiation in JeWells with the deep learning 3D detection of dead/mitosis cells (left, YoloV5) and of nuclei (right, Stardist). N= 3 batches of 40 organoids each. Right : at D8, the total number of cells per organoids are relatively similar for Batch 1 and 2 seeded at the same cell density (1.105 cells/ml) and reduced by half in Batch3 seeded with 0.5.105 cells/ml. The bar represents the average value, The box the std and the whiskers the min-max.Organoids (Batch1 and 2) filling the JeWells or smaller organoids (Batch3) hence display similar growth dynamics. b : left: representative single plane raw image of a HCT116 tumoroid grown in JeWells (yellow stripes: direction of the light sheet illumination). Right: Integrative z-projection of the binarized segmentation the nuclei in the organoid using deep learning 3D segmentation (Stardist). Each pixel value hence represent the value of the integrated nuclear volume over the z axis for each x,y coordinate. Right: Representative lateral profiles from 3 different organoids of nuclei volume along the x axis (yellow line in top panel). We measured a homogeneous distribution of nuclei along the light sheet direction despite the gradient in optical contrast. The homogeneous detection results from the training of Stardist with nuclei with different contrasts covering the whole gradient range in raw images. N=20 organoids c. Comparison of the same ‘B shape’ Neuroectoderm organoid imaged from the left and right side of the JeWell. Both single sided illuminations revealed the same left right asymmetry of the distribution of sox2 positive cells within the organoids. It demonstrates that a single side illumination sufficed to perform quantitative measurement of transcription factor gradients within the organoids. Multi-side (up to 4) illumination is possible with the soSPIM but was not implemented in an automatically manner. d. 3D segmentation comparison between left-side and right-side illumination images. Left: Single plane intensity overlay between left-side (cyan) and right-side (magenta) illumination. Middle: Cross-sections overlays of 3D nuclei segmentation computed from left-side (cyan) and right-side (magenta) illumination data. Objects are the maximum intensity projections of the nuclei intersecting the cross-sections in (x,y), (x,z) and (y,z) directions. Right: Quantification of the difference between the absolute number of detected nucleii in the two illumination directions (green, N = 32), and of the number of nuclei that overlaps (orange, N = 64). Box plots indicate the mean (bar) and Std. The butterfly represents the distribution over all the organoids in each condition.
