Fig. 1.

Distinction between melanosomes, lipofuscin and melanolipofuscin granules in microscopy. a-d) Ultrastructural and elemental identification of pigment granules in an 18-month-old Abca4^−/− mouse RPE cell. a) Routine transmission electron microscopic image of the RPE showing melanosomes, lipofuscin and melanolipofuscin granules. b) Scanning transmission electron microscopic image of an unstained section of the same eye. c) Superimposition of the 4 × 4 binned EDX maps for nitrogen (N), sulphur (S) (both a marker for melanin) and phosphorus (P) (a marker for lipofuscin) maps of the same area as shown in (b). The box marks a region of interest that crosses several melanosomes and lipofuscin granules. d) Intensity profile of the region of interest shown in (c) highlighting the differing elemental composition of the respective pigment granules. e) Light and fluorescence microscopical identification of RPE pigment granules in a 68-year-old human donor eye. Staining with Toluidine blue yields dark blue to black melanosomes and blue lipofuscin granules. Without Toluidine blue staining, only melanosomes are readily visible in bright field images, however, lipofuscin can be identified in the same section by its characteristic SW-AF. Since SW-AF is confined to the lipofuscin moiety of melanolipofuscin, also regularly shaped melanolipofuscin granules can be identified (arrows). baslab: basal labyrinth; BF: bright field; BM: Bruch's membrane; cp: cytoplasm; ec: extra cellular; M: melanosome; ML: melanolipofuscin; L: lipofuscin; OS: outer segment; RPE: retinal pigment epithelium; STEM: scanning transmission electron microscopy; TEM: transmission electron microscope; SW: short wavelength.