Different fluorescence emission spectra of Nile Red-labelled LROs and lipid droplets. (A) Nile Red labelled structures in live wild-type animals as revealed by channel mode confocal images. (B) These labelled structures (pseudo-colored in red) displayed the same emission spectrum (Spec-1) as revealed by linearly un-mixing lambda mode confocal images. (C) Nile Red labelled structures in live dhs-28 mutants as revealed by channel mode confocal images. (D) Nile Red labelled both Spec-1 structures (pseudo-colored in red) and Spec-2 structures (pseudo-colored in green) in live dhs-28 mutants as revealed by linearly un-mixing lambda mode confocal images. Spec-2s included enlarged lipid droplets (arrowheads) and putative small lipid droplets (arrows). To aid visualization of Spec-2 structures, brightness of the original un-mixed image (inset) was enhanced. The same was applied to F and J. (E and F) Spec-1 structures were mostly marked by the LRO marker GLO-1::GFP (arrows). (G) Emission spectrum profiles of Spec-1s in wild-type and dhs-28 animals and Spec-2s in dhs-28 animals. Intensity values were normalized. (H and I) LRO-specific GLO-1::GFP structures were lost in glo-4 mutants. (J) In daf-22; glo-4 mutants, LRO-specific Spec-1s were lost while enlarged (arrowheads) and small (arrows) lipid droplets-specific Spec-2s remained. All images were single confocal slices. Scale bars, 10 μm.