Figure 3
Perturbation of the crypt base pattern was followed by coordinated motion which restored the crypt base pattern and removed the debris from ablation. (a) Time-lapse, two-photon microscopy images of ablation of a single Paneth cell showing two imaging planes. White arrow indicates the position of the ablation laser focus. In the upper layer, the white dashed ellipse indicates the circumference of crypt lumen at baseline. Scale bar: 30 µm. (b) Time-lapse images of ablation of multiple stem cells by focusing laser in three positions indicated by white arrows. White dotted ellipse shows the inner circumference at the baseline. Scale bar: 30 µm. (c) Schematic showing the strategy for describing the alternating pattern using network graphs. Each Paneth cell is a node (black). Edges were defined when nearest Paneth cells were separated by Lgr5+ ISCs. (d) Quantification of change in alternating ISC and Paneth cell pattern at crypt base using rearrangement score based on adjacency matrices calculated from networks defined in (c). **p = 0.0051, unpaired t-test. Orange indicates ablation of a single stem cell, pink is of a single Paneth cell, red is of multiple stem cells and purple is of multiple Paneth cells. (e) Mean and standard deviation of fluorescent intensity from regions manually chosen in Lgr5+ ISCs and Paneth cells over time. (f) Measurement of inner circumference over time at different depths along the crypt lumen. Each value was normalized to the baseline. Mean and SD are plotted with dots representing individual crypts.
