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What are Fluorescent Dyes?
Fluorescent dyes are tools used throughout biotechnology and medicine that offer a unique method of detecting and quantifying the presence of a target molecule, cell, or tissue within an even more complex biological sample. They are used within many realms of research and development, and are often conjugated to other molecules as micro- or nanocarriers for bioassays. In a clinical setting, they can be used to monitor drug delivery to target tissues or for precise imaging and diagnostics applications.
By nature, each fluorescent dye exhibits a unique absorbance and emission spectra, resulting from the change in energy states of electrons when the fluorophore is excited. These absorbance and emission spectra can not only exist within the visible spectrum, but also in ultraviolet (UV) and near infrared (NIR). Additionally, fluorescent dyes are highly sensitive, selective, and have extremely low toxicity which makes them useful for in vivo, in vitro, and in situ applications.
Fluorescent Detection Compared to Other Methods
Outside of fluorescence, colorimetry is another method of detecting target compounds or cells within a sample. In colorimetric assays, the target substrate or activity often produces a colored byproduct, which is quantifiable by absorbance spectroscopy and which is directly related to the presence and/or concentration of the target within the sample.
One application of colorimetric assays is to assess the total cell viability within a sample. For example, in the trypan blue exclusion assay, cells that have lost membrane integrity will appear blue through the binding of trypan blue to macromolecules and intracellular proteins, highlighting non-viable cells. The CCK-8 (cell counting kit-8) is another cell viability assay; it relies on the reduction of a tetrazolium salt, WST-8, by dehydrogenase to produce a yellow-colored formazan dye. Since active dehydrogenases are involved, it correlates with viable cells. In a similar vein, the MTT assay can also be used to indirectly determine cell viability by assessing cell metabolism. This assay relies on the oxidoreductase of NADH in cells to quantitate total metabolic activity within a cell population.
Another common use of colorimetric assays includes total protein quantification. For example, the BCA assay is one that combines the reduction of Cu2+ into Cu1+ by proteins in an alkaline medium, with the detection of the cuprous cation by bicinchoninic acid (BCA). A second, related assay is the Bradford assay, which relies on the reaction between acidified Coomassie blue to proteins to undergo a quantifiable color change.
Although colorimetric and fluorometric assays may be used for similar purposes, the mechanisms by which these assays function are not the same. In a colorimetric assay, the concentration of a target compound is determined based on the absorbance of a colored substrate. In fluorometric assays, on the other hand, the concentration of a target is determined by the kinetic activity of the light absorbed and emitted by a reaction, not a color. Similarly for both assays, the quantification of the color (colorimetric) or light (fluorometric) is directly proportional to the amount of the target compound present.
While both techniques come with their own considerations, fluorescent-based assays have a number of benefits when compared to colorimetric methods. Fluorescent dyes can be used on fixed, permeabilized samples as well as live cells. This means that a cell population can be better studied as it would be in a natural, active state, for example in fluorescent lifetime imaging (FLIM). Fluorescent detection is more sensitive than colorimetric means and can be used for a wider range of analyte concentration. This attribute is particularly useful when a target molecule or cell is especially rare or sparse within the solution. Under optimized conditions fluorescent dyes are increasingly stable, although the effects of photobleaching can never totally be nullified. The results of fluorescent assays can also be interpreted with many different types of equipment, including fluorescence microscopy, flow cytometry, spectrometer and microarray readers.
It should also be mentioned that fluorescent detection assays, in many instances, may be more suitable for a particular experiment than chemiluminescent assays. For instance, there are cell viability assays which rely on the luciferase/D-luciferin system to quantify ATP as a metric of cell health. While sensitive, due to very low background, these assays are challenging to multiplex to measure an array of cell factors simultaneously.
Common Fluorometric Stains and Assays
Various fluorescent dyes have been used as a tool to assess cell viability. A common dye, propidium iodide (PI), is a red-fluorescent nuclear and chromosomal counterstain that binds to DNA. It is not cell-permeant and is used to assess dead cells in a population. Another dye, calcein AM (acetomethoxy), has lipophilic properties which make it cell-permeant. Calcein AM exhibits a bright green color upon binding to intracellular free calcium ions, and the solubility of this AM ester may be improved by Pluronic F-127. These two dyes are often combined into a single kit, such as a live/dead assay, to be used in tandem to indicate live cells, dead cells, and/or DNA. Depending on the fluorescent tag used, live/dead assays function through the membrane integrity of a cell, esterase activity, metabolic activity, or structural segmentation of a protein or peptide.
Fluorescent dyes and assays may also be geared towards detecting and/or quantifying nucleic acids within a sample. For example, 4′,6-diamidino-2-phenylindole (DAPI) binds strongly to adenine-thymine (A-T) rich regions of DNA. DAPI is often used to quantify DNA, or as a nuclear counterstain to mark cells that have lost membrane integrity. In 5′-bromo-2′-deoxyuridine (BrdU) assays, cell proliferation rates are assessed by detecting the rate of DNA or RNA synthesis. This technique is performed through the use of a fluorescent dye and an anti-BrdU antibody. Alternatively, acridine orange is a cell-permeant dye that emits green when bound to dsDNA and red when bound to ssDNA or RNA. It is also commonly used in cell-cycle studies and can be used for lysosomal detection. 7-aminoactinomycin D (7-AAD), also exhibits a strong affinity for dsDNA, particularly guanine-cytosine (G-C) rich regions, and is used in many chromosome banding studies.
Fluorescent dyes may also be used to detect apoptosis and for cytotoxicity studies. Annexin V belongs to a family of calcium-dependent phospholipid binding proteins and has a high affinity for phosphatidylserine. It can detect early stage apoptotic change, generally in combination with a viability dye (e.g., 7-AAD or PI). When conjugated with fluorescein isothiocyanate (FITC), this dye labels phosphatidylserine sites on membrane surfaces, and is used to assess active apoptosis. Hoeschst stains, like Hoeschst 33342, are cell membrane-permeant stains that bind to the minor groove of dsDNA and are often used as a nuclear counterstain for cell cycle studies. Hoeschst stains dye condensed chromatin in apoptotic cells brighter than chromatin in viable cells.
Perhaps one of the most widespread uses for fluorescent dyes comes in protein characterization, including studies that focus on characterization, aggregation, intracellular signaling, structure, or even binding. Cyanine dyes, including Cy7, Cy5, Cy5.5 and Cy3, come in reactive NHS ester forms, which allow for attachment to free amine groups on proteins and antibodies. These dyes range from orange to NIR in fluorescence. Another example is phycoerythrin (PE), a protein in the phycobiliprotein family, present in cyanobacteria and red algae. It is used to label antibodies and cell receptors in microscopy, immunity- and DNA-based assays. A third popular dye is 5-carboxyfluorescein (5-FAM), which exhibits a green fluorescence and is most commonly used for in situ labeling of peptides, proteins, and nucleotides. Some fluorescent dyes have even more specialized functions, for example, thioflavin T is a cell permeable benzothiazole dye that binds to amyloid fibrils and is used to monitor stacked β sheet aggregates. Likewise phalloidin stains are most used to quantitate F-actin, and can be conjugated with bright photostable fluorescent dyes in fixed and/or permeabilized samples.
Fluorescent dyes can also be used for more specific applications like calcium signaling. For example, fluo-4 exhibits fluorescence upon binding to Ca2+. It is used to image the spatial dynamics of Ca2+, determine secondary messengers, in neurotransmitter studies, and cell-based pharmacological screening. Another fluorescent dye, the JC-1 dye, is a cationic carbocyanine compound that enters and accumulates in the mitochondria. At low concentrations, which indicate a low membrane potential, the dye fluoresces green while at higher concentrations, and therefore higher potential, the dye appears red. Whatever the case, it is easy to see that uses for fluorescent dyes are extensive. Importantly, dyes are often not specific to only one type of assay. For example, the same dye that can detect and quantify dsDNA may also be perfect for assessing cell viability depending on the goals and aims of an experiment. The fluorescent dyes and assays mentioned here merely scrape the surface of those that have been used across the literature.
Key Takeaways About Fluorescent Dyes
The use of fluorescent dyes throughout research and development is highly versatile. The multi-faceted nature of fluorescent probes is in part due to their wide commercial availability and diverse catalog. Additionally, fluorescent-based protocols require very few hands on steps, limiting the potential for contamination. They can be used in a standalone assay, or incorporated into an experiment in tandem with other detection methods. These attributes make fluorescent dyes a useful and practical addition to many scientific endeavors.