In vivo autofluorescence lifetime imaging at the fundus of the human eye

Changes in cellular metabolism are considered first signs of fundus diseases, e.g. of age-related macular degeneration. Changes in the metabolism can potentially be detected by measuring the autofluorescence of the fundus. The fundus contains a wide variety of fluorophores in different binding and quenching states. The fluorescence signals cannot be clearly discriminated by commonly used steady state imaging techniques, even when these are combined with spectral resolution and excitation wavelength multiplexing. A considerable improvement is obtained by fluorescence lifetime imaging (FLIM). FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Due to its high sensitivity and high time resolution, its capability to resolve multi-exponential decay functions, and its easy combination with fast scanning we use multi-dimensional time-correlated single photon counting for fundus imaging. By analyzing the spectral properties of the expected fluorophores in the fundus, we show that improved discrimination of fluorophores is obtained by FLIM in combination with selected excitation wavelength and emission wavelength. As demonstrated in lifetime histograms of 40° fundus images, several fluorophores are excited at 446 nm, but predominantly lipofuscin at 468 nm excitation. Simultaneous detection of lifetime images in two emission ranges 500 nm to 560 nm and 560 nm to 700 nm improves further the discrimination of fluorophores.