Nile blue
Encyclopedia
Nile blue is a stain
used in biology
and histology
. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei.
It may also be used in conjunction with fluorescence microscopy to stain for the presence of polyhydroxybutyrate
granules in prokaryotic or eukaryotic cells. Boiling a solution of Nile blue with sulfuric acid
produces Nile red
(Nile blue oxazone).
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 10−3–10−8 mol/L.
s (triglyceride
s, cholesteryl ester
s, steroid
s) which are stained pink and acids (fatty acid
s, chromolipids, phospholipid
s) which are stained blue.
The Nile blue staining, according to Kleeberg, uses the following chemicals:
in formaldehyde
, then immersed for 20 minutes in the Nile blue solution and rinsed with water. For better differentiation, it is dipped in 1% acetic acid for 10–20 minutes 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 a microscope slide
and excess water is removed. The sample can be embedded in glycerol
or glycerol gelatin
.
s and various fatty substances and fat mixtures are purple blue.
, at a 1000 times magnification; under 450 nm excitation wavelength they show a strong orange fluorescence.
. 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.
of malignant tumors. These dyes aggregate in the tumor cells, especially in the lipid membranes, and/or are sequestered and concentrated in subcellular organelle
s.
With the Nile blue derivative N-Ethyl-Nile Blue (EtNBA), normal and premalignant tissues in animal experiments can be distinguished by fluorescence spectroscopy
in fluorescence imaging. EtNBA shows no phototoxic
effects.
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...
used in biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...
and histology
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei.
It may also be used in conjunction with fluorescence microscopy to stain for the presence of polyhydroxybutyrate
Polyhydroxybutyrate
Polyhydroxybutyrate is a polyhydroxyalkanoate , a polymer belonging to the polyesters class that was first isolated and characterized in 1925 by French microbiologist Maurice Lemoigne. PHB is produced by microorganisms apparently in response to conditions of physiological stress...
granules in prokaryotic or eukaryotic cells. Boiling a solution of Nile blue with sulfuric acid
Sulfuric acid
Sulfuric acid is a strong mineral acid with the molecular formula . Its historical name is oil of vitriol. Pure sulfuric acid is a highly corrosive, colorless, viscous liquid. The salts of sulfuric acid are called sulfates...
produces Nile red
Nile red
Nile red is a lipophilic stain. It is produced by boiling a solution of Nile blue with sulfuric acid. As can be seen from the structural formulae, this process replaces an amino group with a carbonyl group. Nile red stains intracellular lipid droplets red...
(Nile blue oxazone).
Chemical and physical properties
Nile blue is a fluorescent dye. The fluorescenceFluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
shows especially in nonpolar solvents with a high quantum yield
Quantum yield
The quantum yield of a radiation-induced process is the number of times that a defined event occurs per photon absorbed by the system. The "event" may represent a chemical reaction, for example the decomposition of a reactant molecule:...
.
The absorption
Absorption (electromagnetic radiation)
In physics, absorption of electromagnetic radiation is the way by which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed to other forms of energy for example, to heat. The absorption of light during wave propagation is...
and emission maxima of Nile blue are strongly dependent on pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
and the solvents used:
| Solvent | Absorption λ max (nm) | Emission λ max (nm) |
|---|---|---|
| Toluene Toluene Toluene, formerly known as toluol, is a clear, water-insoluble liquid with the typical smell of paint thinners. It is a mono-substituted benzene derivative, i.e., one in which a single hydrogen atom from the benzene molecule has been replaced by a univalent group, in this case CH3.It is an aromatic... |
493 | 574 |
| Acetone Acetone Acetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory... |
499 | 596 |
| Dimethylformamide Dimethylformamide Dimethylformamide is an organic compound with the formula 2NCH. Commonly abbreviated as DMF , this colourless liquid is miscible with water and the majority of organic liquids. DMF is a common solvent for chemical reactions... |
504 | 598 |
| Chloroform Chloroform Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous... |
624 | 647 |
| 1-Butanol | 627 | 664 |
| 2-propanol | 627 | 665 |
| Ethanol Ethanol Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a... |
628 | 667 |
| Methanol Methanol Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol... |
626 | 668 |
| Water Water Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a... |
635 | 674 |
| 1.0 N hydrochloric acid Hydrochloric acid Hydrochloric acid is a solution of hydrogen chloride in water, that is a highly corrosive, strong mineral acid with many industrial uses. It is found naturally in gastric acid.... (pH = 1.0) |
457 | 556 |
| 0.1 N sodium 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 10−3–10−8 mol/L.
Nile blue staining
Nile blue is used for histological staining of biological preparations. It highlights the distinction between neutral lipidLipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
s (triglyceride
Triglyceride
A triglyceride is an ester derived from glycerol and three fatty acids. There are many triglycerides, depending on the oil source, some are highly unsaturated, some less so....
s, cholesteryl ester
Cholesteryl ester
A cholesteryl ester is, as its name would imply, an ester of cholesterol. The ester bond is formed between the carboxylate group of a fatty acid and the hydroxyl group of cholesterol. Cholesteryl Esters have a lower solubility in water than Cholesterol and, in other words, are more hydrophobic...
s, steroid
Steroid
A steroid is a type of organic compound that contains a characteristic arrangement of four cycloalkane rings that are joined to each other. Examples of steroids include the dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone.The core...
s) which are stained pink and acids (fatty acid
Fatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
s, chromolipids, phospholipid
Phospholipid
Phospholipids are a class of lipids that are a major component of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride, a phosphate group, and a simple organic molecule such as choline; one exception to this rule is sphingomyelin, which is derived from...
s) which are stained blue.
The Nile blue staining, according to Kleeberg, uses the following chemicals:
- Nile Blue A
- 1% acetic acidAcetic acidAcetic acid is an organic compound with the chemical formula CH3CO2H . It is a colourless liquid that when undiluted is also called glacial acetic acid. Acetic acid is the main component of vinegar , and has a distinctive sour taste and pungent smell...
- GlycerolGlycerolGlycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
or glycerol gelatinGelatinGelatin is a translucent, colorless, brittle , flavorless solid substance, derived from the collagen inside animals' skin and bones. It is commonly used as a gelling agent in food, pharmaceuticals, photography, and cosmetic manufacturing. Substances containing gelatin or functioning in a similar...
Workflow
The sample or frozen sections is/are fixatedFixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...
in formaldehyde
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
, then immersed for 20 minutes in the Nile blue solution and rinsed with water. For better differentiation, it is dipped in 1% acetic acid for 10–20 minutes 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 a microscope slide
Microscope slide
A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured on the slide, and then both are inserted together in the microscope for viewing...
and excess water is removed. The sample can be embedded in glycerol
Glycerol
Glycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
or glycerol gelatin
Gelatin
Gelatin is a translucent, colorless, brittle , flavorless solid substance, derived from the collagen inside animals' skin and bones. It is commonly used as a gelling agent in food, pharmaceuticals, photography, and cosmetic manufacturing. Substances containing gelatin or functioning in a similar...
.
Results
Unsaturated glycerides are pink, nuclei and elastins(?) dark, fatty acidFatty acid
In chemistry, especially biochemistry, a fatty acid is a carboxylic acid with a long unbranched aliphatic tail , which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from...
s 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 immersionOil immersion
In light microscopy, oil immersion is a technique used to increase the resolution of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.Immersion oils are...
, 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 electrophoresisElectrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
. As it does not require UV trans-illumination in order to be visualised in an agarose gel as with ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
, 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 in oncology
Derivatives of Nile blue are potential photosensitizers in photodynamic therapyPhotodynamic therapy
Photodynamic therapy is used clinically to treat a wide range of medical conditions, including malignant cancers, and is recognised as a treatment strategy which is both minimally invasive and minimally toxic...
of malignant tumors. These dyes aggregate in the tumor cells, especially in the lipid membranes, and/or are sequestered and concentrated in subcellular organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....
s.
With the Nile blue derivative N-Ethyl-Nile Blue (EtNBA), normal and premalignant tissues in animal experiments can be distinguished by fluorescence spectroscopy
Fluorescence spectroscopy
Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit...
in fluorescence imaging. EtNBA shows no phototoxic
Phototoxic
Phototoxicity is a chemically induced skin irritation requiring light . The skin response resembles an exaggerated sunburn. The involved chemical may enter into the skin by topical administration or it may reach the skin via systemic circulation following ingestion or parenteral administration...
effects.