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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Nat Cell Biol. 2021 Apr 1;23(4):377–390. doi: 10.1038/s41556-021-00654-5

Extended Data Fig. 5. Geometric effects on raw FRET signals in 3D organoid models.

Extended Data Fig. 5

a, PDO model expressing the non-phosphorylatable (hence ERK-insensitive) control sensor EKAREV(TA) was FRET-imaged to assess non-biological geometry effects on raw signals in 3D organoid FRET microscopy. YFP/CFP ratios can differ between organoids situated either far away or close to the objective (distance difference ~150 μm). Experiment performed once, this direct comparison representing general observations.

b, Performing FRET acquisition, Turq2 and YPet emissions were determined from all cells of a bulky organoid (>200 cells) and plotted against their z-coordinates. YFP/CFP ratios increase subtly with increasing depth within the organoid, likely due to differential scattering-induced loss of fluorescence between the two fluorophores. Experiment performed twice with same outcome.