Fluorescein

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Fluorescein
Other names Fluorescein, resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, soap yellow, Pyoverdin
Identifiers
CAS number [2321-07-5]
PubChem 16850
EINECS number 219-031-8
MeSH Fluorescein
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.

Contents

[edit] Chemical and physical properties

Fluorescein under UV illumination
Fluorescein under UV illumination

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 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.

[edit] 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.

Fluorescein isothiocyanate

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.

[edit] 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 a Lewis acid and as the catalyst. This route has a high yield under milder conditions. [1] [2]

[edit] Applications

[edit] 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.[citation needed]

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.

[edit] 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 [3].

[edit] Health care applications

Fluorescein sodium is used extensively as a diagnostic tool in the field of ophthalmology, and increasingly during surgery for brain tumors.

[edit] Ophthalmology

Fluorescein in dropper used for eye examination.
Fluorescein in dropper used for eye examination.
 This article may require cleanup to meet Wikipedia's quality standards.
Please improve this article if you can. (December 2007)

In ophthalmology, fluorescein is applied topically in the form of an eye drop, given orally, or injected intravenously to produce a fluorescein angiogram.

Main article: Fluorescein angiography

In the process of removing foreign bodies from the eye, fluorescein-containing paper strips are employed by the practitioner to check the effectiveness of his operative procedure. Typically, metal foreign bodies will leave a "rust ring" and this must be removed from the surface of the eye. These procedures refer solely to bodies which contact the cornea.

A type of curette called a "spud" is used to scrape the surface of the cornea until it is clean. Of course penetrating injuries do not fall into this kind of procedure which is typically employed in the practitioner's office. An ultraviolet source is used to fluoresce the dye, which is later lavaged from the eye after successful completion of the procedure.

A similar process is used to examine an eye which has sustained a "brushing" injury where a foreign object is not seen to enter the cornea, but rather cause an abrasion on the cornea. These usually heal completely in one or more days depending upon the severity of the abrasion.

It is apparent that the dye property of fluorescein is an important tool in a practitioner's office. It may be called into frequent use if the physician serves where industries employ any kind of grinding or similar operations which generate the kinds of missiles found to cause these injuries of the eye.

So it can be seen that topical fluorescein is a useful tool 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.

[edit] Safety

Topical, oral, and intravenous use of fluorescein can cause adverse reactions including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction,[4] cardiac arrest,[5] and sudden death.[6][7] 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,[8][9] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop.[5] Reported rates of adverse reactions vary from 1% to 6%[10][11][12][13] 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.[12] The risk can be reduced with prior (prophylactic) use of antihistamines[14] and prompt emergency management of any ensuing anaphylaxis.[15] A simple prick test may help to identify persons at greatest risk of adverse reaction.[13]

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.[citation needed] 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.

[edit] See also

[edit] References

  1. ^ 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.
  2. ^ Yuichiro Ueno; Jiao, G.-S.; Burgess, K., Preparation of 5- and 6-Carboxyfluorescein. Practical Synthetic Procedures 2004, 31, (15), 2591-2593.
  3. ^ Noga E. J., Udomkusonsri, P. (2002). "Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish". Vet Pathol 39: 726-731(6). doi:10.1354/vp.39-6-726. PMID 12450204. 
  4. ^ The Diagnosis and Management of Anaphylaxis - XXI. Anaphylactoid reactions to fluorescein. J Allergy Clin Immunol 1998;101:S465-528. Free full text; see also The diagnosis and management of anaphylaxis: an updated practice parameter. from the National Guideline Clearinghouse.
  5. ^ a b el Harrar N, Idali B, Moutaouakkil S, el Belhadji M, Zaghloul K, Amraoui A, Benaguida M (1996). "[Anaphylactic shock caused by application of fluorescein on the ocular conjunctiva]" (in French). Presse Med 25 (32): 1546–7. PMID 8952662. 
  6. ^ Fineschi V, Monasterolo G, Rosi R, Turillazzi E (1999). "Fatal anaphylactic shock during a fluorescein angiography.". Forensic Sci. Int. 100 (1-2): 137–42. doi:10.1016/S0379-0738(98)00205-9. PMID 10356782. 
  7. ^ Hitosugi M, Omura K, Yokoyama T, Kawato H, Motozawa Y, Nagai T, Tokudome S (2004). "An autopsy case of fatal anaphylactic shock following fluorescein angiography: a case report.". Med Sci Law 44 (3): 264–5. PMID 15296251. 
  8. ^ Kinsella FP, Mooney DJ (1988). "Anaphylaxis following oral fluorescein angiography.". Am. J. Ophthalmol. 106 (6): 745–6. PMID 3195657. 
  9. ^ Gómez-Ulla F, Gutiérrez C, Seoane I (1991). "Severe anaphylactic reaction to orally administered fluorescein.". Am. J. Ophthalmol. 112 (1): 94. PMID 1882930. 
  10. ^ Kwan AS, Barry C, McAllister IL, Constable I (2006). "Fluorescein angiography and adverse drug reactions revisited: the Lions Eye experience.". Clin. Experiment. Ophthalmol. 34 (1): 33–8. doi:10.1111/j.1442-9071.2006.01136.x. PMID 16451256. 
  11. ^ Jennings BJ, Mathews DE (1994). "Adverse reactions during retinal fluorescein angiography.". J Am Optom Assoc 65 (7): 465–71. PMID 7930354. 
  12. ^ a b Kwiterovich KA, Maguire MG, Murphy RP, Schachat AP, Bressler NM, Bressler SB, Fine SL (1991). "Frequency of adverse systemic reactions after fluorescein angiography. Results of a prospective study.". Ophthalmology 98 (7): 1139–42. PMID 1891225. 
  13. ^ a b Matsuura M, Ando F, Fukumoto K, Kyogane I, Torii Y, Matsuura M (1996). "[Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]" (in Japanese). Nippon Ganka Gakkai Zasshi 100 (4): 313–7. PMID 8644545. 
  14. ^ Ellis PP, Schoenberger M, Rendi MA (1980). "Antihistamines as prophylaxis against side reactions to intravenous fluorescein.". Trans Am Ophthalmol Soc 78: 190–205. PMID 7257056. 
  15. ^ Yang CS, Sung CS, Lee FL, Hsu WM (2007). "Management of anaphylactic shock during intravenous fluorescein angiography at an outpatient clinic.". J Chin Med Assoc 70 (8): 348–9. PMID 17698436. 

[edit] External links