Fluorescein

Chembox new
ImageFile = Fluoresceine.svg
ImageSize =
ImageFile2 = Fluorescein-sample.jpg
IUPACName =
OtherNames = Fluorescein, resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, soap yellow, Pyoverdin
Section1 = Chembox Identifiers
CASNo = 2321-07-5
EINECS = 219-031-8
PubChem = 16850
SMILES = OC(C1=C(C(C(C=CC(O)=C3)
=C3O2)=C4C2=CC(C=C4)=O)C=CC=C1)=O
MeSHName = Fluorescein
Section2 = Chembox Properties
Formula = C₂₀H₁₂O₅
MolarMass = 332.306 g/mol
Appearance =
Density =
MeltingPt = 314 - 316 °C
BoilingPt =
Solubility = Slightly
Section3 = Chembox Hazards
MainHazards =
FlashPt =
RPhrases =
SPhrases = S26 S36
Autoignition =

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 pK_a of 6.4 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 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.

ynthesis

Fluorescein was first synthesized by Adolf von Baeyer in 1871. It 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 a Lewis acid and as the catalyst. This route has a high yield under milder conditions. [Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P., Synthesis of Fluorinated Fluoresceins. Journal of Organic Chemistry 1997, 62, (19), 6469-6475.] [Yuichiro Ueno; Jiao, G.-S.; Burgess, K., Preparation of 5- and 6-Carboxyfluorescein. Practical Synthetic Procedures 2004, 31, (15), 2591-2593.]

Applications

Uses in river systems

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 the thousands of goldfish that populate the river. Fact|date=March 2008

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.

Biochemical 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 [cite journal | author = Noga E. J., Udomkusonsri, P. | date = 2002 | title = Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish | journal = Vet Pathol | volume = 39 | issue = | pages = 726–731(6) | pmid = 12450204 | doi = 10.1354/vp.39-6-726| url = http://www.vetpathology.org/cgi/reprint/39/6/726.pdf | accessdate = 2007-07-16] .

Health care applications

Fluorescein sodium is used extensively as a diagnostic tool in the field of ophthalmology, where topical fluorescein is used in the diagnosis of corneal abrasions, corneal ulcers, herpetic corneal infections. Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders in e.g. legs, including retinal disease macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors , and increasingly during surgery for brain tumors.

afety

Topical, oral, and intravenous use of fluorescein can cause adverse reactions including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction, [The Diagnosis and Management of Anaphylaxis - XXI. Anaphylactoid reactions to fluorescein.J Allergy Clin Immunol 1998;101:S465-528. [http://www.jcaai.org/PP/anaph_21_ana_reacs.asp Free full text] ; see also [http://www.guideline.gov/summary/summary.aspx?ss=15&doc_id=6887&nbr=4211 The diagnosis and management of anaphylaxis: an updated practice parameter.] from the National Guideline Clearinghouse.] cardiac arrest,cite journal
author = el Harrar N, Idali B, Moutaouakkil S, el Belhadji M, Zaghloul K, Amraoui A, Benaguida M
title = [Anaphylactic shock caused by application of fluorescein on the ocular conjunctiva]
language = French
journal = Presse Med
volume = 25
issue = 32
pages = 1546–7
year = 1996
pmid = 8952662
doi =
issn = ] and sudden death.cite journal
author = Fineschi V, Monasterolo G, Rosi R, Turillazzi E
title = Fatal anaphylactic shock during a fluorescein angiography
journal = Forensic Sci. Int.
volume = 100
issue = 1-2
pages = 137–42
year = 1999
pmid = 10356782
doi = 10.1016/S0379-0738(98)00205-9 ] cite journal
author = Hitosugi M, Omura K, Yokoyama T, Kawato H, Motozawa Y, Nagai T, Tokudome S
title = An autopsy case of fatal anaphylactic shock following fluorescein angiography: a case report
journal = Med Sci Law
volume = 44
issue = 3
pages = 264–5
year = 2004
pmid = 15296251
doi =
issn = ] 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,cite journal
author = Kinsella FP, Mooney DJ
title = Anaphylaxis following oral fluorescein angiography
journal = Am. J. Ophthalmol.
volume = 106
issue = 6
pages = 745–6
year = 1988
pmid = 3195657
doi =
issn = ] cite journal
author = Gómez-Ulla F, Gutiérrez C, Seoane I
title = Severe anaphylactic reaction to orally administered fluorescein
journal = Am. J. Ophthalmol.
volume = 112
issue = 1
pages = 94
year = 1991
pmid = 1882930
doi =
issn = ] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop. Reported rates of adverse reactions vary from 1% to 6%cite journal
author = Kwan AS, Barry C, McAllister IL, Constable I
title = Fluorescein angiography and adverse drug reactions revisited: the Lions Eye experience
journal = Clin. Experiment. Ophthalmol.
volume = 34
issue = 1
pages = 33–8
year = 2006
pmid = 16451256
doi = 10.1111/j.1442-9071.2006.01136.x ] cite journal
author = Jennings BJ, Mathews DE
title = Adverse reactions during retinal fluorescein angiography
journal = J Am Optom Assoc
volume = 65
issue = 7
pages = 465–71
year = 1994
pmid = 7930354
doi =
issn = ] cite journal
author = Kwiterovich KA, Maguire MG, Murphy RP, Schachat AP, Bressler NM, Bressler SB, Fine SL
title = Frequency of adverse systemic reactions after fluorescein angiography. Results of a prospective study
journal = Ophthalmology
volume = 98
issue = 7
pages = 1139–42
year = 1991
pmid = 1891225
doi =
issn = ] cite journal
author = Matsuura M, Ando F, Fukumoto K, Kyogane I, Torii Y, Matsuura M
title = [Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]
language = Japanese
journal = Nippon Ganka Gakkai Zasshi
volume = 100
issue = 4
pages = 313–7
year = 1996
pmid = 8644545
doi =
issn = ] 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. The risk can be reduced with prior (prophylactic) use of antihistaminescite journal
author = Ellis PP, Schoenberger M, Rendi MA
title = Antihistamines as prophylaxis against side reactions to intravenous fluorescein
journal = Trans Am Ophthalmol Soc
volume = 78
issue =
pages = 190–205
year = 1980
pmid = 7257056
doi =
issn = ] and prompt emergency management of any ensuing anaphylaxis.cite journal
author = Yang CS, Sung CS, Lee FL, Hsu WM
title = Management of anaphylactic shock during intravenous fluorescein angiography at an outpatient clinic
journal = J Chin Med Assoc
volume = 70
issue = 8
pages = 348–9
year = 2007
pmid = 17698436
doi =
issn = ] A simple prick test may help to identify persons at greatest risk of adverse reaction.cite journal
author = Matsuura M, Ando F, Fukumoto K, Kyogane I, Torii Y, Matsuura M
title = [Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]
language = Japanese
journal = Nippon Ganka Gakkai Zasshi
volume = 100
issue = 4
pages = 313–7
year = 1996
pmid = 8644545
doi =
issn = ]

The most common adverse reaction is nausea, due to a difference in the pH from the body and the pH of the sodium fluorescein dye.Fact|date=December 2007 The nausea usually is transient and subsides quickly. Hives can range from a minor annoyance to severe. A single dose of antihistamine may give complete relief. Anaphylactic shock and subsequent cardiac arrest and sudden death are very rare but because they occur within minutes, a health care provider who uses fluorescein should be prepared to perform emergency resuscitation.

ee 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

External links

* [http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein(EtOH).html Absorption and Emission Spectra of Fluorescein in Ethanol] and [http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein-dibase.html Basic Ethanol] at OGI School of Science and Engineering
* [http://probes.invitrogen.com/handbook/figures/0571.html Fluorescein Ionization Equilibria] at Invitrogen
* [http://physchem.ox.ac.uk/MSDS/FL/fluorescein.html MSDS at Oxford University]
* [http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein-dibase.html Absorption spectra and fluorescence emission spectra]