Purpose: Autofluorescent (AF) material within drusen has rarely been described and there is little knowledge about origin and formation of these particles. Drusen formation is still a relatively unknown process and analysis of AF inclusions might be important for the understanding of fundamental processes. Here we present a detailed analysis of drusen containing AF material using Structured Illumination Microscopy (SIM), which provides a lateral resolution twice as high as conventional fluorescence microscopy. Methods: Eight histological RPE sections obtained from eight human donor eyes (76 ± 4 years) were examined by SIM using laser light of different wavelengths (488 nm, 568 nm). Drusen were studied regarding size and shape. AF material within drusen was analyzed in terms of size, shape, AF behavior, and distribution across drusen. Results: A total of 441 drusen was found, of which 101 contained AF material (22.9%). 90.1% of these drusen were smaller than 63 Am (mean: 35.65 Am ± 2.38 Am) regardless of whether classified as hard or soft drusen. AF particles (n= 190) within drusen show similar spectra compared to lipofuscin (LF) granules in RPE cells. Up to 11 particles were found within one single druse. Nearly all particles were located in the outer 2/3 of the drusen 85.94%). Conclusions: Structured Illumination Microscopy (SIM) allows studying AF particles within 20 drusen on a higher resolution level compared to conventional fluorescence microscopy, multi-photon or even confocal microscopy and therefore provides detailed insights in drusen. Shape and autofluorescence analysis of the material embedded in drusen suggest that these particles originate from the overlaying RPE cells.