Fig 2. Photo-bleaching experiments using acridine orange.

(a) Single-photon and two-photon excited widefield images of lens tissue fibres stained with 120 μM acridine orange in PBS, taken at image acquisition rates of 1 Hz, 10 Hz and 100 Hz with continuous irradiation for 600 seconds. The normalised fluorescence intensities, averaged over 30 ROIs from 10 recordings made using 5 specimens for each image acquisition rate are plotted over time in (b) for single-photon excitation and in (c) for two-photon excitation. Photo-bleaching rates were similar for single-photon and two-photon excitation at image acquisition rates of 1 Hz and 10 Hz, but with an image acquisition rate of 100 Hz, photo-bleaching is markedly reduced with widefield two-photon excitation, with the normalised average fluorescence intensity having levelled off at 75% the initial value, while with single-photon excitation the fluorescence was at 45% after 600 seconds, and still decreasing. Scale bar = 15 μm.