Names | |
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Pronunciation | /flʊəˈrɛsi.ɪn,flʊəˈrɛsiːn/ |
IUPAC name 3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one | |
Other names Fluorescein, resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, soap yellow | |
Identifiers | |
3D model (JSmol) | |
ChEBI | |
ChEMBL | |
ChemSpider | |
DrugBank | |
ECHA InfoCard | 100.017.302 |
EC Number |
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KEGG | |
MeSH | Fluorescein |
PubChem CID | |
UNII | |
CompTox Dashboard (EPA) | |
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Properties | |
C20H12O5 | |
Molar mass | 332.311 g·mol−1 |
Melting point | 314 to 316 °C (597 to 601 °F; 587 to 589 K) |
Slightly | |
Pharmacology | |
S01JA01 ( WHO ) | |
Hazards | |
GHS labelling: | |
Warning | |
H319 | |
P305, P338, P351 | |
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). |
Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications. [1]
The color of its aqueous solutions is green by reflection and orange by transmission (its spectral properties are dependent on pH of the solution), [2] as can be noticed in bubble levels, for example, in which fluorescein is added as a colorant to the alcohol filling the tube in order to increase the visibility of the air bubble contained within. More concentrated solutions of fluorescein can even appear red (because under these conditions nearly all incident emission is re-absorbed by the solution).
It is on the World Health Organization's List of Essential Medicines. [3]
Fluorescein sodium, the sodium salt of fluorescein, is used extensively as a diagnostic tool in the field of ophthalmology and optometry, where topical fluorescein is used in the diagnosis of corneal abrasions, corneal ulcers and herpetic corneal infections. It is also used in rigid gas permeable contact lens fitting to evaluate the tear layer under the lens. It is available as sterile single-use sachets containing lint-free paper applicators soaked in fluorescein sodium solution. [4]
The thyroxine ester of fluorescein is used to quantify the thyroxine concentration in blood. [1]
Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8.
Fluorescein is a precursor to the red dye eosin Y by bromination. [1]
Oral and intravenous use of fluorescein can cause adverse reactions, including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction, [5] [6] causing cardiac arrest [7] and sudden death due to anaphylactic shock. [8] [9]
Intravenous use has the most reported adverse reactions, including sudden death, but this may reflect greater use rather than greater risk. Both oral and topical uses have been reported to cause anaphylaxis, [10] [11] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop. [7] Reported rates of adverse reactions vary from 1% to 6%. [12] [13] [14] [15] The higher rates may reflect study populations that include a higher percentage of persons with prior adverse reactions. The risk of an adverse reaction is 25 times higher if the person has had a prior adverse reaction. [14] The risk can be reduced with prior (prophylactic) use of antihistamines [16] and prompt emergency management of any ensuing anaphylaxis. [17] A simple prick test may help to identify persons at greatest risk of adverse reaction. [15]
The fluorescence of this molecule is very intense; peak excitation occurs at 495 nm and peak emission at 520 nm. Values for the deprotonated form in basic solution.
Fluorescein has a pKa of 6.4, [2] and its ionization equilibrium leads to pH-dependent absorption and emission over the range of 5 to 9. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns, which allows for pH determination from nonintensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry. Upon exhaustive irradiation with visible light fluorescein decomposes to release phthalic and formic acids and carbon monoxide, effectively acting as a photoCORM. [18]
Fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm.
Many derivatives of fluorescein are known. Examples are:
In oligonucleotide synthesis, several phosphoramidite reagents containing protected fluorescein, e.g. 6-FAM phosphoramidite 2, [19] are used for the preparation of fluorescein-labeled oligonucleotides.
The extent to which fluorescein dilaurate is broken down to yield lauric acid can be detected as a measure of pancreatic esterase activity.
Approximately 250 tons/y were produced in the year 2000. The method involves the fusion of phthalic anhydride and resorcinol, [1] similar to the route described by Adolf von Baeyer in 1871. [20] In some cases, acids such as zinc chloride and methanesulfonic acid are employed to accelerate the Friedel-Crafts reaction. [21] [22]
Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and emission maximum of 512 nm (in water). The major derivatives are fluorescein isothiocyanate (FITC) and, in oligonucleotide synthesis, 6-FAM phosphoramidite.
In cellular biology, the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications (for example, flow cytometry). Additional biologically active molecules (such as antibodies) may also be attached to fluorescein, allowing biologists to target the fluorophore to specific proteins or structures within cells. This application is common in yeast display.
Fluorescein can also be conjugated to nucleoside triphosphates and incorporated into a probe enzymatically for in situ hybridisation. The use of fluorescein amidite, shown below right, allows one to synthesize labeled oligonucleotides for the same purpose. Yet another technique termed molecular beacons makes use of synthetic fluorescein-labeled oligonucleotides. Fluorescein-labelled probes can be imaged using FISH, or targeted by antibodies using immunohistochemistry. The latter is a common alternative to digoxigenin, and the two are used together for labelling two genes in one sample. [23]
Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders including retinal disease, macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors. It is also being used increasingly during surgery for brain and spine tumors. [24]
Diluted fluorescein dye has been used to localise multiple muscular ventricular septal defects during open heart surgery and confirm the presence of any residual defects. [25]
Fluorescein is used as a rather conservative flow tracer in hydrological tracer tests to help in understanding of water flow of both surface waters and groundwater. The dye can also be added to rainwater in environmental testing simulations to aid in locating and analyzing any water leaks, and in Australia and New Zealand as a methylated spirit dye.
As fluorescein solution changes its color depending on concentration, [26] it has been used as a tracer in evaporation experiments.
One of its more recognizable uses was in the Chicago River, where fluorescein was the first substance used to dye the river green on St. Patrick's Day in 1962. In 1966, environmentalists forced a change to a vegetable-based dye to protect local wildlife. [27]
Fluorescein dye solutions, typically 15% active, are commonly used as an aid to leak detection during hydrostatic testing of subsea oil and gas pipelines and other subsea infrastructure. Leaks can be detected by divers or ROVs carrying an ultraviolet light.
Fluorescein has often been used to track water movement in groundwater to study water flow and observe areas of contamination or obstruction in these systems. The fluorescence that is created by the dye makes problem areas more visible and easily identified. A similar concept can be applied to plants because the dye can make problems in plant vasculature more visible. In plant science, fluorescein, and other fluorescent dyes, have been used to monitor and study plant vasculature, particularly the xylem, which is the main water transportation pathway in plants. This is because fluorescein is xylem-mobile and unable to cross plasma membranes, making it particularly useful in tracking water movement through the xylem. [28] Fluorescein can be introduced to a plant's veins through the roots or a cut stem. The dye is able to be taken up into the plant the same way as water and moves from the roots to the top of the plant due to a transpirational pull. [29] The fluorescein that has been taken up into the plant can be visualized under a fluorescent microscope.
Anaphylaxis is a serious, potentially fatal allergic reaction and medical emergency that is rapid in onset and requires immediate medical attention regardless of use of emergency medication on site. It typically causes more than one of the following: an itchy rash, throat closing due to swelling that can obstruct or stop breathing; severe tongue swelling that can also interfere with or stop breathing; shortness of breath, vomiting, lightheadedness, loss of consciousness, low blood pressure, and medical shock. These symptoms typically start in minutes to hours and then increase very rapidly to life-threatening levels. Urgent medical treatment is required to prevent serious harm and death, even if the patient has used an epipen or has taken other medications in response, and even if symptoms appear to be improving.
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
Angiography or arteriography is a medical imaging technique used to visualize the inside, or lumen, of blood vessels and organs of the body, with particular interest in the arteries, veins, and the heart chambers. Modern angiography is performed by injecting a radio-opaque contrast agent into the blood vessel and imaging using X-ray based techniques such as fluoroscopy.
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.
Fluorescein angiography (FA), fluorescent angiography (FAG), or fundus fluorescein angiography (FFA) is a technique for examining the circulation of the retina and choroid using a fluorescent dye and a specialized camera. Sodium fluorescein is added into the systemic circulation, the retina is illuminated with blue light at a wavelength of 490 nanometers, and an angiogram is obtained by photographing the fluorescent green light that is emitted by the dye. The fluorescein is administered intravenously in intravenous fluorescein angiography (IVFA) and orally in oral fluorescein angiography (OFA). The test is a dye tracing method.
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks, but they lack the lightfastness for fabric dyeing.
Iodinated contrast is a form of water-soluble, intravenous radiocontrast agent containing iodine, which enhances the visibility of vascular structures and organs during radiographic procedures. Some pathologies, such as cancer, have particularly improved visibility with iodinated contrast.
The Alexa Fluor family of fluorescent dyes is a series of dyes invented by Molecular Probes, now a part of Thermo Fisher Scientific, and sold under the Invitrogen brand name. Alexa Fluor dyes are frequently used as cell and tissue labels in fluorescence microscopy and cell biology. Alexa Fluor dyes can be conjugated directly to primary antibodies or to secondary antibodies to amplify signal and sensitivity or other biomolecules.
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (−N=C=S), replacing a hydrogen atom on the bottom ring of the structure. It is typically available as a mixture of isomers, fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. It was synthesized by Robert Seiwald and Joseph Burckhalter in 1958.
Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.
IAEDANS is an organic fluorophore. It stands for 5-({2-[ amino]ethyl}amino)naphthalene-1-sulfonic acid. It is widely used as a marker in fluorescence spectroscopy.
Rhodamine B is a chemical compound and a dye. 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 detected easily and inexpensively with fluorometers.
Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.
Rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is a stain. Rose bengal belongs to the class of organic compounds called xanthenes. Its sodium salt is commonly used in eye drops to stain damaged conjunctival and corneal cells and thereby identify damage to the eye. The stain is also used in the preparation of Foraminifera for microscopic analysis, allowing the distinction between forms that were alive or dead at the time of collection.
6-Carboxyfluorescein (6-FAM) is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm. A carboxyfluorescein molecule is a fluorescein molecule with a carboxyl group added. They are commonly used as a tracer agents. It is used in the sequencing of nucleic acids and in the labeling of nucleotides.
Dye tracing is a method of tracking and tracing various flows using dye as a flow tracer when added to a liquid. Dye tracing may be used to analyse the flow of the liquid or the transport of objects within the liquid. Dye tracking may be either qualitative, showing the presence of a particular flow, or quantitative, when the amount of the traced dye is measured by special instruments.
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular biology.
BODIPY is the technical common name of a chemical compound with formula C
9H
7BN
2F
2, whose molecule consists of a boron difluoride group BF
2 joined to a dipyrromethene group C
9H
7N
2; specifically, the compound 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene in the IUPAC nomenclature. The common name is an abbreviation for "boron-dipyrromethene". It is a red crystalline solid, stable at ambient temperature, soluble in methanol.
Indocyanine green (ICG) is a cyanine dye used in medical diagnostics. It is used for determining cardiac output, hepatic function, liver and gastric blood flow, and for ophthalmic and cerebral angiography. It has a peak spectral absorption at about 800 nm. These infrared frequencies penetrate retinal layers, allowing ICG angiography to image deeper patterns of circulation than fluorescein angiography. ICG binds tightly to plasma proteins and becomes confined to the vascular system. ICG has a half-life of 150 to 180 seconds and is removed from circulation exclusively by the liver to bile.
Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.
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