Difference between revisions of "Fluorescein" - New World Encyclopedia

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! {{chembox header}}| '''Fluorescein''' <!-- replace if not identical with the article name —>
 
! {{chembox header}}| '''Fluorescein''' <!-- replace if not identical with the article name —>
 
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| align="center" colspan="2" bgcolor="#ffffff" | [[Image:Fluoresceine.svg|200px|Fluorescein]] <!-- replace if not identical with the pagename —>
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| align="center" colspan="2" bgcolor="#ffffff" | [[Image:Fluoresceine.svg|150px|Fluorescein]] <!-- replace if not identical with the pagename —>
 
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| [[Simplified molecular input line entry specification|SMILES]]
 
| [[Simplified molecular input line entry specification|SMILES]]
| <small>OC(C1=C(C(C(C=CC(O)=C3)=C3O2)=C4C2=CC(C=C4)=O)C=CC=C1)=O</small>
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|<small>c1ccc2c(c1)C(=O)OC23c4ccc(cc4Oc5c3ccc(c5)O)O</small>  
 
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! {{chembox header}}| '''Fluorescein Isothiocyanate'''
 
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| align="center" colspan="2" bgcolor="#ffffff" | [[Image:Fluorescein Isothiocyanate.gif|200px|Fluorescein Isothiocyanate]]
 
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| [[IUPAC nomenclature|Systematic name]]
 
| Fluorescein Isothiocyanate
 
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| [[Chemical formula]]
 
| C<sub>21</sub>H<sub>11</sub>NO<sub>5</sub>S
 
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| [[Molecular mass]]
 
| xx.xx g/mol
 
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| [[Density]]
 
| x.xxx g/cm<sup>3</sup>
 
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| [[Melting point]]
 
| >360 °C
 
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| xx.x °C
 
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| [[CAS registry number|CAS number]]
 
| [27072-45-3]
 
|-
 
| [[Simplified molecular input line entry specification|SMILES]]
 
| <small>xxxxx</small>
 
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| {{chembox header}} | <small>[[wikipedia:Chemical infobox|Disclaimer and references]]</small>
 
 
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'''Fluorescein''' (chemical formula C<sub>20</sub>H<sub>12</sub>O<sub>5</sub>) is a highly [[fluorescence|fluorescent]] substance, absorbing light mainly in the blue range and emitting light mainly in the green range. It is 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''' (chemical formula C<sub>20</sub>H<sub>12</sub>O<sub>5</sub>) is a highly [[fluorescence|fluorescent]] substance, absorbing light mainly in the blue range and emitting light mainly in the green range. This substance and its derivatives are 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 is known as the color additive named [[Federal Food, Drug, and Cosmetic Act|D&C]] Yellow no. 7, and its disodium salt is called D&C Yellow no. 8.
 +
{{toc}}
  
 
==Chemical and physical properties==
 
==Chemical and physical properties==
[[Image:Fluorescein.jpg|thumb|120px|left|Fluorescein under [[Ultraviolet|UV]] illumination]]
+
[[Image:Fluorescein.jpg|thumb|180px|left|Fluorescein under [[Ultraviolet|UV]] illumination.]]
 
 
Fluorescein has an [[Absorption (optics)|absorption]] maximum at 490 nanometers (nm) and an [[Fluorescence|emission]] maximum of 514 nm (in water). Also, fluorescein has an isoabsorptive point (equal absorption for all pH values) at 460 nm.
 
 
 
The fluorescence of this molecule is very high, and excitation occurs at 494 [[Nanometre|nm]] and emission at 525 nm. 
 
  
Fluorescein has a [[Acid dissociation constant|pK<sub>a</sub>]] at 6.4 and multiple ionization equilibria. This leads to pH dependent [[Absorption (optics)|absorption]] and [[Fluorescence|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.
+
Fluorescein has an [[Absorption (optics)|absorption]] maximum at 494-nanometer (nm) wavelength, and an [[Fluorescence|emission]] maximum of 521 nm (in water). It has an [[acid dissociation constant]] (pK<sub>a</sub>) at 6.4 and multiple ionization equilibria. This leads to pH dependent absorption and emission over the range of 5 to 9. The wavelength of 460 nm is its "isosbestic point"—that is, absorption at this wavelength is equal for all [[pH]] values.
  
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. Other derivatives include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein. These have been tailored for various chemical and biological applications where higher [[Photobleaching|photostability]], different spectral characteristics, or different attachment groups are needed.
+
Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately three and four nanoseconds (ns). This property can be used to determine pH from non-intensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry.
 +
[[File:FITC-2D-skeletal.png|200px|thumb|Fluorescein Isothiocyanate]]
 +
There are many fluorescein derivatives, for example ''fluorescein isothiocyanate'', often abbreviated as ''FITC''. In the FITC molecule, an [[isothiocyanate]] group ('''-N=C=S''') replaces a [[hydrogen]] atom on the bottom ring of the fluorescein molecule. This derivative is reactive towards [[amine]] groups on proteins inside cells. Other derivatives include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein. These have been tailored for various chemical and biological applications where higher [[Photobleaching|photostability]], different spectral characteristics, or different attachment groups are needed.
  
 
==Synthesis==
 
==Synthesis==
Fluorescein can be prepared from [[phthalic anhydride]] and [[resorcinol]] in the presence of [[zinc chloride]] via the [[Friedel-Crafts reaction]].
+
Fluorescein can be prepared from [[phthalic anhydride]] and [[resorcinol]] in the presence of [[zinc chloride]] ([[catalyst]]) via the [[Friedel-Crafts reaction]].
 
[[Image:ZnCl2 fluorescein.png|center|400px]]
 
[[Image:ZnCl2 fluorescein.png|center|400px]]
  
 
+
A second method to prepare fluorescein uses [[methanesulfonic acid]] as the [[catalyst]].
A second method to prepare fluorescein uses [[methanesulfonic acid]] as the catalyst.
 
  
 
==Applications==
 
==Applications==
 
===Uses in river systems===
 
===Uses in river systems===
[[Image:Chicago River, dye travelling upstream.jpg|thumb|left|Fluorescein used in the [[Chicago River]]]]
+
[[Image:Chicago River, dye travelling upstream.jpg|thumb|right|250px|Fluorescein used in the [[Chicago River]].]]
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]].
+
 
 +
One of Fluorescein's 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.
+
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===
 
===Biological research===
In biology, the [[isothiocyanate]] derivative of fluorescein is often used to label and track [[cell (biology)|cells]] in [[fluorescence]] microscopy applications. Additional biologically active molecules (such as [[antibody|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]].
+
In [[biology]], the [[isothiocyanate]] derivative of fluorescein is often used to label and track [[cell (biology)|cells]] in [[fluorescence]] microscopy applications. Fluorescein may also be attached to biologically active molecules (such as [[antibody|antibodies]]), 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 triphosphate|nucleoside triphosphates]] and incorporated into a [[hybridization probe|probe]] for [[in situ hybridisation]]. Fluorescein-labelled probes can be imaged using [[fluorescent in situ hybridization|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.
+
Fluorescein can also be linked to [[nucleoside triphosphate|nucleoside triphosphates]] and incorporated into a [[hybridization probe|probe]] for [[in situ hybridization]]. Fluorescein-labeled probes can be imaged using a technique known as FISH ([[fluorescent in situ hybridization]]), or targeted by [[antibodies]] using [[immunohistochemistry]]. The latter is a common alternative to [[digoxigenin]], and the two are used together for labeling two genes in one sample.
  
 
===Ophthalmic applications===
 
===Ophthalmic applications===
 
[[Image:Fluorescin in dropper.jpg|250px|thumb|Fluorescein in dropper used for eye examination.]]  
 
[[Image:Fluorescin in dropper.jpg|250px|thumb|Fluorescein in dropper used for eye examination.]]  
  
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.
+
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 abrasion]]s, [[corneal ulcer]]s, [[herpetic corneal infection]]s, and [[dry eye]]. [[Fluorescein angiography]] is used to diagnose and categorize [[macular degeneration]], [[diabetic retinopathy]], inflammatory intraocular conditions, and intraocular [[tumor]]s.
+
Topical fluorescein is useful in the diagnosis of [[corneal abrasion]]s, [[corneal ulcer]]s, [[herpetic corneal infection]]s, and [[dry eye]]. [[Fluorescein angiography]] is used to diagnose and categorize [[macular degeneration]], [[diabetic retinopathy]], inflammatory intraocular conditions, and intraocular [[tumor]]s.
  
 
== See also ==
 
== See also ==
 
 
* [[Fluorescence]]
 
* [[Fluorescence]]
 
* [[Ultraviolet]]
 
* [[Ultraviolet]]
  
 
== References ==
 
== References ==
 
+
* Lakowicz, Joseph R. 2006. ''Principles of Fluorescence Spectroscopy''. Second Edition. New York, NY: Springer Science. ISBN 0387312781
* Lakowicz, Joseph R., 2006. ''Principles of Fluorescence Spectroscopy'' (Second Edition). New York, NY: Springer Science. ISBN 0387312781 (ISBN-13: 978-0387312781).
+
* Stevens, Rosalind A., Patrick J. Saine, and Marshall E. Tyler. 1999. ''Stereo Atlas of Fluorescein and Indocyanine Green Angiography''. 1st edition. Woburn, MA: Butterworth-Heinemann. ISBN 0750670010
 
+
* Valeur, Bernard. 2001. ''Molecular Fluorescence: Principles and Applications''. Weinheim, Federal Republic of Germany: Wiley-VCH Verlag GmbH. ISBN 352729919X
* Stevens, Rosalind A., Patrick J. Saine, and Marshall E. Tyler, 1999. ''Stereo Atlas of Fluorescein and Indocyanine Green Angiography''. (1st edition). Woburn, MA: Butterworth-Heinemann. ISBN 0750670010 (ISBN-13: 978-0750670012).
 
 
 
* Valeur, Bernard, 2001. ''Molecular Fluorescence: Principles and Applications''. Weinheim, Federal Republic of Germany: Wiley-VCH Verlag GmbH. ISBN 352729919X (ISBN-13: 978-3527299195).
 
  
 
==External links==
 
==External links==
 +
All links retrieved April 14, 2017.
 +
*[http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein(EtOH).html Absorption and Emission Spectra of Fluorescein in Ethanol]
 +
*[http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein-dibase.html Absorption and Emission Spectra of Fluorescein in Basic Ethanol]
  
*[http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein(EtOH).html Absorption and Emission Spectra of Fluorescein in Ethanol]
 
*[http://omlc.ogi.edu/spectra/PhotochemCAD/html/fluorescein-dibase.html Absorption and Emission Spectra of Fluorescein in Basic Ethanol]
 
*[http://probes.invitrogen.com/handbook/figures/0571.html Fluorescein Ionization Equilibria]
 
  
 
[[Category:Physical sciences]]
 
[[Category:Physical sciences]]
 
[[Category:Chemistry]]
 
[[Category:Chemistry]]
[[Category:Chemical technology]]
 
  
 
{{credit|108832531}}
 
{{credit|108832531}}

Revision as of 19:50, 14 April 2017

Fluorescein
Fluorescein
Systematic name Fluorescein
Chemical formula C20H12O5
Molecular mass 332.32 g/mol
Density 1.602 g/mL
Melting point 314-316°C
Boiling point
CAS number [2321-07-5]
SMILES c1ccc2c(c1)C(=O)OC23c4ccc(cc4Oc5c3ccc(c5)O)O

Fluorescein (chemical formula C20H12O5) is a highly fluorescent substance, absorbing light mainly in the blue range and emitting light mainly in the green range. This substance and its derivatives are 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 is known as the color additive named D&C Yellow no. 7, and its disodium salt is called D&C Yellow no. 8.

Chemical and physical properties

Fluorescein under UV illumination.

Fluorescein has an absorption maximum at 494-nanometer (nm) wavelength, and an emission maximum of 521 nm (in water). It has an acid dissociation constant (pKa) at 6.4 and multiple ionization equilibria. This leads to pH dependent absorption and emission over the range of 5 to 9. The wavelength of 460 nm is its "isosbestic point"—that is, absorption at this wavelength is equal for all pH values.

Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately three and four nanoseconds (ns). This property can be used to determine pH from non-intensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry.

Fluorescein Isothiocyanate

There are many fluorescein derivatives, for example fluorescein isothiocyanate, often abbreviated as FITC. In the FITC molecule, an isothiocyanate group (-N=C=S) replaces a hydrogen atom on the bottom ring of the fluorescein molecule. This derivative is reactive towards amine groups on proteins inside cells. Other derivatives include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein. These 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 (catalyst) via the Friedel-Crafts reaction.

ZnCl2 fluorescein.png

A second method to prepare fluorescein uses methanesulfonic acid as the catalyst.

Applications

Uses in river systems

Fluorescein used in the Chicago River.

One of Fluorescein's 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 biology, the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications. Fluorescein may also be attached to biologically active molecules (such as antibodies), allowing biologists to target the fluorophore to specific proteins or structures within cells. This application is common in yeast display.

Fluorescein can also be linked to nucleoside triphosphates and incorporated into a probe for in situ hybridization. Fluorescein-labeled probes can be imaged using a technique known as FISH (fluorescent in situ hybridization), or targeted by antibodies using immunohistochemistry. The latter is a common alternative to digoxigenin, and the two are used together for labeling two genes in one sample.

Ophthalmic applications

Fluorescein in dropper used for eye examination.

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.

See also

References
ISBN links support NWE through referral fees

  • Lakowicz, Joseph R. 2006. Principles of Fluorescence Spectroscopy. Second Edition. New York, NY: Springer Science. ISBN 0387312781
  • Stevens, Rosalind A., Patrick J. Saine, and Marshall E. Tyler. 1999. Stereo Atlas of Fluorescein and Indocyanine Green Angiography. 1st edition. Woburn, MA: Butterworth-Heinemann. ISBN 0750670010
  • Valeur, Bernard. 2001. Molecular Fluorescence: Principles and Applications. Weinheim, Federal Republic of Germany: Wiley-VCH Verlag GmbH. ISBN 352729919X

External links

All links retrieved April 14, 2017.

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