Fluorescein Dyes: Definition, Structure, Synthesis and Uses

Fluorescein, a synthetic organic compound, has become an indispensable tool in biological research due to its fluorescent properties. Its bright green fluorescence under ultraviolet or blue light makes it ideal for various labeling applications in cell biology, molecular biology, and immunology. The versatility of fluorescein and its derivatives, particularly fluorescein isothiocyanate (FITC), allows for the specific labeling of cells, proteins, antibodies, and nucleic acids, providing crucial insights into the structure and function of biological systems.

What is Fluorescein?

Fluorescein is a synthetic organic dye that belongs to the family of xanthene-based dyes and is formally classified as a triarylmethine compound. It is a deep orange/red powder with the molecular formula C₂₀H₁₂O₅. Fluorescein is renowned for its intense fluorescence, making it widely used as a tracer and dye in bioanalytical and medical fields. It is characterized by its ability to emit visible green light when exposed to ultraviolet or blue light. In more concentrated solutions, fluorescein may appear red due to the reabsorption of emitted light by the solution itself. The basic principle of fluorescein's fluorescence emission is that, after being excited by laser light of a certain wavelength (excitation wavelength), the electrons in its outer atomic shell absorb energy and transition from their ground state orbitals to excited state orbitals. When the electrons return from the excited state to the ground state, they release energy and emit fluorescence at a specific wavelength (emission wavelength).

Fluorescein Structure

Fluorescein is a fluorescent dye that can undergo tautomeric isomerism, existing in both an open-ring quinonoid structure (I) and a closed-ring lactone structure (II). The phenyl ring at position 9 of fluorescein is perpendicular to the oxygenated anthracene ring in both the quinonoid and lactone structures, thus it does not participate in conjugation. The quinonoid structure of fluorescein exhibits strong absorption and fluorescence in the visible light region, while the lactone structure shows relatively poor molecular conjugation, resulting in only limited absorption and fluorescence in the ultraviolet region, which is typically difficult to observe. Consequently, the fluorescence properties of fluorescein vary significantly with pH, as the ionization of its phenolic groups alters its electronic configuration. This pH sensitivity makes fluorescein highly suitable for applications that require pH-dependent fluorescence, such as biological assays and as a pH indicator.

Fluorescein Excitation and Emission

Fluorescein dye has many advantages, such as good water solubility, high fluorescence quantum efficiency, and a large molar absorptivity. With these advantages, even though the excitation and emission wavelengths of fluorescein are both in the visible light range, fluorescein is widely applied in biochemical, modern biological, and medical research. One of the most prominent features of fluorescein is its strong fluorescence. When dissolved in water, fluorescein exhibits a maximum absorption around 494 nm and a maximum emission around 520 nm. Fluorescein is relatively stable under normal conditions, but it is prone to photodegradation upon prolonged exposure to light, resulting in the breakdown into phthalic acid, formic acid, and other products. Additionally, the fluorescence intensity of the molecule is strongly influenced by its environment, particularly the pH of the solution. Fluorescein has a pKa of 6.4, allowing it to exist in both protonated and deprotonated forms. This ionization equilibrium leads to pH-dependent absorption and emission characteristics in the range of pH 5 to 9.

Fluorescein Synthesis

Fluorescein is one of the first synthetic fluorescent dyes created by humans, and it has been extensively studied. In 1871, Bayer synthesized fluorescein, a typical dye, from resorcinol and phthalic anhydride through acylation and cyclodehydration. The reaction begins with resorcinol dissolving in sodium hydroxide, which acts as a catalyst. Phthalic anhydride is then added to the mixture. The resulting condensation reaction forms a dihydroxy derivative, which subsequently undergoes cyclization and oxidation to produce fluorescein. The synthesis can be divided into several steps. First, resorcinol and phthalic anhydride are heated together, typically in a solvent such as ethanol or water to promote the reaction. The mixture is then refluxed for a period to ensure the reaction is complete. After cooling, the reaction mixture is acidified (usually with hydrochloric acid) to precipitate fluorescein. The precipitate is then filtered, washed, and dried to obtain the final product. In some cases, acids such as zinc chloride and methanesulfonic acid can be used to accelerate the Friedel-Crafts reaction.

Fluorescein Dye

Over the years, numerous fluorescein derivatives have been developed, leading to diverse applications. The most common fluorescein derivatives include FITC, carboxyfluorescein, 5/6-carboxyfluorescein succinimidyl ester, fluorescein amidite (FAM), and fluorescein diacetate.

• Fluorescein Isothiocyanate

Fluorescein isothiocyanate (FITC) is one of the most widely used fluorescein derivatives. It has the typical properties of fluorescein derivatives, being prone to degradation in water and unsuitable for long-term storage. Among its forms, 5-FITC is used more often than 6-FITC. FITC is compatible with the 488 nm spectral line of an Argon-ion laser, with absorption/emission maxima at 492/519 nm (at pH 9.0). The isothiocyanate group of FITC can react with amino groups, allowing it to label amino-modified DNA, producing highly stable products. FITC also binds strongly to proteins. Additionally, FITC-oligos are commonly used in hybridization probes; FITC-peptides are used in Edman degradation for protein sequencing, and FITC is frequently utilized in protein electrophoresis detection and fluorescence resonance energy transfer assays.

• Carboxyfluorescein

Carboxyfluorescein (FAM) is another fluorescein derivative, with 5-FAM being used more frequently than 6-FAM. Carboxyfluorescein-5-succinimidyl ester, or 5-FAM (NHS), is widely present in fluorescent labeling kits. Compared to FITC, FAM reacts more quickly with amino groups, and the resulting products are more stable, although FITC binds a larger quantity of protein, and the reaction process is easier to control. FAM is also compatible with the 488 nm spectral line of an Argon-ion laser, with absorption/emission maxima at 492/518 nm (at pH 9.0), and is stable in water, maintaining the common properties of fluorescein derivatives. 5-FAM is mainly used in automated DNA sequencing, for labeling d/ddCTP, and is also commonly used for PCR product quantification and nucleic acid probes.

• Tetrachloro Fluorescein

Tetrachloro fluorescein (TET) is a fluorescein derivative. Both TET and HEX are modifications of FAM, with chlorine atoms inducing a red shift in the absorption and emission maxima of FAM, and reducing pH sensitivity to some extent. TET is also compatible with Argon-ion laser excitation sources, with absorption/emission maxima at 521/536 nm. TET, HEX, FAM, and TAMRA are used together in automated DNA sequencing, with TET labeling d/ddATP and HEX labeling d/ddGTP or d/ddATP.

What is Fluorescein Used For?

Although fluorescein has intense fluorescence and vivid color, it is unsuitable as a textile dye because of its high solubility in water, making it difficult to firmly adhere to fibers. However, in the field of analysis, fluorescein is an important fluorescent reagent. This is because, despite being highly soluble in water, fluorescein also has good affinity for cell membrane lipids, allowing it to easily penetrate cells for bioimaging. Through molecular design, specific receptors can be attached to fluorescein that respond selectively to certain analytes, and the different emission properties of fluorescein in its various forms can be utilized for effective recognition of target substances. As a result, fluorescein is widely used in the field of fluorescent probes. When fluorescein is attached to proteins as a fluorescent probe, conformational changes in the protein cause changes in the microenvironment around the fluorescein, which alters its spectral properties. Thus, changes in the spectral position, intensity, and fluorescence lifetime of fluorescein can be used to detect protein conformational changes.

• Medical and Diagnostics

One of the most important uses of fluorescein is in medical diagnostics. Sodium fluorescein is widely used in ophthalmology to diagnose corneal abrasions, corneal ulcers, and other eye conditions. It is applied topically to the eye, staining damaged areas of the corneal epithelium, which can then be easily identified under blue light. Fluorescein angiography is another important application, where fluorescein is injected intravenously to visualize retinal blood vessels, aiding in the diagnosis of retinal diseases such as diabetic retinopathy, macular degeneration, and vascular occlusion. Fluorescein is also used in cardiac surgery to locate ventricular septal defects. Additionally, fluorescein-labeled thyroxine esters are used for the quantitative determination of thyroxine levels in the blood.

• Biological Research

In cell and molecular biology, fluorescein and its derivatives are widely used to label cells, proteins, and nucleic acids. FITC is commonly used in immunofluorescence and flow cytometry to label antibodies, allowing researchers to detect specific cellular components. Fluorescein-labeled probes are also used in fluorescence in situ hybridization (FISH) and other nucleic acid detection methods. Molecular beacons are used to detect specific nucleic acid sequences, often employing fluorescein as the reporter dye. Fluorescein's ability to bind to antibodies or nucleotides makes it an indispensable tool for studying gene expression and protein localization.

• Cell Labeling

Fluorescein and its derivatives are extensively used to label cells for imaging and flow cytometry analysis. Cell membranes or specific intracellular compartments can be targeted using fluorescein-based dyes, allowing for the visualization of cell morphology, the detection of cell surface markers, and the differentiation of cell types. For example, fluorescein diacetate (FDA) is a non-fluorescent derivative that penetrates live cell membranes and is converted by intracellular esterases into fluorescein, allowing for the study of cell viability and metabolic activity.

• Protein Labeling

Fluorescein-labeled proteins are used to study protein localization and dynamics within cells. By attaching fluorescein molecules to specific proteins, researchers can track their movement and determine their interactions in real time using fluorescence microscopy. This approach has been particularly valuable in understanding signaling pathways and protein-protein interactions, helping to elucidate complex cellular processes.

• Antibody Labeling

One of the most significant applications of fluorescein is in immunofluorescence assays, where it is used to label antibodies. FITC-conjugated antibodies are commonly used in both direct and indirect immunofluorescence to detect target antigens in cells or tissue samples. In direct immunofluorescence, the primary antibody is directly labeled with FITC, enabling the specific binding of the antibody to the antigen, which can then be visualized by fluorescence microscopy. In indirect immunofluorescence, a secondary FITC-labeled antibody is used to bind a primary antibody, amplifying the fluorescent signal and improving detection sensitivity. These techniques are widely employed in diagnostics, as well as in research to understand the distribution and quantity of specific proteins.

• Nucleic Acid Labeling

Fluorescein and its derivatives are also used to label nucleic acids for hybridization-based detection techniques. Fluorescein-labeled oligonucleotide probes are used in FISH to detect specific DNA or RNA sequences within cells. The fluorescein tag allows researchers to visualize hybridization events, which is critical for identifying gene expression patterns, chromosomal abnormalities, or the presence of specific pathogens. Molecular beacons, which are short oligonucleotide sequences labeled with a fluorophore and a quencher, often utilize fluorescein as the reporter dye. These molecular beacons can form stem-loop structures that emit fluorescence upon hybridization to target nucleic acids, enabling highly specific detection of sequences in real time.

• Other Applications

In environmental science, fluorescein is widely used as a tracer to study water movement in hydrological tracer tests, helping to understand the flow of surface water and groundwater. In industry, fluorescein is used to detect leaks in pipelines, boilers, and other sealed systems, as its strong fluorescence makes it easy to detect leaks even under challenging conditions. In forensic science, fluorescein is used to detect latent blood stains. When applied to a suspected surface, fluorescein binds with hemoglobin and emits a bright fluorescence under ultraviolet light, helping investigators detect blood stains that might be invisible to the naked eye. In plant biology, fluorescein is used to study water movement in plant tissues. It cannot cross the plasma membrane, making it suitable for monitoring xylem transport. Researchers introduce fluorescein into the roots or cut stems of plants to trace water movement through the xylem under a fluorescence microscope.

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