Optimization and characterization of calcein AM fluorescence for in-vivo imaging.

<p>(A) Calcein AM fluorescence shows a linear relationship with the incubation dose in cultured human corneal endothelial cells. (B) Peak fluorescence is seen at 2 hours after incubation. (C) Fluorscence dimishes rapidly over the first 24hrs in cells returned to culture. (D) Fluorescence dimishes rapidly over the first 24 hours (mean fluorescence drops from 109 to 36) in whole corneas stained with calcein AM and then returned to organ culture at 37°C, making descrimination between viable and non-viable areas not possible. For tissue stored in Optisol at 4°C, fluorescence contrast remains high at 7 days post incubation, with little change in fluorescence intensity (99 vs 89).