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Nile red-stained, lipid droplet-filled macrophages exhibit greater fluorescence intensity than does Nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Better selectivity for cytoplasmic lipid droplets is obtained when the cells are viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm).
Nile red is strongly fluorescent, but only in the presence of a hydrophobic environment. Nile red is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution.
Spectral and physicochemical properties of the lipophilic dye Nile red induce a yellow-gold-spectral shift in its excitation-emission peak, allowing it to fluoresce in the green emission spectrum only when in a lipid-rich environment, but not in more polar environments.

When Nile red-stained Caenorhabditis elegans is viewed for green fluorescence, discrete lipid bodies can be observed throughout the intestine and other tissues either in clusters or evenly dispersed, depending on the animal's genotype or experimental treatment.