Figure 3.

Label-free optical spatial analysis characterized cellular organization in EVPOME constructs and reliably distinguished control from stressed constructs. (a) En-face, optically sectioned NAD(P)H images revealed cellular organization of the EVPOME constructs. In control constructs, differentiating cells (top) were characterized by large, loosely packed cells. Alternatively, basal layer cells (bottom) were characterized by small, closely packed cells. In thermally-stressed constructs, cell morphologies appeared disorganized in both layers. In metabolically-stressed constructs, EVPOMEs grew thin cellular layers (no distinct differentiating and basal layers). Therefore, only the basal layer image was acquired, which appeared disorganized. Scale bar: 20 μm. (b) Cellular organization was quantified by spatial analysis of optical images to extract a Hurst parameter (H). For both stressing experiments, the Hurst parameter significantly distinguished between stressed and control EVPOMEs (*n = 4 batches with 11 H value differences extracted from control and n = 3 batches with 8 from thermally-stressed, P-value = 0.004; ** n = 4 batches with 12 H values extracted from control and n = 3 batches with 10 from metabolically-stressed, P-value <0.001).