High-speed two-photon excited autofluorescence imaging of ex vivo human retinal pigment epithelial cells toward age-related macular degeneration diagnostic.

Age-related macular degeneration (AMD) is among the major concerns in ophthalmology, as it is the primary cause for irreversible blindness in developed countries. Nevertheless, there is poor understanding of the origins and mechanisms that trigger this important ocular disease. In common clinical pratice, AMD is monitored by autofluorescence imaging of the retinal pigment epithelial (RPE) cells through a confocal scanning laser ophthalmoscope. The RPE cells derive their dominant autofluorescence from the lipofuscin granules that accumulate in the cytoplasm with increasing age and disease. We explored a different approach to retinal RPE imaging using two-photon excited autofluorescence, offering intrinsic three-dimensional resolution, larger sensing depth and reduced photodamage compared to single-photon excited fluorescence ophthalmoscopy. A two-photon microscope, based on the architecture of a conventional scanning laser ophthalmoscope (HRT, Heidelberg Engineering, Germany), was designed for autofluorescence imaging on retina samples from postmortem human-donor eyes. We were able to visualize at video-rate speed single RPE lipofuscin granules, demonstrating the potential to develop this method toward clinical practice for patients with RPE-related retinal disease like AMD.