Endogenous Fluorophores Enable Two-Photon Imaging of the Primate Eye

PURPOSE. Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes. METHODS. Two-photon microscopy (TPM) was used to visualize excised retina from monkey eyes. Retinoid composition and content in human and monkey eyes were quantified by HPLC and mass spectrometry (MS). RESULTS. Clear images of inner and outer segments of rods and cones were obtained in primate eyes at different eccentricities. Fluorescence spectra from outer segments revealed a maximum emission at 480 nm indicative of retinols and their esters. In cynomolgus monkey and human retinal extracts, retinyl esters existed predominantly in the 11-cis configuration along with notable levels of 11-cis-retinol, a characteristic of cone-enriched retinas. Average amounts of di-retinoid-pyridinium-ethanolamine (A2E) in primate and human eyes were 160 and 225 pmol/eye, respectively. CONCLUSIONS. These data show that human retina contains sufficient amounts of retinoids for two-photon excitation imaging. Greater amounts of 11-cis-retinyl esters relative to rodent retinas contribute to the fluorescence signal from both monkey and human eyes. These observations indicate that TPM imaging found effective in mice could detect early age-and disease-related changes in human retina.