Investigation of lipofuscin photooxidation extent using visible spectroscopy and fluorescence lifetime imaging microscopy

The study is devoted to the development of an early preclinical method for the diagnosis of age-related macular degeneration using timecorrelated fluorescence microscopy and visible spectroscopy data for the analysis of fluorescence lifetimes and spectra for lipofuscin granules. Lipofuscin granules were obtained from cells of the retinal pigment epithelium of human cadaveric eyes without signs of pathology. To research the fluorescence lifetimes of lipofuscin granules we used the fluorescence excitation method with time-correlated single photon counting technique and the fluorescence lifetimes imaging microscopy (FLIM) method in combination with a superconducting single-photon detector. Photo-oxidized granules were used as a model of age-related macular degeneration. A comparative analysis of the fluorescence lifetimes and spectra before and after photooxidation of lipofuscin granules showed significant differences in the characteristic lifetimes, as well as a shift of the maximum of the fluorescence spectrum to the short-wavelength region. Analysis of the obtained data confirms the possibility of developing a comprehensive method for the spectral and lifetime characterization of fundus autofluorescence. It is necessary to take into account the changes in the contribution of the τlifetime and the shift in the peak of the fluorescence spectrum. This approach will make it possible to determine diagnostic criteria for norm and 2 and τ3 components of the decomposition of the lifetimes curve of fluorescence decay with the longest characteristic pathology in the diagnosis of degenerative diseases of the retina and retinal pigment epithelium. Analysis of the data obtained showed that the FLIM method might become promising for the development of an early preclinical method for the diagnosis of age-related macular degeneration of the retina.

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