Different labelling properties of Nile Red and BODIPY in live animals. (A and B) Nile Red labelled intracellular structures in gut epithelial cells in wild-type (WT) animals but not in glo-4 mutants. Stained midlines were gut lumen (the same in other images if not otherwise indicated). (C and D) No autofluorescence in unstained wild-type or glo-4 animals was detected under the same Nile Red imaging condition. (E) BODIPY labelled a population of structures with high intensity (arrowheads) and labelled another population of structures with low intensity (white arrows) in gut epithelia cells in wild-type animals. (F) In glo-4 animals, only the low-intensity BODIPY structures remained in gut epithelial cells (arrows). Red arrows point to structures in hypodermal cells (E, F and J). (G and H) LRO-specific autofluorescence (arrows) was detected under the same BODIPY imaging condition in unstained wild-type animals but not in unstained glo-4 animals. (I and J) Nile Red and BODIPY labelled both enlarged lipid droplets (arrowheads) and putative small lipid droplets (white arrows) in live daf-22; glo-4 mutants. To better visualize Nile Red-stained structures, brightness of the original image (I, inset) was enhanced. All animals were late stage L4 if not other otherwise indicated. All images were 3-D projections of 9 μm confocal stacks. Gridlines, 10 μm.