Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescenttracer for many applications.[1]
The color of its aqueous solutions is green by reflection and orange by transmission (its spectral properties are dependent on pH of the solution),[2] as can be noticed in bubble levels, for example, in which fluorescein is added as a colorant to the alcohol filling the tube in order to increase the visibility of the air bubble contained within. More concentrated solutions of fluorescein can even appear red (because under these conditions nearly all incident emission is re-absorbed by the solution).
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 solution.[4]
The thyroxine ester of fluorescein is used to quantify the thyroxine concentration in blood.[1]
Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8.
Fluorescein is a precursor to the red dye eosin Y by bromination.[1]
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,[10][11] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop.[7] Reported rates of adverse reactions vary from 1% to 6%.[12][13][14][15] 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.[14] The risk can be reduced with prior (prophylactic) use of antihistamines[16]
and prompt emergency management of any ensuing anaphylaxis.[17] A simple prick test may help to identify persons at greatest risk of adverse reaction.[15]
Chemistry
Fluorescein under UV illuminationFluorescence excitation and emission spectra of fluorescein
The fluorescence of this molecule is very intense; peak excitation occurs at 495 nm and peak emission at 520 nm. Values for the deprotonated form in basic solution.
Fluorescein has a pKa of 6.4,[18] 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. Upon exhaustive irradiation with visible light fluorescein decomposes to release phthalic and formic acids and carbon monoxide, effectively acting as a photoCORM.[19]
Fluorescein isothiocyanate and 6-FAM phosphoramidite
Many derivatives of fluorescein are known. Examples are:
fluorescein isothiocyanate1, often abbreviated as FITC, features an isothiocyanate group (−N=C=S) substituent. FITC reacts with the amine groups of many biologically relevant compounds including intracellular proteins to form a thiourea linkage.
succinimidyl ester modified fluorescein, i.e. NHS-fluorescein, is another common amine-reactive derivative, yielding amide adducts that are more stable than the aforementioned thioureas.
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 enzymatically for in situ hybridisation. The use of fluorescein amidite, shown below right, 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.[24]
Fluorescein drops being instilled for an eye examination
Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders including retinal disease, macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors. It is also being used increasingly during surgery for brain and spine tumors.[25]
Diluted fluorescein dye has been used to localise multiple muscular ventricular septal defects during open heart surgery and confirm the presence of any residual defects.[26]
The Gemini 4 spacecraft releases dye into the water, to aid location after splashdown, June 1965.
Earth sciences
Fluorescein is used as a rather conservative flow tracer in hydrological tracer tests to help in understanding of water flow of both surface waters and groundwater. The dye can also be 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.
As fluorescein solution changes its color depending on concentration,[27] it has been used as a tracer in evaporation experiments.
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.[28]
Fluorescein dye solutions, typically 15% active, are commonly used as an aid to leak detection during hydrostatic testing of subseaoil and gaspipelines and other subsea infrastructure. Leaks can be detected by divers or ROVs carrying an ultraviolet light.
Plant science
Fluorescein has often been used to track water movement in groundwater to study water flow and observe areas of contamination or obstruction in these systems. The fluorescence that is created by the dye makes problem areas more visible and easily identified. A similar concept can be applied to plants because the dye can make problems in plant vasculature more visible. In plant science, fluorescein, and other fluorescent dyes, have been used to monitor and study plant vasculature, particularly the xylem, which is the main water transportation pathway in plants. This is because fluorescein is xylem-mobile and unable to cross plasma membranes, making it particularly useful in tracking water movement through the xylem.[29] Fluorescein can be introduced to a plant's veins through the roots or a cut stem. The dye is able to be taken up into the plant the same way as water and moves from the roots to the top of the plant due to a transpirational pull.[30] The fluorescein that has been taken up into the plant can be visualized under a fluorescent microscope.
See also
Chemical derivatives of fluorescein:
Eosin, group of dibromo, or tetrabromo, derivatives of fluorescein
Calcein, fluorescent dye and complexometric indicator
Fluorescein amidite (FAM), synthetic equivalents of fluorescein used in oligonucleotide synthesis
Merbromin, or mercurochrome, organomercuric antiseptic
^World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
^ abEl 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". Presse Médicale. 25 (32): 1546–7. PMID8952662.
^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. PMID10356782.
^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. doi:10.1258/rsmmsl.44.3.264. PMID15296251. S2CID71681503.
^Gómez-Ulla F, Gutiérrez C, Seoane I (1991). "Severe anaphylactic reaction to orally administered fluorescein". Am. J. Ophthalmol. 112 (1): 94. doi:10.1016/s0002-9394(14)76222-1. PMID1882930.
^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. PMID16451256. S2CID32809716.
^Jennings BJ, Mathews DE (1994). "Adverse reactions during retinal fluorescein angiography". J Am Optom Assoc. 65 (7): 465–71. PMID7930354.
^ abMatsuura M, Ando F, Fukumoto K, Kyogane I, Torii Y, Matsuura M (1996). "[Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]". Nippon Ganka Gakkai Zasshi (in Japanese). 100 (4): 313–7. PMID8644545.
^Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. (1997). "Synthesis of Fluorinated Fluoresceins". The Journal of Organic Chemistry. 62 (19): 6469–6475. doi:10.1021/jo9706178.
^Burgess, Kevin; Ueno, Yuichiro; Jiao, Guan-Sheng (2004). "Preparation of 5- and 6-Carboxyfluorescein". Synthesis. 2004 (15): 2591–2593. doi:10.1055/s-2004-829194.
