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Pulsed-SBS imaging of mouse mammary gland organoids. a, Brightfield image of an organoid 48 h post seeding singularized mammary epithelial cells into basement membrane matrix. b,c, Brillouin shift image (b) and Brillouin linewidth (c) image of the organoid in a. In b and c, the pixel steps in x and y directions are both 0.5 µm and the pixel time is 20 ms. d, Brightfield image of an organoid 70 h post seeding. e,f, Brillouin shift image (e) and Brillouin linewidth (f) image of the organoid in d. The average Brillouin linewidth in the lumen in f (FWHM 488 MHz) is 200 MHz decreased compared to c (FWHM 688 MHz) (representative of n = 3 organoids). Arrows in e highlight a dividing cell in cytokinesis. Dashed lines in b–f demarcate organoid lumen. In e and f, the pixel steps in x and y directions are both 0.25 µm and the pixel time is 20 ms. g, Brightfield image of an organoid 80 h post seeding. h,i, Brillouin shift image (h) and Brillouin linewidth (i) image of the organoid in g. j,k, Brillouin shift image (j) and Brillouin linewidth (k) image of the x–z plane marked with a dashed yellow line in g and h. The average Brillouin shift of the bended rim regions (black dashed boxes in j, average 5.56 GHz) shows a ~100 MHz up-shift compared to the average shift of the upper and lower cap of the epithelium (red dashed boxes in j, average 5.46 GHz) (representative of n = 4 organoids). A piece of debris, potentially a dead, extruded cell is marked with an arrow in j. In h–k, the pixel steps in x and y directions are 0.5 µm and the pixel time 20 ms. Scale bars in all the images are 10 µm.
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