Nile red (also known as Nile blue oxazone) is a lipophilic stain. Nile red stains intracellular lipid droplets yellow. In most polar solvents, Nile red will not fluoresce; however, when in a lipid-rich environment, it can be intensely fluorescent, with varying colors from deep red (for polar membrane lipid) to strong yellow-gold emission (for neutral lipid in intracellular storages). The dye is highly solvatochromic and its emission and excitation wavelength both shift depending on solvent polarity and in polar media will hardly fluoresce at all. Nile red has applications in cell biology, where it can be used as a membrane dye which can be readily visualized using an epifluorescence microscope with excitation and emission wavelengths usually shared with red fluorescent protein. Nile red has also been used as part of a sensitive detection process for microplastics in bottled water. Additionally, nile red is a remarkable candidator in fabricating membrane for different sensors to detect environmental changes, such as taste, gas, pH, etc. In triglycerides (a neutral lipid), Nile red has an excitation maximum of about 515 nm (green), and emission maximum of about 585 nm (yellow-orange). In contrast, in phospholipids (polar lipids), Nile red has an excitation maximum of about 554 nm (green), and an emission maximum of about 638 nm (red). Synthesis Nile red can be prepared through acid hydrolysis by boiling a solution of Nile blue with sulfuric acid. This process replaces an iminium group with a carbonyl group. Alternatively, Nile red and its analogs (naphthooxazine dyes) can be prepared by acid-catalyzed condensation of corresponding 5-(dialkylamino)-2-nitrosophenols with 2-naphthol. The yields are generally moderate as no co-oxidant is used in this procedure. Since the reaction to generate Nile red does not usually completely exhaust the supply of Nile blue, additional separation steps are required if pure Nile red is needed. References Oxazine dyes Vital stains Diethylamino compounds