What is a fluorescent dye?
Fluorescent dyes are tools used throughout biotechnology and medicine, providing unique ways to detect and quantify the presence of target molecules, cells, or tissues within more complex biological samples. They are used in many areas of research and development and are often coupled to other molecules as micro- or nanocarriers for bioassays. In clinical practice, it can be used to monitor drug delivery to target tissues and for precise imaging and diagnostic applications.
In nature, each fluorochrome exhibits a unique absorbance and emission spectrum due to the change in the energy state of its electrons when the fluorochrome is excited. These absorbance and emission spectra exist not only within the visible spectrum, but also in the ultraviolet (UV) and near-infrared (NIR). In addition, fluorescent dyes have high sensitivity, selectivity, and extremely low toxicity, making them useful for in vivo, in vitro, and in situ applications.
Comparison of fluorescence detection with other methods
Besides fluorescence, colorimetry is another method to detect target compounds or cells within a sample. In colorimetric assays, the target substrate or activity often produces a colored byproduct, which can be quantified by absorbance spectroscopy and is directly related to the presence and/or concentration of the target in the sample.
One application of colorimetric assays is to assess total cell viability within a sample. For example, in trypan blue exclusion assays, cells that have lost membrane integrity appear blue due to trypan blue binding. trypan blue Analyzes macromolecules and intracellular proteins to highlight non-viable cells. of CCK-8 (Cell Counting Kit-8) is another cell viability assay. It relies on the reduction of the tetrazolium salt WST-8 by dehydrogenase to produce a yellow formazan dye. Correlates with living cells due to the involvement of active dehydrogenases. Similarly, MTT This assay can also be used to indirectly determine cell viability by assessing cell metabolism. This assay relies on intracellular NADH oxidoreductase enzymes to quantify total metabolic activity within a cell population.
Another common application of colorimetric assays involves the quantification of total protein. for example, BCA assay It combines protein-mediated reduction of Cu2+ to Cu1+ in alkaline medium and detection of cuprous cations by bicinchoninic acid (BCA). A second related assay is bradford assayIt relies on the reaction of acidified Coomassie Blue with proteins to cause a quantifiable color change.
Although colorimetric and fluorescent assays can serve similar purposes, the mechanisms by which these assays work are not the same. Colorimetric assays determine the concentration of a target compound based on the absorbance of a colored substrate. In fluorometric assays, on the other hand, the concentration of the target is determined not by its color but by the kinetic activity of the light absorbed and emitted by the reaction. Similarly, in both assays, the quantification of color (colorimetric) or light (fluorometric) is directly proportional to the amount of target compound present.
Although both techniques have their own considerations, fluorescence-based assays have many advantages when compared to colorimetric methods. Fluorescent dyes can be used on fixed and permeabilized samples as well as live cells. This means that cell populations can be better studied as if they were in their natural active state, for example with Fluorescence Lifetime Imaging (FLIM). Fluorescence detection is more sensitive than colorimetric means and can be used over a wide range of analyte concentrations. This attribute is particularly useful when target molecules or cells are particularly rare or sparse in solution. Under optimized conditions, fluorescent dyes are increasingly stable, but the photobleaching effect cannot be completely abolished. Fluorescence assay results can also be interpreted using different types of equipment, including fluorescence microscopy, flow cytometry, spectrometers, and microarray readers.
It should also be mentioned that in many cases, fluorescence detection assays may be more suitable for certain experiments than chemiluminescence assays. For example, there are cell viability assays that rely on luciferase.D-luciferin A system that quantifies ATP as an indicator of cellular health. Although these assays are sensitive, they have very low background and are difficult to multiplex to measure a range of cellular factors simultaneously.
Common fluorescent stains and assays
Various fluorescent dyes have been used as tools to assess cell viability. Although it is a common dye, propidium iodide (PI) is a red fluorescent nuclear and chromosomal counterstain that binds to DNA. It is not cell permeable and is used to assess dead cells within a population. Another dye is Calcein AM (acetomethoxy) has lipophilic properties that result in cell permeability. Calcein AM exhibits a bright green color when bound to free calcium ions in cells, and the solubility of this AM ester may be improved by the following methods. Pluronic F-127. These two dyes are often combined into a single kit, such as in a live-dead assay, and used in parallel to indicate live cells, dead cells, and/or DNA. Depending on the fluorescent tag used, live/dead assay It functions through cell membrane integrity, esterase activity, metabolic activity, or structural segmentation of proteins and peptides.
Fluorescent dyes and fluorescent assays can also be directed to the detection and/or quantification of nucleic acids within a sample. For example, 4′,6-diamidino-2-phenylindole (DAPI) binds strongly to adenine thymine (AT)-rich regions of DNA. DAPI It is often used to quantify DNA or as a nuclear counterstain to mark cells that have lost membrane integrity. 5′-bromo-2′-deoxyuridine (BrdU) assay assesses cell proliferation rate by detecting the rate of DNA or RNA synthesis. This technique is performed using a fluorescent dye and an anti-BrdU antibody. or, acridine orange It is a cell-permeable dye that emits green light when bound to dsDNA and red light when bound to ssDNA or RNA. It is also commonly used in cell cycle studies and can be used to detect lysosomes. 7-Aminoactinomycin D (7-AAD) also shows a strong affinity for dsDNA, especially guanine-cytosine (GC)-rich regions, and is used in many chromosome banding studies.
Fluorescent dyes can also be used to detect apoptosis and study cytotoxicity. Annexin V It belongs to the family of calcium-dependent phospholipid-binding proteins and has a high affinity for phosphatidylserine. Commonly combined with viability dyes (7-AAD or PI), early stage apoptotic changes can be detected. When combined with fluorescein isothiocyanate (FITC), this dye labels phosphatidylserine sites on the membrane surface and is used to assess active apoptosis. Hesyst’s dirt, Heshust 33342a cell membrane-permeable stain that binds to the minor groove of dsDNA and is often used as a nuclear counterstain in cell cycle studies. Hoeshust staining stains condensed chromatin in apoptotic cells more brightly than chromatin in living cells.
Perhaps one of the most widely used applications of fluorescent dyes is protein characterization, including studies focused on characterization, aggregation, intracellular signaling, structure, and even binding. Contains cyanine pigment Cy7, Cy5, Cy5.5 and Cy3is provided in the form of a reactive NHS ester, allowing conjugation to free amine groups on proteins and antibodies. The fluorescence range of these dyes ranges from orange to near-infrared. Another example is phycoerythrin (PE), a protein of the phycobiliprotein family, present in cyanobacteria and red algae. Used to label antibodies and cell receptors in microscopy, immunology and DNA-based assays. The third most popular dye is 5-carboxyfluorescein (5-FAM), exhibits green fluorescence and is the most commonly used. on site 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. Similarly Phalloidin The stain is most commonly used to quantify F-actin and can be combined with bright photostable fluorescent dyes in fixed and/or permeabilized samples.
Fluorescent dyes can also be used for more specialized applications such as calcium signaling. for example, fluo-4 It exhibits fluorescence when bound to Ca2+. It is used for imaging the spatial dynamics of Ca2+, determining second messengers, studying neurotransmitters, and cell-based pharmacological screening. Another fluorescent dye is JC-1 The dye is a cationic carbocyanine compound that enters and accumulates in the mitochondria. At low concentrations, indicating a low membrane potential, the dye fluoresces green, but at higher concentrations, the potential is higher and the dye appears red. In any case, it is easy to see that fluorescent dyes have a wide range of applications. Importantly, dyes are often not specific to just one type of assay. For example, the same dyes that can detect and quantify dsDNA may also be ideal for assessing cell viability, depending on the purpose and purpose of the experiment. The fluorescent dyes and assays mentioned here only scratch the surface of those used throughout the literature.
Important points about fluorescent dyes
The use of fluorescent dyes throughout research and development is extremely versatile. The pleiotropic nature of fluorescent probes is due, in part, to their wide commercial availability and diverse catalog. Furthermore, fluorescence-based protocols require few manual steps, thus limiting the potential for contamination. These can be used in standalone assays or incorporated into experiments in parallel with other detection methods. These properties make fluorescent dyes useful and practical for many scientific endeavors.
