Figure 4.

Confluent cultures of human RPE cells containing endogenous lipofuscin and subjected to lethal blue light irradiation. (A) Fluorescence micrograph of a representative culture imaged to show lipofuscin. Three cells containing abundant lipofuscin are indicated (white arrowheads). (B) Merged phase contrast and PI fluorescence images of the same field shown in (A) illustrated before light treatment (0 hours) and at two time points during irradiation (4 and 8 hours) to show the time-dependent increase in PI-positive nuclei. The same high lipofuscin cells indicated in (A) are highlighted (white arrowheads), which became PI-positive early in the irradiation time course (by 4 hours). Three low lipofuscin cells also are indicated (white arrows) with nuclei that remain PI-negative through 8 hours. (C) Survival curves for RPE cells grouped by lipofuscin content (% cell area occupied by thresholded autofluorescence) showing the time of onset of nuclear PI fluorescence as a real-time measure of cell death during blue light irradiation over 15 hours. Each dot represents an individual cell. All survival curves for cells with different lipofuscin content differ significantly from each other (GraphPad Prism, Comparison of Survival Curves function, P < 0.0001). Age of RPE donor: 37 years.