Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy

The emission spectra of single lipofuscin granules are examined using spect rally resolved confocal microscopy and near-field scanning optical microsco py (NSOM). The emission spectrum varies among the granules examined reveali ng that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spe ctra of human retinal pigment epithelium cells. NSOM measurements reveal th at the shape of the spectrum does not vary with position within the emissiv e regions of single lipofuscin granules. These results suggest that the rel ative distribution of fluorophores within the emissive regions of an indivi dual granule is homogeneous on the spatial scale similar to 150 nm.