Fig. 3: Proof-of-concept experiment on well-known conditions of morphological modifications to validate multi-scale morphological parameters.
a, Cell roundness was compared under isotonic and hypertonic conditions. Representative actin images are shown. The cell roundness of two representative spheroids (one per condition) are plotted in the histoplot, and filtering was applied using the normalized histoplot with a red bar indicating a high cell roundness threshold. The percentages of cells exhibiting high roundness are displayed (red) on 2D and 3D segmentation. Scale bar, 30 µm. Red arrows highlight cells of high roundness in the core of the spheroid. Violin plots show the cell roundness for isotonic and hypertonic conditions. b, The relative position of nuclei to the organoid border was compared between isotonic and hypertonic conditions. Representative images with DAPI and phalloidin staining are displayed, with cyan contours representing nuclei near the border and red contours marking nuclei farther from the border. Filtering was applied to classify nuclei into two groups based on their relative position to the organoid border. Red arrows point to nuclei in the long-distance class. Scale bar, 10 µm. The histoplot presents the relative position of nuclei for two representative spheroids (one per condition), while the violin plots show the distribution of nuclei positions for five spheroids per condition. c, Chromatin compaction was evaluated by the CV of DAPI staining under isotonic and hypertonic conditions. Clickable nuclei contours, marked by red dots and white hands, link the images to the corresponding histoplot (two spheroids represented, one per condition). Filtering was applied to identify nuclei with high CV values. Violin plots show the distribution of CV values for both conditions. For all violin plots, mean values are represented by blue bars. ****P < 0.0001, using a two-tailed unpaired t-test with Welch’s correction; analysis based on ten organoids (five organoids per condition). Scale bars, 15 µm and 5 µm (zoomed images).
