Difference between revisions of "Fluorescein" - New World Encyclopedia
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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. 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]]. | ||
==Chemical and physical properties== | ==Chemical and physical properties== | ||
| − | [[Image:Fluorescein.jpg|thumb|120px|left|Fluorescein under [[Ultraviolet|UV]] illumination]] | + | [[Image:Fluorescein.jpg|thumb|120px|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. | 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. | + | 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 | + | 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 five to nine. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately three and four 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. |
| − | There are many fluorescein derivatives, for example | + | 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. |
==Synthesis== | ==Synthesis== | ||
| Line 82: | Line 81: | ||
[[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|left|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 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. | + | 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. | + | 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]]. |
| − | Fluorescein can also be conjugated to [[nucleoside triphosphate|nucleoside triphosphates]] and incorporated into a [[hybridization probe|probe]] for [[in situ hybridisation]]. | + | 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. |
===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]]. | + | 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]]. | + | 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 == | ||
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== References == | == References == | ||
| − | * Lakowicz, Joseph R. | + | * 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 | |
| − | * Stevens, Rosalind A., Patrick J. Saine, and Marshall E. Tyler | + | * Valeur, Bernard. 2001. ''Molecular Fluorescence: Principles and Applications''. Weinheim, Federal Republic of Germany: Wiley-VCH Verlag GmbH. ISBN 352729919X |
| − | |||
| − | * Valeur, Bernard | ||
==External links== | ==External links== | ||
| − | *[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(EtOH).html Absorption and Emission Spectra of Fluorescein in Ethanol] Retrieved November 12, 2007. |
| − | *[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-dibase.html Absorption and Emission Spectra of Fluorescein in Basic Ethanol] Retrieved November 12, 2007. |
| − | *[http://probes.invitrogen.com/handbook/figures/0571.html Fluorescein Ionization Equilibria] | + | *[http://probes.invitrogen.com/handbook/figures/0571.html Fluorescein Ionization Equilibria] Retrieved November 12, 2007. |
[[Category:Physical sciences]] | [[Category:Physical sciences]] | ||
[[Category:Chemistry]] | [[Category:Chemistry]] | ||
| − | |||
{{credit|108832531}} | {{credit|108832531}} | ||
Revision as of 05:35, 13 November 2007
| Fluorescein | |
|---|---|
| |
| Systematic name | Fluorescein |
| Chemical formula | C20H12O5 |
| Molecular mass | 332.32 g/mol |
| Density | x.xxx g/cm3 |
| Melting point | 314-316°C |
| Boiling point | xx.x °;l/ol |
| CAS number | [2321-07-5] |
| SMILES | OC(C1=C(C(C(C=CC(O)=C3)=C3O2)=C4C2=CC(C=C4)=O)C=CC=C1)=O |
| Disclaimer and references | |
| Fluorescein Isothiocyanate | |
|---|---|
| Fluorescein Isothiocyanate | |
| Systematic name | Fluorescein Isothiocyanate |
| Chemical formula | C21H11NO5S |
| Molecular mass | xx.xx g/mol |
| Density | x.xxx g/cm3 |
| Melting point | >360 °C |
| Boiling point | xx.x °C |
| CAS number | [27072-45-3] |
| SMILES | xxxxx |
| Disclaimer and references | |
Fluorescein (chemical formula C20H12O5) is a highly 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.
Chemical and physical properties
Fluorescein has an absorption maximum at 490 nanometers (nm) and an 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 nm and emission at 525 nm.
Fluorescein has a pKa at 6.4 and multiple ionization equilibria. This leads to pH dependent absorption and emission over the range of five to nine. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately three and four 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.
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 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 biology, the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications. 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.
See also
ReferencesISBN 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
- Absorption and Emission Spectra of Fluorescein in Ethanol Retrieved November 12, 2007.
- Absorption and Emission Spectra of Fluorescein in Basic Ethanol Retrieved November 12, 2007.
- Fluorescein Ionization Equilibria Retrieved November 12, 2007.
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