Investigations of the fine structure of RPE cells by volume electron microscopy revealed lipofuscin and melanolipofuscin granules in the apical 3/4 of cell bodies as well as mitochondria in the basal 3/4, occupying large fractions of the cell volume, whereas the apical processes are populated by melanosomes.
27 Further, using structured illumination microscopy (SIM), Bermond et al. identified nine different phenotypes of lipofuscin, melanin, and melanolipofuscin granules, showing hyper – or hypo-autofluorescence at 510 to 750 nm upon excitation at 488 nm.
28 Whereas the number of lipofuscin granules increased with age, that of melanolipofuscin did not change. Furthermore, Ach et al.
29 found differences in granule count for retinal areas defined by differences in rod:cone ratio (fovea, peri-fovea, and near periphery
30). In AMD eyes, granule aggregation was observed in parallel with loss of autofluorescent granules, resulting in regions of high signal and no signal within the same cells, viewed en face.
29,31 As these investigations on RPE flatmounts used high-resolution microscopy without spectral or lifetime resolution, only fluorescence intensity could be observed. Bindewald-Wittich et al. showed for RPE flatmounts a 36-nm hypsochromic shift of fluorescence in cells with large lipofuscin granules with two-photon imaging with spectral resolution.
32 Similarly, Han et al. demonstrated a shift of autofluorescence to shorter wavelengths of large lipofuscin granules in dysmorphic (enlarged) RPE cells on RPE flatmounts in an 80-year-old subject.
33 Although we did not investigate single granules, the similarities in spectral changes in dysmorphic cells with a high load of lipofuscin might indicate that shorter emission wavelengths are associated with pathologic cell alterations. The observed hypsochromic shift of EMW by 10 to 20 nm over the development of pathology from very nonuniform RPE to intraELM cells agrees well with a highly significant 12 nm difference of the peak emission wavelength between aged healthy subjects and patients with AMD, observed in vivo by FLIO.
34