Figure 1. Multi–photon excitation of a 6–month–old wild type (WT) mouse eye at 730 and 910 nm produced emission spectra indicating more than one fluorophore.

(a) A series of TPM images of an intact mouse eye were obtained along the axis perpendicular to the RPE layer with an excitation wavelength of 730 nm. The main box reveals the enface image of RPE cells; the two cross–section images, one shown at the bottom and the other at the right edge, were assembled from a series of z-slice images. The yellow outlined rectangle represents the region from which fluorescence was collected for spectral analysis with the excitation light focused on the RPE. Scale bar, 75 μm. (b) Fluorescence emission spectra from the RPE of an intact mouse eye through the sclera (ts) and from flat–mounted (fm) mouse RPE are super–imposable. The second harmonic signal (SH) exhibits a sharp maximum at half of the 910 nm excitation wavelength. (c) A series of TPM images of an intact mouse eye obtained with an excitation wavelength of 910 nm. In the region of the blue outlined rectangle, a strong second harmonic signal from the sclera was dominant, as the curvature of the eye brought the sclera more into focus. Scale bar, 75 μm. The yellow outlined rectangle represents the region from which fluorescence was collected for spectral analysis shown in b. (d) TPM image of flat–mounted ex vivo RPE. Part of the RPE is folded over, exposing a sagittal view of retinosomes, indicated with the yellow arrow. Scale bar, 20 μm.