Nile blue is a fluorescent dye. The fluorescence shows especially in nonpolar solvents with a high quantum yield. The absorption and emission maxima of Nile blue are strongly dependent on pH and the solvents used: The duration of Nile blue fluorescence in ethanol was measured as 1.42 ns. This is shorter than the corresponding value of Nile red with 3.65 ns. The fluorescence duration is independent on dilution in the range 10−3 to 10−8 mol/L.
Nile blue staining
Nile blue is used for histological staining of biological preparations. It highlights the distinction between neutral lipids which are stained pink and acids which are stained blue. The Nile blue staining, according to Kleeberg, uses the following chemicals:
The sample or frozen sections is/are fixated in formaldehyde, then immersed for 20 minutes in the Nile blue solution or 30 sec in nile blue A and rinsed with water. For better differentiation, it is dipped in 1% acetic acid for 10–20 minutes or 30 sec until the colors are pure. This might take only 1–2 minutes. Then the sample is thoroughly rinsed in water. Afterwards, the stained specimen is taken on a microscope slide and excess water is removed. The sample can be embedded in glycerol or glycerol gelatin.
Results
Unsaturated glycerides are pink, nuclei and elastins are dark, fatty acids and various fatty substances and fat mixtures are purple blue.
Example: Detection of poly-β-hydroxybutyrate granules (PHB)
The PHB granules in the cells of Pseudomonas solanacearum can be visualized by Nile blue A staining. The PHB granules in the stained smears are observed with an epifluorescence microscope under oil immersion, at a 1000 times magnification; under 450 nm excitation wavelength they show a strong orange fluorescence.
Nile blue in DNA Electrophoresis
Nile blue is also apparently used in a variety of commercial DNA staining formulations used for DNA electrophoresis. As it does not require UV trans-illumination in order to be visualised in an agarose gel as with ethidium bromide, it can be used to observe DNA as it is separated and also as a dye to aid in gel-extraction of DNA fragments without incurring damage by UV-irradiation.
Nile Blue and related naphthoxazinium dyes can be prepared by acid-catalyzed condensation of either 5--2-nitrosophenols with 1-naphthylamine, 3-phenols with N-alkylated 4-nitroso-1-naphtylamines, or N,N-dialkyl-1,4-phenylenediamines with 4--1,2-naphthoquinones. Alternatively, the product of an acid-catalyzed condensation of 4-nitroso-N,N-dialkylaniline with 2-naphthol can be oxidized in the presence of an amine, installing a second amino substituent in 5-position of the benzophenoxazinium system. The following scheme illustrates the first of these four approaches, leading to Nile Blue perchlorate:
