Fig. 6.

Correlative fluorescence and electron microscopy of the RPE of an 80-year-old human donor. a) A 150 nm thick section was first investigated by BF and for NIR-AF (red) and SW-AF (green) and subsequently investigated by electron microscopy. To compensate for low signal to noise ratio, 5 individual images of BF, NIR-AF and SW-AF were averaged, respectively. In the NIR-/SW-AF overlay image, lipofuscin granules appear yellow due to the presence of both NIR- and SW-AF. The EM image shown is a composite image of 20 individual images taken at x12,000 magnification. Contrast is low due to complete lack of heavy-metal staining, however, melanosomes and (melano-)lipofuscin granules are still readily identifiable (compare to SI S5 that shows a heavy-metal stained section from the same eye). Magnified EM and fluorescence pictures of the boxed area are shown in (b). The full resolution version of the EM composite image can be accessed under DOI:10.6084/m9.figshare.7951097. b) Melanosomes (arrowheads) show exclusively NIR-AF, but no SW-AF. However, NIR-AF intensity varies between high (black arrowheads) and low (white arrowheads). Lipofuscin granules (short arrows) appear ultrastructurally indistinguishable and show similar SW-AF intensities, but their NIR-AF intensities vary between high (short black arrows) and low (short white arrows). Virtually all lipofuscin granules present NIR-AF. Melanolipofuscin granules (long arrows) can be identified by their NIR-AF positive melanin core and a SW-AF and NIR-AF positive lipofuscin shell or protrusions. BF: bright field; EM: electron microscopy; N: nucleus; NIR: near-infrared; SW: short wavelength.