In-vivo and in-vitro investigations of retinal fluorophores in age - related macular degeneration by fluorescence lifetime imaging

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). However, a deeper understanding of the generation of single compounds contributing to the lipofuscin as well as of the role of other fluorophores such as FAD, glycated proteins, and collagen needs their discrimination by fluorescence lifetime imaging (FLIM). FLIM at the ocular fundus is performed using a scanning laser ophthalmoscope equipped with a picosecond laser source (448nm or 468nm respectively, 100ps, 80 MHz repetition rate) and dual wavelength (490-560nm and 560-7600nm) time-correlated single photon counting. A three - exponential fit of the fluorescence decay revealed associations of decay times to anatomical structures. Disease - related features are identified from alterations in decay times and amplitudes. The in-vivo investigations in patients were paralleled by experiments in an organ culture of the porcine ocular fundus. Photo - oxidative stress was induced by exposure to blue light (467nm, 0.41 mW/mm boolean AND 2). Subsequent analysis (fluorescence microscopy, HPLC, LC-MS) indicated the accumulation of the pyridinium bis-retinoid A2E and its oxidation products as well as oxidized phospholipids. These compounds contribute to the tissue auto - fluorescence and may play a key role in the pathogenesis of AMD. Thus, FLIM observation at the ocular fundus in vivo enhances our knowledge on the etiology of AMD and may become a diagnostic tool.