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| Names | |
|---|---|
| IUPAC name [9-(diethylamino)benzo[a]phenoxazin-5-ylidene]azanium sulfate | |
| Other names Nile blue A, Nile blue sulfate | |
| Identifiers | |
3D model (JSmol) | |
| ChEMBL |
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| ChemSpider |
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| ECHA InfoCard | 100.020.757 |
| UNII | |
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| Properties | |
| C20H20ClN3O | |
| Molar mass | 353.845 g/mol |
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). | |
Nile blue (orNile blue A) is astain used inbiology andhistology. It may be used with live or fixed cells, and imparts a blue colour to cellnuclei.
It may also be used in conjunction with fluorescence microscopy to stain for the presence ofpolyhydroxybutyrate granules in prokaryotic or eukaryotic cells. Boiling a solution of Nile blue withsulfuric acid producesNile red (Nile blue oxazone).
Nile blue is afluorescent dye. Thefluorescence shows especially innonpolar solvents with a highquantum yield.[1]
Theabsorption andemission maxima of Nile blue are strongly dependent onpH and the solvents used:[1]
| Solvent | Absorption λ max (nm) | Emission λ max (nm) |
|---|---|---|
| Toluene | 493 | 574 |
| Acetone | 499 | 596 |
| Dimethylformamide | 504 | 598 |
| Chloroform | 624 | 647 |
| 1-Butanol | 627 | 664 |
| 2-propanol | 627 | 665 |
| Ethanol | 628 | 667 |
| Methanol | 626 | 668 |
| Water | 635 | 674 |
| 1.0 Mhydrochloric acid (pH = 1.0) | 457 | 556 |
| 0.1 Msodium hydroxide solution (pH = 11.0) | 522 | 668 |
| Ammonia water (pH = 13.0) | 524 | 668 |
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.[1]
Nile blue is used forhistological staining of biological preparations. It highlights the distinction between neutrallipids (triglycerides,cholesteryl esters,steroids) which are stained pink and acids (fatty acids,chromolipids,phospholipids) which are stained blue.[2]
The Nile blue staining, according to Kleeberg, uses the following chemicals:
The sample or frozen sections is/arefixated informaldehyde, then immersed for 20 minutes in the Nile blue solution or 30 sec in nile blue A (1% w/v in distilled water) 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 (for one to two hours). Afterwards, the stained specimen is taken on amicroscope slide and excess water is removed. The sample can be embedded inglycerol or glycerolgelatin.
Unsaturated glycerides are pink, nuclei and elastins are dark,fatty acids and various fatty substances and fat mixtures are purple blue.[3]
ThePHB granules in the cells ofPseudomonas solanacearum can be visualized by Nile blue A staining. The PHB granules in the stained smears are observed with anepifluorescence microscope underoil immersion, at a 1000 times magnification; under 450 nm excitation wavelength they show a strong orange fluorescence.[4]
Nile blue is also used in a variety of commercial DNA staining formulations used for DNAelectrophoresis.[5] As it does not require UV trans-illumination in order to be visualised in anagarose gel as withethidium 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.
Derivatives of Nile blue are potentialphotosensitizers inphotodynamic therapy ofmalignant tumors. These dyes aggregate in the tumor cells, especially in thelipid membranes, and/or are sequestered and concentrated in subcellularorganelles.[6]
With the Nile blue derivativeN-ethyl-Nile blue (EtNBA), normal and premalignant tissues in animal experiments can be distinguished byfluorescence spectroscopy in fluorescence imaging. EtNBA shows nophototoxic effects.[7]
Nile Blue and related naphthoxazinium dyes can be prepared by acid-catalyzed condensation of either 5-(dialkylamino)-2-nitrosophenols with1-naphthylamine, 3-(dialkylamino)phenols withN-alkylated 4-nitroso-1-naphtylamines, orN,N-dialkyl-1,4-phenylenediamines with 4-(dialkylamino)-1,2-naphthoquinones. Alternatively, the product of an acid-catalyzed condensation of 4-nitroso-N,N-dialkylaniline with2-naphthol (a salt of 9-(dialkylamino)benzo[a]phenoxazin-7-ium) can be oxidized in the presence of an amine, installing a second amino substituent in 5-position of the benzo[a]phenoxazinium system.[8] The following scheme illustrates the first of these four approaches, leading to Nile Blue perchlorate:
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