Autofluorescent lipofusin inclusions in muscle biopsies from LOPD. (a) LOPD fiber (pt. NBSL9a) viewed in fluorescence (top) and transmitted light (bottom) shows autofluorescent inclusions directly surrounded by myofibrils. Fluorescence was excited at 488 nm and collected from 467 to 499 nm. Transmitted light is with DIC contrast. Bar: 25 μm. (b) LOPD fiber (pt. NBSL2) with prominent inclusions. The fiber was stained with LAMP2 (lysosomes: green) and LC3 (autophagosomes: red). Some inclusions are seen within the lysosome or autolysosome (arrows) whereas others appear free in the cytoplasm (asterisks). Bar: 10 μm. (c) Autofluorescent inclusions stain positive for Oil Red. The fiber (isolated from muscle biopsy of pt. D3) was also stained with LAMP2 (green). Bar: 10 μm. (d) Sudan Black B staining demonstrates lipofuscin accumulation in a fiber from pt. D3. Bar: 10 μm.