Fluorescent Labeling: Definition, Principles, Types and Applications

Fluorescence labeling technology originated in the 1940s, when fluorescently labeled antibodies were used to detect some corresponding antigens. With the development of molecular biology, modern medicine, and the application of various advanced fluorescence detection technologies and instruments, fluorescence labeling as a new, non-radioactive labeling technology has received great attention and has achieved extremely rapid development. It has been widely used in the detection of substances inside and outside cells, nucleic acid detection, and early diagnosis of diseases, and has played an important role in the fields of biological research and medical research.

What is Fluorescent Labeling?

Fluorescent labeling (also known as fluorescent tag or fluorescent probe) is a widely used technique in molecular biology and biotechnology to detect and analyze biological molecules such as proteins, antibodies, and amino acids. This technique involves chemically attaching a fluorescent molecule, known as a fluorophore, to a target molecule, enabling it to emit light at a specific wavelength when exposed to an excitation source. Fluorophores selectively bind to specific regions or functional groups on the target molecules, allowing them to be visualized under a fluorescence microscope or detected in various assays.

Fig. 1. Methods for fluorescent labeling of proteins (J Chem Biol. 2013, 6(3): 85-95).

Fluorescent labeling can be achieved through chemical reactions, such as covalent bonding, or through biological interactions. Common fluorescent probes include ethidium bromide, fluorescein, and green fluorescent protein (GFP). For instance, fluorescein derivatives, such as fluorescein isothiocyanate (FITC), can be covalently attached to proteins through their amino groups, while GFP can be expressed genetically for in vivo imaging of live cells. This technique is frequently used to label antibodies, proteins, nucleic acids, and peptides, providing a sensitive and specific way to detect molecular targets. Fluorescent labeling allows researchers to study biological processes in real-time and in multiple colors, facilitating multiplex analysis by employing different fluorophores with distinct spectral properties.

How Does Fluorescent Tagging Work?

Fluorescence refers to a light-induced luminescence phenomenon. The compounds that fluorescent labeling relies on are called fluorophores. Fluorophores are compounds with conjugated double-bond systems in their chemical structure, which can transition to an excited state when exposed to appropriate excitation light. When returning from the excited state to the ground state, they emit fluorescence. By covalently binding or physically adsorbing fluorophores onto specific functional groups of the molecules under study, their fluorescent properties can be used to provide information about the target of interest.

Fig. 2. Excitation and emission of a fluorophore (J Chem Biol. 2013, 6(3): 85-95).

Certain small fluorophores, such as fluorescein or biotin, can be activated and react specifically with primary amine groups on proteins or peptides within a specific pH and temperature range, forming stable amide bonds, thereby achieving the conjugation of labels with proteins or antibodies. Additionally, some activated fluorophores can react specifically with other chemical reactive groups, either naturally present or introduced into the protein or antibody, forming stable chemical bonds and thus achieving conjugation. In summary, the principle of fluorescent labeling is that activated fluorophores react with active groups on proteins or antibodies under certain conditions, thereby achieving the conjugation of labels with proteins or antibodies. Fluorescent labeling technology has the advantages of no radioactive contamination and ease of operation, which contribute to its increasingly widespread application.

Factors Affecting Fluorescent Labeling

• Interference from Inorganic Salt Components

The presence of interfering components in the protein, such as glycine, imidazole, tris, sodium azide, thimerosal, and proclin, can impact the final labeling outcome. If the protein sample to be labeled contains these components, they should be removed prior to labeling through dialysis, desalting, or multiple rounds of ultrafiltration.

• Interference from Carrier Proteins

Carrier proteins like BSA (bovine serum albumin) or gelatin, which are sometimes present in the initial protein sample or added as stabilizers during storage, can also bind to fluorophores. This reduces the binding efficiency of dye molecules to the target protein. If carrier proteins are mixed with the sample, they need to be removed by affinity purification or other chromatographic methods, or a BSA removal kit can be used. Accurate quantification of protein concentration is required after removal before proceeding with labeling.

• Inappropriate Experimental Operation

Improper procedures, such as incomplete mixing of dye and protein or other handling errors, can affect the final fluorescent labeling results. It is recommended to strictly follow the protocol instructions during the labeling process.

• Inaccurate Initial Protein Concentration

The protein concentration should be accurately controlled before labeling, and the dye amount should be calculated accordingly. If the initial protein concentration is inaccurate, it may lead to insufficient or excessive labeling, which can negatively impact subsequent experimental results.

• Improper Dye Storage

Some dyes are prone to hydrolysis due to moisture and should be stored at -20 °C or -80 °C with a desiccant. To prevent water condensation, dyes should be brought to room temperature before use in subsequent operations.

What are the Different Types of Fluorescent Labels?

Fluorescent labeling reagents are chemicals that provide fluorophores. In fluorescence labeling technology, commonly used reagents include rhodamines and fluoresceins. Other fluorescent labeling reagents include polycyclic aromatic compounds, aromatic heterocyclic compounds, and some chelates of rare earth elements. Recently developed novel fluorescent labeling reagents have also received considerable attention.

• Fluorescein

Fluorescein-based labeling reagents include standard fluorescein and its derivatives. Fluorescein is one of the standard fluoresceins, and structural modifications can generate a series of fluorescein derivatives. Fluorescein is suitable for Argon-ion Laser's 488 nm spectral line, with relatively high fluorescence absorption, good fluorescence yield, and excellent water solubility. It typically does not cause protein precipitation when labeling proteins. Like other fluorescein derivatives, fluorescein has drawbacks such as high photobleaching rates, high pH sensitivity, and broad emission spectra. It is primarily used in confocal laser scanning microscopy and flow cytometry applications. Fluorescein derivatives include fluorescein isothiocyanate, tetrachlorofluorescein, and hydroxyfluorescein. They are widely used in hybridization probes, protein degradation sequencing, and antibody labeling. The primary structure of fluorescein-based labeling reagents contains a large carboxyl group on the benzene ring, maintaining the aromatic ring perpendicular to the chromophore, which improves the fluorescence quantum yield. However, fluorescein derivatives share some common drawbacks, such as high pH sensitivity, high photobleaching rates, and broad emission spectra.

• Rhodamine

Rhodamine 110 is one of the standard fluoresceins, and structural modifications can produce a series of rhodamine derivatives. The main structure of rhodamine reagents contains active groups such as -NCS and -SO2X at positions R2 and R3. In labeling reactions, these active groups mainly react with -NH2. Compared to fluorescein derivatives, rhodamine-based fluorophores have stronger photostability, higher fluorescence yield, and lower pH sensitivity. Rhodamine-based fluorophores are highly suitable for labeling oligonucleotides, but proteins can cause fluorescence quenching for most rhodamine-based fluorophores, making them unsuitable for protein labeling. Additionally, Rhodamine 110 can be used for automated DNA sequencing.

• Cyanine

Cyanine is currently the brightest fluorophore available among organic dyes. Although cyanine dyes generally have low quantum yields (≤0.25), they exhibit very high extinction coefficients. The chemical structure of cyanine derivatives contains a conjugated polymethine chain with quaternary nitrogen, allowing researchers to significantly adjust the photophysical properties of the fluorophore by modifying functional groups and adjusting the conjugation length. However, the use of cyanine dyes may be limited by photobleaching, and their small Stokes shifts may also be a limitation (relevant for super-resolution microscopy). Furthermore, Cy5 and Cy7 derivatives are prone to oxidation in the presence of oxygen and ozone, limiting their application in experiments requiring long-term measurements. Recently, researchers have developed new derivatives to improve their quantum yield and oxidation resistance, partially overcoming the issues associated with cyanine dyes. Consequently, cyanine dyes have been developed as fluorescent probes to detect and label mercury, DNA, and RNA in live cells, among other applications. With advancements in single-molecule localization microscopy, cyanine dyes have been increasingly used in biological imaging.

• Coumarin

Coumarin family compounds contain a 2H-chromen-2-one group, making them one of the largest groups among small organic dyes. Coumarin is widely used in life sciences due to its excellent properties, such as photostability, high quantum yield, and significant Stokes shift. Its blue emission makes it an ideal choice for multicolor imaging and can be used with green-to-infrared dyes. However, in cellular imaging, coumarin's short-wavelength excitation may overlap with autofluorescence signals, which is one of its drawbacks. Coumarin's brightness is also limited by weak absorption, as seen in AF350. Over the past few decades, coumarin fluorescent molecules have been widely used in life sciences.

• BODIPY

Boron-dipyrromethene compounds, or BODIPYs, are popular for their outstanding photophysical properties. They exhibit high quantum yields, excellent brightness, narrow full-width-at-half-maximum (FWHM) absorption and emission spectra, and small Stokes shifts. The main limitation of BODIPY dyes is their susceptibility to oxidation. Various BODIPY derivatives cover a wide range of the visible spectrum, from green (BODIPY FL) to red (BODIPY 650/665). BODIPY-based fluorescent probes are widely used in biomedical and environmental sciences. For instance, BODIPY dyes are used to trace intracellular calcium ion concentration changes, revealing neuronal activity or cell signaling pathways. They are also used as markers for drug delivery, combining drugs with BODIPY to monitor drug release, which has significant value in drug development and helps optimize drug delivery systems. These applications highlight the importance of BODIPY fluorescent probes in life sciences and medical research and their potential in developing innovative solutions.

• Phycoerythrin

Phycoerythrin (PE) is found in phycoerythrin complexes in algae, located in chlorophyll reaction centers. In its natural structure, phycoerythrin absorbs light energy and converts it into energy for growth. Once isolated and purified, phycoerythrin exhibits strong fluorescence, absorbing light at different wavelengths and emitting bright red fluorescence. At this stage, it no longer accepts incoming light or transfers energy. Phycoerythrin has a broad absorption range, with a maximum absorption wavelength of 566 nm and maximum emission wavelength of 574 nm. It exhibits strong long-wavelength excitation and emission, avoiding interference from other biological materials' fluorescence, high quantum yield, excellent water solubility, and stable crosslinking to multiple sites on various biological or synthetic materials.

• Peridinin-Chlorophyll-Protein Complex

The peridinin-chlorophyll-protein complex (PerCP) is found in the photosynthetic apparatus of dinoflagellates and thin-walled dinoflagellates. It is a protein complex with a molecular weight of approximately 35 kD, with its peak maximum excitation wavelength near 490 nm and peak emission wavelength around 677 nm when excited by 488 nm argon-ion laser.

• Other Fluorescent Reagents

Other fluorescent labeling reagents include polycyclic aromatic compounds such as indole, naphthalene, anthracene, pyrene, and aromatic heterocycles like acridine, and phenanthridine, as well as chelates of lanthanides, phycoerythrin N, pyronin, and chromomycin A3. Indole, pyronin, and chromomycin A3 are mainly used to label biological nucleic acids. These traditional fluorescent reagents have been widely used but have some drawbacks, such as photobleaching, which can lead to unstable fluorescence signals. Additionally, conventional fluorescent labeling of biomolecules can only attach a limited number of fluorescent molecules to the active groups of biomolecules, resulting in limited analysis sensitivity. In recent years, the emergence of nanoscale fluorescent probes has opened up new development opportunities for biological labeling. Nanoscale fluorescence has advantages such as (1) broad excitation spectra, (2) continuous distribution, (3) symmetrical and narrow-width emission spectra, and (4) tunable colors.

What is the Use of Fluorescent Labeling?

Fluorescent labeling is a versatile tool in molecular biology and biotechnology, facilitating the detailed study of biological processes at both cellular and molecular levels. Its application spans across genomics, proteomics, cell biology, and drug discovery, offering powerful capabilities for visualizing and quantifying biomolecular interactions, studying cellular processes, and advancing our understanding of biological systems. As technologies advance, fluorescent labeling continues to be an indispensable component in both fundamental research and clinical diagnostics.

• Genetic Labeling

In genomics, fluorescent labeling is extensively used in DNA sequencing and in situ hybridization. During DNA sequencing, fluorescent dyes label the nucleotides, allowing automatic sequencing systems to read DNA sequences based on fluorescence signals. In fluorescence in situ hybridization (FISH), labeled DNA or RNA probes are used to detect specific nucleic acid sequences within cells or tissues, making it a valuable tool for studying chromosomal abnormalities, gene mapping, and diagnostics.

• Protein Labeling

In proteomics, fluorescent labeling aids in the detection, quantification, and localization of proteins. Techniques like fluorescence resonance energy transfer (FRET) use fluorescently labeled proteins to study protein-protein interactions, providing detailed insights into molecular mechanisms in living cells. Immunofluorescence is another key method, where antibodies are conjugated with fluorophores to specifically label target proteins within cells or tissues. This allows researchers to study protein distribution and expression patterns, helping in disease research and understanding cellular functions.

• Cell Labeling

Flow cytometry is a widely utilized technique in cell biology that heavily relies on fluorescent labeling. By tagging cells with fluorescent markers that bind to specific cell surface or intracellular molecules, flow cytometry can rapidly analyze cell populations for a variety of features, such as cell type, function, and viability. This is particularly useful in immunology for sorting immune cell types and understanding immune responses, as well as in cancer research for characterizing tumor cells.

• Enzyme Labeling

Fluorescent labeling is widely used to study enzyme activity, localization, and interactions. Enzymes tagged with fluorescent markers can be monitored in real-time to observe their catalytic activities and their role in biological pathways. This allows researchers to analyze enzyme kinetics, understand the effect of inhibitors, and visualize enzyme localization within cells. Fluorescently labeled enzymes are also used in various assays to detect substrate transformation, making them valuable tools in diagnostic applications and drug discovery.

• Chemical Labeling

Chemical labeling with fluorescent markers involves attaching a fluorescent tag to a molecule through a chemical reaction. This approach helps track small molecule interactions, reaction pathways, and intracellular dynamics. For example, fluorescent labeling is commonly used to study metabolic pathways by labeling metabolites. In drug discovery, chemical compounds can be labeled to observe their distribution, localization, and binding behavior in living cells. This helps elucidate the mechanism of drug action and assess pharmacokinetics.

• Antibody Labeling

One of the most widespread applications of fluorescent labeling is antibody conjugation. Fluorescently labeled antibodies are used to detect specific antigens in techniques such as immunofluorescence and flow cytometry. In immunofluorescence, antibodies labeled with fluorescent tags bind to target proteins within cells or tissues, allowing for the visualization of protein localization and expression levels. This is particularly useful in diagnostic procedures to detect disease markers, as well as in research to understand the role of proteins in cellular processes. Flow cytometry, on the other hand, relies on fluorescently labeled antibodies to analyze cell surface or intracellular molecules across large cell populations, aiding in cell sorting and characterization.

• Amino Acid Labeling

Fluorescent labeling of amino acids is a key technique in protein research, used for studying protein synthesis, folding, and interactions. Fluorescent amino acids or their analogs can be incorporated into proteins during translation, allowing researchers to track protein dynamics in real-time. This is especially useful in analyzing protein-protein interactions through fluorescence resonance energy transfer (FRET). The incorporation of labeled amino acids helps understand protein conformational changes, binding events, and complex formation in living cells. Additionally, fluorescent tagging of specific amino acids within proteins enables the investigation of post-translational modifications.

• Lipid Labeling

Lipid labeling with fluorescent probes is used to investigate the organization, dynamics, and function of lipids within biological membranes. Fluorescent lipid analogs are used to study the distribution and mobility of lipids in cell membranes, providing insights into membrane fluidity, lipid rafts, and membrane protein interactions. This has significant implications in understanding membrane-related processes such as signaling, transport, and membrane fusion. Fluorescently labeled lipids are also useful for tracking lipid metabolism and the intracellular trafficking of lipoproteins.

• Carbohydrate Labeling

Fluorescent labeling of carbohydrates helps in studying glycan structures and their biological roles. Glycans can be labeled to monitor their distribution, interactions, and role in cell recognition processes. For example, fluorescently labeled lectins are used to bind specific sugar moieties on cell surfaces, aiding in the study of cell-cell communication and pathogen-host interactions. Additionally, fluorescent carbohydrate probes are used to investigate glycosylation patterns and changes associated with disease states, providing insights into the role of glycans in health and disease.

Reference:

1. Toseland, C.P. Fluorescent labeling and modification of proteins. J Chem Biol. 2013, 6(3): 85-95.

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