Clinical FAF, excited at 488 nm, is thought to originate from lipofuscin and melanolipofuscin in RPE cells.
15–17 In our investigation, as in prior studies,
10 we found histologic autofluorescence also from Bruch's membrane and sub-RPE deposits, such as basal laminar deposits, as well as basal linear deposits and drusen; however, the RPE autofluorescence was by far the strongest. Furthermore, RPE autofluorescence clearly showed the granular structure, known from previous investigations with two-photon
18,19 as well as super-resolution microscopy,
20 and autofluorescence spectrum similarly reported for lipofuscin and melanolipofuscin.
11,21 We found the peak of the RPE spectrum at 610 nm, as was reported by Delori et al.
15 for human post mortem RPE excited at 510 nm. In vivo, these authors found peak emissions at 631 and 621 nm upon 510 and 470 nm excitation, respectively. Thus, slight differences in the emission peak might be due to the excitation conditions (wavelength and 960 nm two-photon excitation vs. one-photon excitation). A dependence of the peak emission on the excitation wavelength was also found by Marmorstein et al.
10 on histology. These authors measured slightly shorter emission peaks for 488 nm excitation (approximately corresponding with our 960 nm two-photon excitation), which might be due to the fact that they used macular samples exclusively. Also, Delori et al.
15 reported shorter emission peaks for the macula than measured at an eccentricity of 7° upon 470 nm excitation in vivo. In contrast, Ben Ami et al.
21 did not find differences in the spectral signature of foveal, perifovea, and near-periphery histologic RPE. For areas of drusen in vivo, Arend et al.
21 found a hypsochromic spectral shift. They revealed a spectrum peaking at 560 nm, which they attributed to drusen autofluorescence. We measured very similar autofluorescence with a peak at 570 nm. Tong et al.
21 reported a drusen-specific spectrum with a peak most abundantly found at 520 nm (excitation 480 nm). This finding is shorter than what we measured. However, we used histologic cross-sections, whereas Tong et al.
21 applied en face imaging, which could yield in autofluorescence contribution also of Bruch's membrane, which is known to have short-wavelength autofluorescence.