Fluorescein
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| Fluorescein | |
|---|---|
| Image:Fluoresceine.svg | |
| Image:Fluorescein-sample.jpg | |
| Other names | Fluorescein, resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, soap yellow |
| Identifiers | |
| CAS number | |
| PubChem | |
| EINECS number | |
| MeSH | |
| SMILES | OC(C1=C(C(C(C=CC(O)=C3) =C3O2)=C4C2=CC(C=C4)=O)C=CC=C1)=O |
| Properties | |
| Molecular formula | C20H12O5 |
| Molar mass | 332.306 g/mol |
| Melting point |
314 - 316 °C |
| Solubility in water | Slightly |
| Hazards | |
| S-phrases | S26 S36 |
| Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa) Infobox disclaimer and references | |
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 521 nm (in water). Also, fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm. Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as D&C Yellow no. 8.
Chemical and physical properties
The fluorescence of this molecule is very high, and excitation occurs at 494 nm and emission at 521.
Fluorescein has a pKa of 6.4 and multiple ionization equilibria. This 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 non-intensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry.
Derivatives
There are many fluorescein derivatives, for example fluorescein isothiocyanate, often abbreviated as FITC. FITC is the original fluorescein 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 fluorescein core, creating NHS-fluorescein, forms another common amine reactive derivative.
Other derivatives of fluorescein include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein. These derivatives, along with newer fluors such as Alexa 488 and DyLight 488, have been tailored for various chemical and biological applications where higher photostability, different spectral characteristics, or different attachment groups are needed.
Synthesis
Fluorescein can be prepared from phthalic anhydride and resorcinol in the presence of zinc chloride via the Friedel-Crafts reaction.
A second method to prepare fluorescein uses methanesulfonic acid as the catalyst.
Applications
Uses in river systems
One of its more recognizable uses is in the Chicago River, where fluorescein is used to dye the river green on St. Patrick's Day.
Other uses of fluorescein include using it as a water-soluble dye 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.
Biological research
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 for in situ hybridisation. 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.
Ophthalmic applications
Fluorescein sodium is used extensively as a diagnostic tool in the field of ophthalmology. It is applied topically in the form of a drop or it can be injected intravenously to produce a fluorescein angiogram.
Topical fluorescein is useful in the diagnosis of corneal abrasions, corneal ulcers, herpetic corneal infections, and dry eye. Fluorescein angiography is used to diagnose and categorize macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors.
Safety
Topical, oral, and intravenous use of fluorescein can cause adverse reactions. Intravenous use has documented adverse reactions including nausea, vomiting, anaphylaxis and (rarely) death.[1]
See also
- Chemical derivatives of fluorescein
- Eosin
- Fluorescein isothiocyanate
- Erythrosine
- DyLight Fluor, a product line of fluorescent dyes
- Pseudomonas aeruginosa, a bacterium that secretes fluorescein
- Fluorescein diacetate hydrolysis, a biochemistry laboratory test
References
- Noga E. J., Udomkusonsri, P. (2002). "Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish". Vet Pathol 39: 726-731(6). PMID 12450204. Retrieved on 2007-07-16.
External links
- Absorption and Emission Spectra of Fluorescein in Ethanol and Basic Ethanol at OGI School of Science and Engineering
- Fluorescein Ionization Equilibria at Invitrogen
- MSDS at Oxford University
- Absorption spectra and fluorescence emission spectraca:Fluoresceïna
de:Fluorescein eo:Fluoreskeino fr:Fluorescéine ja:フルオレセインsv:Fluorescein
Acknowledgement and Attribution Regarding Sources of Content
Some of the initial content on this page may be incorporated in part from copyleft sources in the public domain including wikis such as Wikipedia and AskDrWiki. Drug information for patients came from the The National Library of Medicine. Infectious disease information may have come from the Centers for Disease Control (CDC). Differential Diagnoses are drawn from clinicians as well as an amalgamation of 3 sources: 1.The Disease Database; 2. Kahan, Scott, Smith, Ellen G. In A Page: Signs and Symptoms. Malden, Massachusetts: Blackwell Publishing, 2004:3; 3. Sailer, Christian, Wasner, Susanne. Differential Diagnosis Pocket. Hermosa Beach, CA: Borm Bruckmeir Publishing LLC, 2002:7 .
