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Fluorescein
Fluorescein
Fluorescein
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Fluorescein
Skeletal formula
Ball-and-stick model
Sample of dark red powder
Names
Pronunciation /flʊəˈrɛsi.ɪn, flʊəˈrɛsiːn/
IUPAC name
3′,6′-Dihydroxy-3H-spiro[2-benzofuran-1,9′-xanthen]-3-one
Other names
Resorcinolphthalein, C.I. 45350, solvent yellow 94, D&C yellow no. 7, angiofluor, Japan yellow 201, soap yellow
Identifiers
3D model (JSmol)
ChEBI
ChEMBL
ChemSpider
DrugBank
ECHA InfoCard 100.017.302 Edit this at Wikidata
EC Number
  • 219-031-8
KEGG
MeSH Fluorescein
UNII
  • InChI=1S/C20H12O5/c21-11-5-7-15-17(9-11)24-18-10-12(22)6-8-16(18)20(15)14-4-2-1-3-13(14)19(23)25-20/h1-10,21-22H checkY
    Key: GNBHRKFJIUUOQI-UHFFFAOYSA-N checkY
  • InChI=1/C20H12O5/c21-11-5-7-15-17(9-11)24-18-10-12(22)6-8-16(18)20(15)14-4-2-1-3-13(14)19(23)25-20/h1-10,21-22H
    Key: GNBHRKFJIUUOQI-UHFFFAOYAZ
  • c1ccc2c(c1)C(=O)OC23c4ccc(cc4Oc5c3ccc(c5)O)O
Properties
C20H12O5
Molar mass 332.311 g·mol−1
Melting point 314 to 316 °C (597 to 601 °F; 587 to 589 K)
Slightly
Pharmacology
S01JA01 (WHO)
Hazards
GHS labelling:
GHS07: Exclamation mark
Warning
H319
P305, P338, P351
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is checkY☒N ?)

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 used as a fluorescent tracer in 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).

It is on the World Health Organization's List of Essential Medicines.[3]

Uses

[edit]
Main article: Fluorescein (medical use)

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 globe rupture,[4] 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.[5]

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]

Safety

[edit]

Oral and intravenous use of fluorescein can cause adverse reactions, including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction,[6][7] causing cardiac arrest[8] and sudden death due to anaphylactic shock.[9][10]

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,[11][12] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop.[8] Reported rates of adverse reactions vary from 1% to 6%.[13][14][15][16] 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.[15] The risk can be reduced with prior (prophylactic) use of antihistamines[17] and prompt emergency management of any ensuing anaphylaxis.[18] A simple prick test may help to identify persons at greatest risk of adverse reaction.[16]

Chemistry

[edit]
Fluorescein under UV illumination
Fluorescence 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.[citation needed]

Fluorescein has a pKa of 6.4,[2] 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. The remaining resorcinol rings react with singlet oxygen formed in situ to give oxidized, ring-opened products.[19]

Fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm.

Derivatives

[edit]
Fluorescein isothiocyanate and 6-FAM phosphoramidite

Many derivatives of fluorescein are known. Examples are:

In oligonucleotide synthesis, several phosphoramidite reagents containing protected fluorescein, e.g. 6-FAM phosphoramidite 2,[20] are used for the preparation of fluorescein-labeled oligonucleotides.

The extent to which fluorescein dilaurate is broken down to yield lauric acid can be detected as a measure of pancreatic esterase activity.

Synthesis

[edit]

Approximately 250 tons were produced in the year 2000. The method involves the fusion of phthalic anhydride and resorcinol,[1] similar to the route described by Adolf von Baeyer in 1871.[21] In some cases, acids such as zinc chloride and methanesulfonic acid are employed to accelerate the Friedel-Crafts reaction.[22][23]

The mechanism of the reaction when undertaken with a strong acid (for ex. ) while heating is as follows.

Research

[edit]

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 512 nm (in water). The major derivatives are fluorescein isothiocyanate (FITC) and, in oligonucleotide synthesis, 6-FAM phosphoramidite.

Biosciences

[edit]
See also: Fluoro-jade stain

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

[edit]

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 subsea oil and gas pipelines and other subsea infrastructure. Leaks can be detected by divers or ROVs carrying an ultraviolet light.

Plant science

[edit]

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

[edit]

References

[edit]
[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Fluorescein

View on Grokipedia
from Grokipedia
Fluorescein is a synthetic belonging to the dye family, first synthesized in 1871 by through the condensation of and . It is characterized by its vivid yellow color in solution and intense green-yellow when exposed to blue or light, making it a cornerstone in diagnostic imaging and fluorescence-based techniques. With the molecular formula \ceC20H12O5\ce{C20H12O5} and a molecular weight of 332.3 g/mol, fluorescein appears as an orange-red crystalline powder that is sparingly soluble in water but dissolves readily in alkaline solutions. In , fluorescein is predominantly employed as a diagnostic , especially in , where it aids in identifying corneal abrasions, foreign bodies, and vascular abnormalities through topical application or intravenous injection for fluorescein . The sodium salt form, sodium fluorescein (\ceC20H10Na2O5\ce{C20H10Na2O5}), enhances its water solubility and is the preferred for clinical use, rapid visualization of and iris vasculature within seconds of administration. Its mechanism relies on excitation at wavelengths of 465-490 nm, producing emission at 520-530 nm, which highlights tissue and leaks in biological barriers without significant in standard doses. Beyond , it supports procedures like gastrointestinal for detecting and serves as a tracer in surgical bioimaging to delineate tumor margins. Fluorescein also plays key roles in non-medical fields, including as a and fluorescent probe, hydrology for tracking water flow, and biological research for labeling proteins and cells in . Derivatives of fluorescein extend its utility in antimicrobial applications, studies, and advanced sensing technologies, underscoring its versatility as a foundational . While generally safe, potential adverse effects include transient nausea, skin reactions, or rare , necessitating careful monitoring during use.

History and Discovery

Discovery and Initial Characterization

Fluorescein emerged during a transformative period in 19th-century chemistry, marked by the rapid advancement of synthetic organic dyes derived from derivatives. Following William Henry Perkin's accidental discovery of in 1856, the dye industry exploded, with German chemists leading innovations in and related compounds for textiles and other applications. , a prominent figure in this field, contributed significantly to understanding structures and synthesis, earning the 1905 for his work on organic dyes and hydroaromatic compounds. This context of industrial and academic pursuit set the stage for Baeyer's exploration of xanthene-based dyes. In 1871, synthesized fluorescein through the of and , typically facilitated by a dehydrating agent such as . This straightforward heating process yielded a reddish-orange crystalline compound, representing a key milestone in dye chemistry as it introduced a novel class of fluorescent materials. Baeyer documented the synthesis in a seminal paper published in the Berichte der Deutschen Chemischen Gesellschaft, highlighting its potential as a coloring agent. The discovery built on his earlier work with , substituting for phenol to produce this structurally analogous yet distinctly fluorescent substance. Baeyer initially named the compound resorcinolphthalein, reflecting its chemical precursors—resorcinol and phthalic anhydride—consistent with naming conventions for phthalein dyes at the time. However, its remarkable fluorescence in alkaline solutions prompted a shift to the standardized name "fluorescein," honoring the phenomenon first systematically described by George Gabriel Stokes in 1852. This renaming underscored the compound's unique optical properties, distinguishing it from non-fluorescent analogs. Early characterizations confirmed its empirical formula as C20_{20}H12_{12}O5_5, determined through elemental analysis shortly after synthesis. The was established as 332.311 g/mol based on this , providing a foundational metric for its identification and further study in the late 19th and early 20th centuries. These initial determinations laid the groundwork for understanding fluorescein as a xanthene derivative, though detailed structural elucidations awaited later advancements in . By the early 1900s, its basic composition was well-accepted in chemical , solidifying its place in .

Historical Applications

Fluorescein was first commercialized as a in the late , with German chemical company industrializing its production shortly after its synthesis in to meet demand for vibrant yellow colorants suitable for and fabrics. Although its intense made it visually striking, the 's tendency to fade under light limited its widespread adoption in textiles compared to more stable synthetic colorants of the era. By the 1920s, fluorescein's high visibility in dilute solutions led to its early adoption in for tracing water flow in rivers, aquifers, and underground systems, leveraging its non-toxicity and detectability even at low concentrations to contamination paths and movement. This application built on pioneering experiments from the late but gained practical prominence in the for environmental and engineering studies. In , fluorescein was first used in 1882 by Swiss ophthalmologist Pflüger to study corneal nourishment, with clinical for epithelial defects and abrasions under blue light becoming a standard diagnostic technique by the early . This technique, evolving from earlier experimental uses, enhanced diagnostic precision in eye examinations during the mid-20th century. A notable cultural application emerged in 1962 when city workers in first used fluorescein to dye the green for the parade, turning an engineering tool for into a festive tradition that highlighted the dye's vivid color in water. The event involved dumping approximately 40 pounds of the substance, which fluoresced under sunlight to create an emerald hue lasting several days.

Chemical Properties

Molecular Structure and Physical Characteristics

Fluorescein is an organic compound with the molecular formula C20_{20}H12_{12}O5_5, characterized by a xanthene core in the form of 3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one, resulting from the linkage of phthalic acid and resorcinol moieties. This tricyclic structure, typically existing in a lactone form, has been confirmed by X-ray crystallography, revealing a planar xanthene ring system essential for its conjugated pi-electron framework. In its solid state, fluorescein presents as a dark orange to crystalline powder, often exhibiting a faint greenish-yellow under reflected . It demonstrates limited in (approximately 0.1 g/L at neutral ), but this solubility markedly increases in alkaline conditions due to , enhancing its ionic character. The of fluorescein ranges from 314 to 316 °C, accompanied by , and it remains stable under ambient conditions away from and strong oxidants. With a key pKa of 6.4 for the phenolic hydroxyl group, fluorescein shifts from a yellow-colored neutral or monoanionic form in acidic media to a dianionic form in basic media, reflecting its pH-responsive protonation equilibrium. This underpins its utility as a fluorescent probe.

Optical and Spectroscopic Properties

Fluorescein exhibits strong absorption in the blue-green region of the , with a peak excitation at approximately 494 nm, leading to green upon emission at around 514 nm in basic conditions. This green emission arises from the chromophore's extended π-conjugation, which facilitates efficient electronic transitions. The resulting , the difference between absorption and emission maxima, is about 20 nm, though values up to 25 nm are reported depending on and . The of fluorescein is high, reaching approximately 0.93 in alkaline environments such as 9.5 buffers, indicating nearly efficient conversion of absorbed photons to emitted light. However, this yield is strongly -dependent, with the dianion form predominant above 8 showing optimal , while below 7 quenches emission due to altered electronic structure and reduced radiative decay. The pKa for the phenolic group is around 6.4, marking the transition where intensity drops markedly. Despite its high , fluorescein demonstrates limited photostability, undergoing through irreversible photochemical reactions under prolonged exposure to excitation light, including UV wavelengths. This degradation involves triplet-state mediated processes and , leading to a multi-exponential decay in intensity, with significant loss observed after extended illumination.

Synthesis

Classical Synthesis Methods

The classical synthesis of fluorescein, first reported by in 1871, involves the condensation of with in the presence of (ZnCl₂) as a Lewis acid catalyst. This reaction is conducted at elevated temperatures of 170–200°C, often in a melt or with concentrated to facilitate the process, resulting in the formation of the core characteristic of fluorescein. The procedure typically begins by grinding equimolar amounts of the reactants and catalyst together, followed by heating in an or furnace for 20–30 minutes until a dark melt forms, after which the mixture is cooled and processed. The mechanism is a variant of the Friedel–Crafts acylation, proceeding via stepwise electrophilic aromatic substitutions on the electron-rich rings. Initially, ZnCl₂ coordinates with the carbonyl oxygen of , generating an acylium electrophile that attacks one molecule at the ortho position to a hydroxyl group, driven by the activating effect of the phenolic moieties. A second electrophilic attack by the intermediate on another occurs, followed by dehydration and cyclization to close the ring, yielding the form of fluorescein and releasing two equivalents of water. This ortho-para directing influence of the hydroxyl groups ensures , with computational studies confirming the energy barriers favor the observed substitution pattern. Yields from this method typically range from 70% to 80% on a scale, depending on reaction time and purity of starting materials. Purification involves dissolving the crude product in aqueous to form the water-soluble dianion, filtering to remove salts, and then acidifying with to precipitate the neutral fluorescein, followed by recrystallization from or water to obtain the orange-red solid. This classical approach has been adapted for industrial production since the early , enabling large-scale manufacturing through batch reactors while maintaining the core high-temperature fusion conditions.

Modern and Alternative Synthesis Approaches

Since the classical synthesis of fluorescein involves prolonged heating of and in the presence of a Lewis acid catalyst, modern approaches prioritize reduced reaction times, higher efficiency, and environmental sustainability. -assisted synthesis has emerged as a key innovation, enabling solvent-free conditions that drastically shorten reaction durations while maintaining high yields. In this method, and are reacted with catalyst under microwave irradiation at 800 W and 90 °C, completing the process in 10 minutes with a 90% yield—compared to the hours required in traditional heating. This approach not only enhances energy efficiency by over 40% but also minimizes solvent use, making it suitable for scalable production. For silicon-containing variants, Si-fluoresceins offer improved photostability and red-shifted emission spectra, synthesized via a general strategy starting from bis(2-bromophenyl) intermediates. Developed in 2017, this involves lithium-bromine exchange followed by condensation with derivatives, yielding stable Si-fluoresceins in good efficiency. Refinements in 2023 extended this protocol using Li/H exchange with 2,3,4,5-tetrafluorobenzoic , achieving 77–94% yields for fluorinated Si-fluorescein analogs under milder conditions, further enhancing their utility in bioimaging probes. Green chemistry routes emphasize waste reduction through reusable solvents and catalysts. A notable example employs deep eutectic solvents (DES), such as and (1:2 ratio), which serve dual roles as catalyst and medium for condensing and , delivering excellent yields in a sustainable manner; the DES can be recycled up to four times without significant loss in performance. While enzymatic remains underexplored for core fluorescein production, these DES-based methods align with principles of and reduced toxicity. In 2025, advancements focused on scalable theranostic modifications have introduced multicomponent reactions (MCRs) for clinical-grade production. Starting from fluorescein scaffolds, esterification followed by Ugi-4CR, Passerini-3CR, or related MCRs generates functionalized derivatives with yields of 31–87%, scalable to over 25 mmol for compounds like a potent 15-LOX-1 inhibitor (: 5.7 μM) that retains and exhibits /metal stability. This strategy facilitates rapid diversification, bridging diagnostic with therapeutic potential in a single entity.

Derivatives

Key Derivatives and Their Structures

Fluorescein derivatives are structural analogs of the parent compound, which features a core fused with a moiety. These modifications typically occur on the xanthene ring or the phenyl to alter reactivity or properties while retaining the core structure. One prominent derivative is (FITC), which incorporates an (-N=C=S) group at the 5-position of the phenyl ring attached to the core. This single-isomer compound, often designated as isomer I, enables covalent attachment to nucleophiles. Another key analog is (6-FAM), featuring a group at the 6-position on the phenyl ring of the fluorescein scaffold. This modification provides a reactive site for esterification, such as with succinimidyl groups, and exists as a pure for consistent reactivity. Oregon Green dyes represent fluorinated variants of fluorescein, with 2',7'-difluoro substitutions on the ring in the case of Oregon Green 488, enhancing stability. Additional fluorination on the phenyl ring occurs in Oregon Green 514, often with a succinimidyl at the 5- or 6-position for further derivatization. Eosin Y is a halogenated derivative characterized by tetrabromination at the 2',4',5',7'-positions of the xanthene ring in the fluorescein structure. Similarly, erythrosin (or erythrosin B) features tetraiodination at the same positions, resulting in a disodium salt form of 2',4',5',7'-tetraiodofluorescein. These heavy-atom substitutions distinguish them from the parent compound.

Functional Modifications and Properties

Fluorescein derivatives are engineered through targeted modifications to its core scaffold, enabling tailored chemical and optical behaviors distinct from the parent molecule. These alterations often involve substitutions at key positions, such as the 5- or 6-position on the ring or the group on the xanthene core, which influence reactivity, solubility, and photophysical efficiency. (FITC), featuring an (-NCS) group at the 5-position, exhibits high reactivity toward primary amines, forming stable linkages that facilitate without disrupting the fluorophore's core. This modification results in a slightly reduced (approximately 0.92 in basic aqueous environments compared to the unmodified fluorescein's 0.95), primarily due to intramolecular by the electron-withdrawing isothiocyanate moiety. In contrast, (6-FAM), with a group at the 6-position, enhances solubility through increased polarity and ionic character, allowing better performance in physiological buffers. This substitution also improves chemical stability against and pH fluctuations, maintaining emission around 520 nm with minimal spectral shifts in neutral to slightly basic conditions. Halogenation of fluorescein, as seen in eosin derivatives like (tetrabrominated at the 2',4',5',7' positions), significantly boosts the triplet yield and rate, elevating production to levels suitable for photochemical applications. The heavy atom effect from atoms facilitates efficient energy transfer to ground-state oxygen, with quantum yields for ¹O₂ generation reaching up to 0.6 in organic solvents, far exceeding that of non-halogenated analogs.

Medical Uses

Diagnostic Applications in Ophthalmology

Fluorescein serves as a vital diagnostic agent in ophthalmology, leveraging its fluorescent properties to highlight ocular abnormalities under specific illumination. When excited by blue light, it emits yellow-green fluorescence, facilitating the visualization of corneal defects and retinal vascular issues without invasive measures. This non-toxic dye has been integral to routine eye examinations since its clinical adoption in the mid-20th century. One primary application is staining, where topical fluorescein reveals epithelial defects and foreign bodies. The procedure involves moistening a sterile fluorescein-impregnated strip with saline and gently applying it to the anesthetized eye, followed by illumination with a light source to observe uptake in damaged areas. This method is essential for diagnosing conditions like , corneal ulcers, and trauma-induced abrasions, as the dye binds to exposed in denuded . Fluorescein angiography represents another cornerstone diagnostic tool, involving intravenous injection to image and choroidal vasculature. The standard adult dosage is 5 mL of a 10% fluorescein sodium solution (500 mg total), administered rapidly into an antecubital vein, with commencing immediately to capture dye circulation phases. This technique detects vascular leaks, non-perfusion, and neovascularization in diseases such as age-related macular degeneration () and , where hyperfluorescence indicates pathology like . The U.S. (FDA) approved fluorescein for diagnostic in 1972, building on its clinical use since the for evaluating disorders. The fluorescein angiography market underscores its enduring clinical relevance, valued at USD 854.9 million in 2024 and projected to grow at a (CAGR) of 8.5% through 2030, driven by rising incidences of diseases and advancements in .

Emerging Therapeutic and Surgical Uses

In recent years, sodium fluorescein has gained traction as an adjunct in fluorescence-guided (FGS) for tumors, particularly high-grade gliomas, by enhancing intraoperative visualization of tumor margins beyond what white-light allows. Administered intravenously at a dose of 5 mg/kg shortly after induction, fluorescein accumulates preferentially in areas of blood-brain barrier disruption, fluorescing under blue light excitation (approximately 465-490 nm) to delineate neoplastic tissue. This approach has been shown to increase the extent of resection while preserving healthy tissue, with studies reporting improved gross total resection rates up to 90% in select cases. In pediatric , fluorescein-guided resection has emerged as a safe and feasible technique for supratentorial intra-axial tumors, including gliomas and ependymomas, where precise margin definition is critical to minimize neurological deficits. A 2022 study of 33 pediatric patients demonstrated that 5 mg/kg fluorescein provided useful (intense or moderate) in 94% of cases, facilitating safer resections without significant adverse events beyond transient yellowing of and skin. Clinical trials have prospectively evaluated its efficacy in pediatric tumors, aiming to quantify improvements in surgical margins and long-term outcomes. These developments build on prior diagnostic applications but extend fluorescein into real-time surgical guidance. The increased availability of fluorescein formulations has further supported these surgical innovations. In 2023, the U.S. FDA approved Nexus Pharmaceuticals' Fluorescein Injection USP (100 mg/mL and 250 mg/mL), addressing prior supply shortages and enabling broader adoption in neurosurgical settings for enhanced tumor delineation during procedures. In April 2025, Nexus Pharmaceuticals launched a 2 mL vial of the 25% solution to further address supply needs and support broader clinical adoption. Combined with advanced microscope filters like the YELLOW 560, this approval facilitates more consistent intraoperative fluorescence, potentially reducing recurrence rates in aggressive brain tumors.

Scientific Applications

Applications in Biosciences

Fluorescein transitioned from its origins as a synthetic , first prepared by in 1871, to a pivotal tool in during the , coinciding with the development of and techniques. This shift was catalyzed by the introduction of (FITC) in 1942 by Albert Coons, who functionalized fluorescein with an isothiocyanate group to enable covalent labeling of antibodies, marking the birth of microscopy. By the early , FITC had become one of the primary fluorescent dyes available for biological applications, alongside , facilitating the visualization of cellular components in research settings. In biosciences, FITC conjugates are widely employed for cell labeling in flow cytometry and immunofluorescence assays, where they bind to specific antigens on cell surfaces or intracellular structures to enable multiparametric of cell populations. For instance, FITC-labeled monoclonal antibodies target surface markers, allowing researchers to quantify cell types, activation states, and interactions in heterogeneous samples with high sensitivity. In immunofluorescence, FITC's green (excitation ~488 nm, emission ~520 nm) permits colocalization studies when combined with other fluorophores, providing insights into protein distributions and cellular dynamics without the need for genetic modification. These applications have been foundational since the 1970s, supporting advancements in and by enabling rapid, quantitative detection of up to thousands of cells per second in flow cytometry. Fluorescein derivatives, such as 6-carboxyfluorescein (6-FAM), serve as fluorescent probes for DNA and RNA analysis in techniques like polymerase chain reaction (PCR) and capillary electrophoresis-based fragment analysis. In PCR-based fragment analysis, one primer is labeled with 6-FAM at the 5' end, allowing the generated amplicons to be detected and sized during electrophoresis, which is essential for genotyping, mutation detection, and short tandem repeat profiling. For example, 6-FAM-labeled primers facilitate the quantification of allele-specific products in real-time PCR or the resolution of DNA fragments differing by as little as one base pair in sequencing workflows. This derivative's stability at pH 7.5–8.5 and compatibility with standard excitation sources have made it a staple in molecular biology since the 1990s, enhancing the throughput of genetic analyses. Fluorescein-based probes also track cellular processes, including and protein localization, by leveraging targeted conjugates for real-time monitoring. In detection, FITC-annexin V binds to externalized on apoptotic cell membranes, enabling flow cytometric or microscopic identification of early-stage with high specificity. This method, validated in various cell lines, quantifies rates by distinguishing viable, early apoptotic, and necrotic cells based on intensity. For protein localization, FITC-conjugated antibodies or ligands visualize subcellular distributions in fixed or live cells via , revealing dynamics such as nuclear translocation or organelle-specific targeting. These approaches, rooted in FITC's established conjugation chemistry, provide conceptual insights into pathways like and have been instrumental in elucidating protein functions since 's inception.

Applications in Environmental and Earth Sciences

Fluorescein serves as an effective fluorescent tracer in environmental and earth sciences, particularly for mapping flow in abiotic systems due to its high in and strong under , which allows for precise detection at low concentrations. In hydrologic studies, it is widely used to trace movement and identify subsurface pathways, providing insights into dynamics without significant environmental disruption. One prominent application is in detecting leaks in dams and tracing , where fluorescein is injected into water sources and monitored downstream using fluorometers to delineate seepage paths. For instance, in 2023, the U.S. Geological Survey (USGS) conducted dye-tracing experiments in Lake Fork Creek, , injecting non-toxic fluorescein to map contributions from historic areas to , revealing flow connections over distances of up to 600 meters. Similar tracer tests have been applied to assess dam integrity, such as at the Bumbuna Dam in , where fluorescein injections helped quantify leakage rates and locate seepage zones in the foundation. In studies of surface water dynamics, fluorescein acts as a non-toxic for simulating and mapping and currents, enabling researchers to visualize dispersion patterns in real-time. Coastal ocean experiments, like those conducted by in 2024, utilized 55 gallons of fluorescein to track deep-water and turbulent mixing to address paradoxes in nutrient distribution off the coast. For systems, aerial imaging of fluorescein tracers has quantified hazards and flow velocities, as demonstrated in field studies along sandy beaches where plumes revealed circulation cells over scales of tens to hundreds of meters. Beyond natural systems, fluorescein finds use in forensic environmental assessments, such as in building and soil permeation tests to evaluate contaminant migration. In urban settings, fluorescent tests trace water leaks from pipes to affected areas, identifying sources in structures through UV illumination of escaped . For , it is employed in column experiments to measure permeability and tracer breakthrough, helping assess how fluids percolate through vadose zones, though results must account for potential interactions. A key limitation of fluorescein as a tracer is its sensitivity, which can quench and reduce detectability in acidic environments, leading to underestimation of flow rates in mine drainage or low- soils. This effect arises because fluorescein's and emission properties shift below 7, necessitating pH adjustments in sample analysis or alternative tracers for such conditions.

Safety and Toxicology

Human Health and Safety Considerations

Fluorescein sodium, when administered intravenously for diagnostic procedures such as , is generally well-tolerated but can elicit adverse reactions in a small of patients. The most frequent side effects are and , reported in 1-6% of cases, typically mild and transient. More serious reactions, including and , are rare, with an overall incidence of severe estimated at 0.05-0.3%. However, the risk of adverse reactions increases substantially—up to 25 times—in individuals with a history of prior exposure to fluorescein. Contraindications for fluorescein use include known to the compound or its components, as this heightens the potential for severe allergic responses. According to current FDA labeling (as of 2021), there is insufficient data on the use of fluorescein in pregnant women to inform a drug-associated for major birth defects and . It should be administered only if the potential benefit justifies the potential to the . Fluorescein sodium is excreted in human milk. Due to the potential for serious adverse reactions in nursing infants, a decision should be made whether to discontinue or the drug, taking into account the importance of the drug to the mother. Patients with a history of allergies, , or previous reactions to fluorescein require careful evaluation prior to administration. In occupational settings, fluorescein is not classified as carcinogenic by the International Agency for Research on Cancer (IARC), with no components identified as probable, possible, or confirmed human carcinogens. However, it acts as an irritant to and eyes upon direct contact, potentially causing redness, discomfort, or ; no specific occupational exposure limits have been established by regulatory bodies like OSHA, but standard is recommended to minimize contact. Safety protocols emphasize close monitoring during intravenous administration, particularly vital signs such as and , to detect early signs of or cardiovascular effects. In cases of overdose or severe reactions like , immediate treatment with epinephrine is indicated, alongside supportive measures such as antihistamines and corticosteroids, with resuscitation equipment readily available.

Environmental Impact and Ecotoxicity

Fluorescein exhibits low ecotoxicity to aquatic organisms at the low concentrations typically used in environmental tracing applications, with toxicity thresholds well above standard injection levels. For instance, the 96-hour LC50 for is reported at 1,372 mg/L, the 48-hour LC50 for cladocerans () at >100 mg/L, and the 72-hour ErC50 for at 209.2 mg/L based on ECHA data. These values indicate minimal risk to aquatic life during hydrologic studies, where fluorescein is injected at concentrations far below these thresholds, often resulting in downstream environmental exposure concentrations (EECs) of 1–2 mg/L or lower. Recent assessments by the U.S. Geological Survey (USGS) affirm fluorescein's safety for environmental releases in water tracing, noting its non-toxicity and rapid degradation in natural conditions. A 2023 USGS study highlighted that fluorescein is environmentally safe and degrades under sunlight exposure, reducing persistence in surface waters. studies confirm that fluorescein breaks down upon solar radiation exposure, with degradation rates influenced by light intensity and exposure duration, further limiting long-term accumulation in aquatic systems. As a synthetic , fluorescein may exhibit some persistence in industrial or municipal effluents if not subject to or treatment, potentially leading to temporary coloration or low-level in receiving waters. Its breakdown is pH-dependent, occurring more readily in alkaline conditions common to many effluents, which facilitates and reduces environmental . Under the European Union's REACH regulation, fluorescein is classified as posing low hazard to the aquatic environment, with safety data sheets confirming no acute or classification for water organisms at relevant exposure levels. Regulatory guidelines for tracing applications recommend injection concentrations below 1 mg/L to ensure negligible ecological impact, aligning with protections for sensitive ecosystems.

Recent Research

Advances in Imaging and Probes

Recent advancements in fluorescein-based probes have focused on enhancing sensitivity, selectivity, and applicability in complex biological environments through innovative molecular designs. In 2023, a fluorescein-based probe, synthesized via condensation of fluorescein with an derivative, demonstrated exceptional performance for Hg(II) ion sensing. This probe exhibits a remarkable 250-fold enhancement at 515 nm upon binding Hg(II), with a low limit of detection of 67.1 nM in an ethanol-water (8:2 v/v) mixture, enabling rapid and selective detection in aqueous media. The mechanism involves the opening of the spirolactam ring, confirmed by spectroscopic and computational analyses, highlighting its potential for environmental and bioimaging applications where high quantum efficiency in water-based systems is crucial. Nanoparticle integrations have further advanced fluorescein's utility in bio by improving specificity and stability. A 2025 review details the use of silica conjugated with (FITC) and (EGFR)-targeting antibodies, which enhance particle detection in tumor microenvironments. These EGFR-FITC-SiO₂ nanoparticles provide superior specificity for head and neck cancers compared to non-targeted FITC alone, as evidenced by stronger signals in and models, reducing off-target accumulation and enabling precise visualization of cellular uptake. This functionalization strategy leverages silica's and FITC's bright emission to achieve high-contrast with minimal . Structural modifications to fluorescein scaffolds have addressed longstanding challenges in solubility and responsiveness for analyte detection. In advancements summarized in a 2024 review covering 2023 developments, probes like acryloyl fluorescein hydrazide incorporate non-hydrogen-bonding groups to boost solubility, achieving a detection limit of 0.001 µM for Hg²⁺ in fully aqueous solutions through PET-inhibited turn-on. Similarly, thiooxo-fluorescein derivatives exhibit 37-fold emission enhancement and improved solubility for ratiometric Hg²⁺ sensing ( 0.039 µM), while pH-sensitive variants with xanthene benzene functionalizations enable precise monitoring of physiological shifts (4.0–7.4) by modulating states. These tweaks, including encapsulation and polar substituents, enhance and selectivity without compromising fluorescein's core xanthene . The synergy between fluorescein and aggregation-induced emission (AIE) luminogens has opened avenues for real-time tracking by mitigating aggregation-caused quenching. A 2023 study elucidated the ACQ-to-AIE transformation in fluorescein derivatives through and steric hindrance, yielding probes with enhanced emission in aggregated states suitable for deep-tissue imaging. Building on this, 2025 applications utilize legumain-activated AIEgen probes for parasite-specific visualization targeting , enabling non-invasive, high-resolution detection of infections via turn-on in biological fluids. This hybrid approach combines fluorescein's with AIE's photostability, facilitating prolonged tracking with minimal invasiveness.

Theranostic and Clinical Developments

Recent research from 2023 to 2025 has advanced fluorescein toward theranostic applications by integrating its diagnostic with therapeutic capabilities, particularly in . A seminal 2025 study detailed the chemical evolution of fluorescein into a theranostic agent through targeted functionalization, synthesizing over 20 derivatives across four scaffolds using multicomponent reactions to inhibit 15-lipoxygenase-1 (15-LOX-1), a key in cancer progression. These modifications enabled cell-permeable compounds, such as derivative 5e, that facilitate real-time fluorescent of cancer cells while delivering (PDT) to induce targeted via reactive oxygen species generation upon light activation. This approach not only preserves fluorescein's utility but also addresses limitations in traditional diagnostics by providing simultaneous therapeutic intervention, demonstrating in live-cell assays for target engagement and tumor visualization. Derivative modifications, such as conjugation with inhibitory moieties, enable this dual functionality without compromising . Ongoing clinical trials are exploring fluorescein's role in fluorescence-guided for head and neck tumors, emphasizing visualization to minimize iatrogenic . A 2025 (NCT06054178) involving six patients undergoing tumor resection administered 1 mg/kg intravenous sodium fluorescein at induction, achieving high-contrast (Weber contrast ratio of 2.5 ± 1.0) with custom near-infrared systems and the Zeiss Kinevo microscope 2-3.5 hours post-injection. This revealed fine branches (1-2 mm) invisible under white light, including (CN VII) structures, with no and compatibility with robotic systems like Da Vinci Xi at higher doses (5 mg/kg). Such advancements fill gaps in precision by reducing postoperative complications like facial paralysis, paving the way for broader adoption in high-risk procedures. Clinical expansions of fluorescein include its application in ureteral and vascular mapping during , enhancing real-time anatomical identification. Updates from procedural studies highlight intravenous sodium fluorescein (0.25-1 mg/kg) for ureteral jet detection and patency assessment via , improving visualization in gynecologic and urologic interventions with minimal adverse effects. In vascular contexts, supports intraoperative blood flow mapping in , delineating perfusion deficits and vessel patency with high sensitivity under blue light excitation. These developments, integrated into existing surgical workflows, reduce operative times and injury risks in complex cases. Regulatory and market milestones underscore fluorescein's theranostic potential. In September 2023, the FDA approved Pharmaceuticals' fluorescein injection, USP (25 mg/mL), a sterile diagnostic solution expanding access for intravenous use in and lymphography. The global theranostics market, encompassing fluorescein-based agents, is projected to grow at a CAGR of 13.6% from 2024 to 2032, driven by demand for precision tools and rising cancer incidences. Fluorescein-specific formulations are anticipated to contribute to this expansion, with reaching approximately USD 150 million by 2025 and a CAGR of 6% through 2033, fueled by innovations in PDT conjugates.

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