الصبغات الفلوريسينية

Fluorescent Dyes

Fluorescence

Fluorescent tag

Fluorescence resonance energy transfer

Immunofluorescence

Fluorophore

Carboxyfluorescein

IAEDANS

Rhodamine

Quenching

Dark quencher

Phycocyanin

Phycoerythrin

2. Rules

There are several rules that deal with fluorescence. The Kasha – Vavilov rule dictates that the quantum yield of luminescence is independent of the wavelength of exciting radiation.

This is not quite true and is violated severely in many simple molecules. A somewhat more reliable statement, although still with exceptions, would be that the fluorescence spectrum shows very little dependence on the wavelength of exciting radiation.

The Jablonski diagram describes most of the relaxation mechanism for excited state molecules.

3. Applications

There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of applications. Some deep-sea animals, such as the Greeneye, use fluorescence.

3.1. Lighting

The common fluorescent tube relies on fluorescence. Inside the glass tube is a partial vacuum and a small amount of mercury.

An electric discharge in the tube causes the mercury atoms to emit light. The emitted light is in the ultraviolet (UV) range and is invisible, and also harmful to living organisms, so the tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs the ultraviolet and re-emits visible light.

Fluorescent lighting is very energy efficient compared to incandescent technology, but the spectra produced may cause certain colours to appear unnatural.

Some claim they may lead to adverse health effects, though that has not been verified. And as with all light sources, over-illumination is possible.

In the mid 1990s, white light-emitting diodes (LEDs) became available, which work through a similar process.

Typically, the actual light-emitting semiconductor produces light in the blue part of the spectrum, which strikes a phosphor compound deposited on the chip; the phosphor fluoresces from the green to red part of the spectrum.

The combination of the blue light that goes through the phosphor and the light emitted by the phosphor produce a net emision of white light.

The modern mercury vapor streetlight is said to have been evolved from the fluorescent lamp.

Glow sticks oxidise phenyl oxalate ester in order to produce light.

Compact fluorescent lighting (CFL) is the same as any typical fluorescent lamp with advantages.

It is self-ballasted and used to replace incandescents in most applications.

They produce
a quarter of the heat per lumen as incandescent bulbs and last about five times as long.

These bulbs contain mercury and must be handled and disposed with care.

3.2. Analytical chemistry

Fluorescence in several wavelenghts can be detected by an array detector, to detect compounds from HPLC flow. Also, TLC plates can be visualized if the compounds or a coloring reagent is fluorescent.

Fingerprints can be visualized with fluorescent compounds such as ninhydrin.

4. Biochemistry and medicine

There is a wide range of applications for fluorescence in this field. Biological molecules can be tagged with a fluorescent chemical group (fluorophore) by a simple chemical reaction, and the fluorescence of the tag enables sensitive and quantitative detection of the molecule. Examples:

• Fluorescence microscopy of tissues, cells or subcellular structures is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labeling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image.

• Automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labeled DNA molecules are separated, the fluorescent label is excited by a UV source, and the identity of the base terminating the molecule is identified by the wavelength of the emitted light.

• DNA detection: the compound ethidium bromide, when free to change its conformation in solution, has very little fluorescence. Ethidium bromide's fluorescence is greatly enhanced when it binds to DNA, so this compound is very useful in visualising the location of DNA fragments in agarose gel electrophoresis. Ethidium bromide can be toxic - a safer alternative is the dye SYBR Green.

• The DNA microarray

• Immunology: An antibody has a fluorescent chemical group attached, and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen, and even quantified, by the fluorescence.

• FACS (fluorescent-activated cell sorting)

• Fluorescence has been used to study the structure and conformations of DNA and proteins with techniques such as Fluorescence resonance energy transfer, which measures distance at the angstrom level. This is especially important in complexes of multiple biomolecules.

• Aequorin, from the jellyfish Aequorea victoria, produces a blue glow in the presence of Ca2+ ions (by a chemical reaction). It has been used to image calcium flow in cells in real time. The success with aequorin spurred further investigation of A. victoria and led to the discovery of Green Fluorescent Protein (GFP), which has become an extremely important research tool. GFP and related proteins are used as reporters for any number of biological events including such things as sub-cellular localization. Levels of gene expression are sometimes measured by linking a gene for GFP production to another gene.

Also, many biological molecules have an intrinsic fluorescence that can sometimes be used without the need to attach a chemical tag. Sometimes this intrinsic fluorescence changes when the molecule is in a specific environment, so the distribution or binding of the molecule can be measured. Bilirubin, for instance, is highly fluorescent when bound to a specific site on serum albumin. Zinc protoporphyrin, formed in developing red blood cells instead of hemoglobin when iron is unavailable or lead is present, has a bright fluorescence and can be used to detect these problems.

As of 2006, the number of fluorescence applications is growing in the biomedical biological and related sciences. Methods of analysis in these fields are also growing, albeit with increasingly unfortunate nomenclature in the form of acronyms such as: FLIM, FLI, FLIP, CALI, FLIE, FRET, FRAP, FCS, PFRAP, smFRET, FIONA, FRIPS, SHREK, SHRIMP, TIRF. Most of these techniques rely on fluorescence microscopes. These microscopes use high intensity light sources, usually mercury or xenon lamps, LEDs, or lasers, to excite fluorescence in the samples under observation. Optical filters then separate excitation light from emitted fluorescence, to be detected by eye, or with a (CCD) camera or other light detectors (photomultiplier tubes, spectrographs, etc). Much research is underway to improve the capabilities of such microscopes, the fluorescent probes used, and the applications they are applied to. Of particular note are confocal microscopes, which use a pinhole to achieve optical sectioning – affording a quantitative, 3D view of the sample.

5. Gemology, mineralogy, geology and forensics

Gemstones, minerals, fibers and many other materials which may be encountered in forensics or with a relationship to various collectibles may have a distinctive fluorescence or may fluoresce differently under short-wave ultraviolet, long-wave ultra violet, or X-rays.

Many types of calcite and amber will fluoresce under shortwave UV. Rubies, emeralds, and the Hope Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation. Fluorescence can also be used to help recognise chirality in minerals [citation needed].

Crude oil (Petroleum) fluoresces in a range of colors, from dull brown for heavy oils and tars through to bright yellowish and bluish white for very light oils and condensates. This phenomenon is used in oil exploration drilling to identify very small amounts of oil in drill cuttings and core sample.

6. Organic liquids

Organic liquids such as mixtures of anthracene in benzene or toluene, or stilbene in the same solvents, fluoresce with ultraviolet or gamma ray irradiation. The decay times of this fluorescence is of the order of nanoseconds since the duration of the light depends on the lifetime of the excited states of the fluorescent material, in this case anthracene or stilbene.

Carboxyfluorescein

Carboxyfluorescein is a fluorescent dye with an excitation and emission of 492/517 nm, respectively. It is commonly used as a tracer agent.

The dye is membrane-impermeant and can be loaded into cells by microinjection or scrape loading. It can be incoporated into liposomes, and allow for the tracking of liposomes as they pass through the body.

In addition, Carboxyfluorescein has been used to track division of cells.[1]

Carboxyfluorescein

Common name

6-FAM

Systematic name

2',7'-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein

Chemical formula

C27H20O11

FW

376.3

CAS number

85138-49-4

IAEDANS

IAEDANS is an organic fluorophore (fluorescent molecule).

It stands for 5-({[(2-iodoacetyl)amino]ethyl}amino)-naphthalene-1-sulphonic acid.

It is widely use like a marker in fluorescence spectroscopy.

The molecular weight of 1,5-IAEDANS is 434.25 g/mol, with a peak excitation wavelength of 336 nm and a peak emission wavelength of 490 nm.

The extinction coefficient of the dye is 5700. It is soluble in dimethylformamide (DMF) above pH 6 and reacts primarily with thiols.

The emission spectrum of IAEDANS overlaps well with the absorption spectra of fluorescein, Alexa Fluor 488, Oregon Green, and BODIPYFL dyes, making it a useful donor for FRET experiments.

Rhodamine

Rhodamine is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be measured easily and inexpensively with instruments called fluorimeters. Rhodamine dyes are used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry and ELISA

Rhodamine dyes are generally toxic, and are soluble in water, methanol, and ethanol.

1. Rhodamine B

Molecular Formula: C28H31N2O3Cl

Molecular Weight: 479.02 grams per mole

CAS Number: 81-88-9

SMILES structure: [Cl-].CCN(CC)c1ccc2c(OC3=CC(C=CC3=C2c4ccccc4C(O)=O)=[N+](CC)CC)c1

Rhodamine B is used in biology as a staining fluorescent dye, sometimes in combination with auramine O, as the auramine-rhodamine stain to demonstrate acid-fast organisms, notably Mycobacterium.

Rhodamine B is tunable around 610 nm when used as a laser dye.

Rhodamine B is also called Rhodamine 610, Basic Violet 10, or C.I. 45170.

2. Rhodamine 6G

Molecular Formula: C28H31N2O3Cl

Molecular Weight: 479.02 g/mol

CAS Number: 989-38-8

SMILES structure: [Cl-].CCNc1cc2OC3=CC(=[NH+]CC)C(=CC3=C(c2cc1C)c4ccccc4C(=O)OCC)C

Rhodamine 6G is often used as a laser dye, and is pumped by the 2nd (532 nm) harmonic from a Nd:YAG laser. The dye has a remarkably high photostability, high quantum yield, low cost, and its lasing range has close proximity to its absorption maximum (approximately 530 nm). The lasing range of the dye is 555 to 585 nm with a maximum at 566 nm.

Rhodamine 6G is also called Rhodamine 590, R6G, Basic Rhodamine Yellow , or C.I. 45160.

3. Rhodamine 123

The laser dye rhodamine 123 is also used in biochemistry to inhibit mitochondrion function.

Rhodamine 123 seems to bind to the mitochondrion membranes and inhibit transport processes, especially the electron transport chain, thus slowing down inner respiration.

It is a substrate of P-glycoprotein (Pgp), which is usually overexpressed in cancer cells. Recent reports indicate that rhodamine 123 may be also a substrate of multidrug resistance-associated protein (MRP), or more specifically, MRP1.

4. Other Rhodamine Derivatives

There are many rhodamine derivatives used for imaging purposes, for example the

tetramethyl rhodamine derivatives

TRITC and TAMRA,

Texas Red and Rhodamine Red.

TRITC is the base rhodamine molecule functionalized with an isothiocyanate group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure.

This derivative is reactive towards amine groups on proteins inside cells.

A succinimidyl-ester functional group attached to the rhodamine core, creating NHS-rhodamine, forms another common amine-reactive derivative.

Other derivatives of rhodamine include newer fluorophores such as

Alexa 546,

Alexa 555

DyLight 549,

have been tailored for various chemical and biological applications where higher photostability, increased brightness, different spectral characteristics, or different attachment groups are needed.

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DYES AND COLORANTS FOR ANITFREEZE/ COOLANT/ DEICERS

a wide range of dyes used to color antifreeze and deicers.

These dyes are highly soluble in water and glycol systems so they can readily be incorporated into formulations.

Dyestuffs used to color antifreeze products must not only be soluble in the coolant formulation and impart the desired color, but they must also be stable at elevated temperatures, have a reduced amount of salts, and must be non corrosive to any of the engine parts.

It is essential to differentiate products from your competition, and the use of color is an economical method to aid in marketing your products.

Color contributes to your brand identity and helps ensure your customers know they are utilizing your products.

Keyacid Green

G-AF Liquid

Product Code: 401-025-42

CI Acid Green 25Chemical

Description Anthraquinone

ShadeBlue Shade Green

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