Fluorescein is a synthetic organic compound available as a dark orange/red powder slightly
soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.
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. The major
derivatives are fluorescein isothiocyanate and, in oligonucleotide synthesis, 6-FAM phosphoramidite.
Fluorescein also has an isosbestic point at 460 nm. Fluorescein is also known as a color
additive. The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8.
The color of its aqueous solution varies from green to orange as a function of the way it
is observed: by reflection or by transmission, as it can be noticed in bubble levels in which
fluorescein is added as a colorant to the alcohol filling the tube to increase the visibility
of the air bubble and the precision of the instrument. More concentrated solutions of
fluorescein can even appear red.
Chemical and physical properties
The fluorescence of this molecule is very intense; peak excitation occurs at 494 nm
and peak emission at 521 nm. 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 nonintensity 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 1, often
abbreviated as FITC, is the original fluorescein molecule functionalized with an isothiocyanate
group, replacing a hydrogen atom on the bottom ring of the structure. This derivative is
reactive towards primary amine groups of biologically relevant compounds including intracellular
proteins to form a thiourea linkage. A succinimidyl ester functional group attached to the fluorescein
core, creating NHS-fluorescein, forms another common amine-reactive derivative, yielding
more stable amide adducts. A carboxyl group added to fluorescein makes carboxyfluorescein,
and carboxyl, fluorescein, and succinimidyl ester groups compose carboxyfluorescein succinimidyl
ester. Pentafluorophenyl esters and tetrafluorophenyl esters are other useful reagents. In oligonucleotide
synthesis, several phosphoramidite reagents containing protected fluorescein, e.g. 6-FAM
phosphoramidite 2, are widely used for the preparation of fluorescein-labeled oligonucleotides.
Other green dyes include Oregon Green, Tokyo Green, SNAFL, and carboxynaphthofluorescein.
These dyes, along with newer fluorophores such as Alexa 488, FluoProbes 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 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 Brønsted acid catalyst.
This route has a high yield under milder conditions. Applications
Biochemical research In cellular biology, the isothiocyanate derivative
of fluorescein is often used to label and track cells in fluorescence microscopy applications.
Additional biologically active molecules 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 enzymatically for in situ hybridisation. The
use of fluorescein amidite shown above allows one to synthesize labeled oligonucleotides
for the same purpose. Yet another technique termed molecular beacons makes use of synthetic
fluorescein-labeled oligonucleotides. 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.
Health care applications
"Fluorescein sodium", the sodium salt of fluorescein, is used extensively as a diagnostic tool in
the field of ophthalmology and optometry, where topical fluorescein is used in the diagnosis
of corneal abrasions, corneal ulcers and herpetic corneal infections. It is also used in rigid
gas permeable contact lens fitting to evaluate the tear layer under the lens. It is available
as sterile single-use sachets containing lint-free paper applicators soaked in fluorescein sodium.
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.
Air Sea Rescue
During World War 2, German aircrew carried small containers of fluorescein. In the event
of parachuting into the sea after being shot down, the dye would be released into the water.
This produced a vivid marking that could be seen from the air over long distances and
aided the Air-sea rescue of the downed crew. This was later adopted by other air forces.
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 local wildlife. 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. Oilfield application
Fluorescein dye solutions, typically 15% active, are commonly used as an aid to leak detection
during hydrostatic testing of subsea oil and gas pipelines and other subsea infrastructure.
Leaks can be detected by divers carrying ultraviolet lights.
Safety
Topical, oral, and intravenous use of fluorescein can cause adverse reactions, including nausea,
vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction, causing
cardiac arrest and sudden death due to anaphylactic shock.
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; a number of other factors, however, are considered
contributors as well. The nausea usually is transient and subsides quickly. Hives can
range from a minor annoyance to severe, and 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. 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, including one
case of anaphylaxis with cardiac arrest following topical use in an eye drop. Reported rates
of adverse reactions vary from 1% to 6%. 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 use of antihistamines and prompt emergency management
of any ensuing anaphylaxis. A simple prick test may help to identify persons at greatest
risk of adverse reaction. See also
Chemical derivatives of fluorescein: Eosin
Carboxyfluorescein Fluorescein isothiocyanate
Fluorescein amidite Merbromin
Erythrosine Rose Bengal
DyLight Fluor, a product line of fluorescent dyes
Fluorescein diacetate hydrolysis, a biochemistry laboratory test
Other dyes: Methylene blue
Laser dyes
References
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 spectra