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What is Fluorochrome?
Fluorochromes are photoreactive chemicals when absorbing light or energy, electrons are raised from the ground state to an excited state. Electrons return to the ground state by a variety of transitions which may involve the emission of a quantum of light. This effect is termed fluorescence. The emitted light will always be of lower energy, and hence longer wavelength, than the exciting light.
As the color of the exciting and emitting light are different, they can be separated from one another by using optical filters. Fluorescent dyes or fluorochromes are commonly used as detection reagents in various applications such as cellular imaging and flow cytometry.
Instruments such as fluorescence microscopes and flow cytometers are equipped with lasers, so they can excite fluorochromes capable of absorbing light at that specific wavelength. Many different fluorochromes have been developed, each one with a particular emission and excitation fluorescence spectra.
Fluorochromes differ in the intensity at which they emit light. Therefore, the fluorochrome brightness will depend on its ability to absorb light and the efficiency at which the absorbed light is converted into emitted light.
Properties of Fluorochrome
The important properties of a fluorochrome are its absorption spectrum, its extinction coefficient at a wavelength convenient for excitation, its emission spectrum, and its quantum efficiency. The properties of a fluorochrome will depend on its environment.
Some fluorochromes, such as fluorescein, are sensitive to pH. If two fluorochromes are closely associated, energy transfer can occur whereby the excitation of one compound causes the other to fluoresce. For energy transfer to occur, the acceptor molecule must have an absorption spectrum that overlaps with the emission spectrum of the donor molecule.
Classes of Fluorochromes
In general, fluorochromes can be divided into 5 classes as discussed below.
The fluorescent proteins can be categorized into two groups. The first group is made of natural proteins that encode their fluorescence in the protein structure. The second class of fluorescent proteins are those derived from the phycobiliproteins found in algae and plants.
Synthetic small molecules
Synthetic molecules are a broad class of relatively small fluorescent compounds that have a long history in flow cytometry. Synthetic dyes are available across the spectrum and come in a variety of configurations that influence solubility and cell permeability. They are also amenable to chemical modifications for fine-tune targeting through conjugation to lipids, antibodies, and other biomolecules or ligands.
The use of quantum dots (QDots) became popular in the late 2000. The QDot is a semiconductor that can be tuned to different emission wavelengths based on the size of the particle. In flow cytometry, QDots are typically excited by the violet laser, although they can be excited by any light below the emission maximum, and so need careful planning when used in panels.
More recently, polymer dyes have become popular. With the introduction of Brilliant Violet, additional polymers for UV excitation (Brilliant UV) and blue excitation (Brilliant Blue) have been developed. The polymer backbone itself is fluorescent and can be coupled to an acceptor fluorochrome, forming a tandem dye.
Tandem dyes are a special class of fluorescent molecule that take advantage of Förster resonance energy transfer (FRET, or fluorescence resonance energy transfer). Tandem dyes are very useful as they can extend the usable spectrum for a given excitation laser. When two fluorochromes are placed in proximity. The emission of the donor molecule must overlap with the excitation of the acceptor molecule.
Fluorochromes in Flow Cytometry
Fluorochromes used in flow cytometry are essentially those that can attach in some way to biologically significant molecules and are excitable by the lasers commonly found on commercial flow cytometers. These fluorochromes can attach to antibodies or proteins, free molecules that become florescent when bound to a target or have other fluorescent characteristics under various biological conditions.
The most common fluorochrome used to label proteins is fluorescein isothiocyanate, the isothiocyanate group reacts with the amino groups on the lysine residues in the protein. The number of labelled proteins that can be used has been greatly extended using tandem dyes. Examples include conjugates of PE and allophycocyanin (APC) with various cyanine dyes.
Brightness plays an important role when considering fluorochromes. Brighter fluorochromes should be reserved for critical markers of low expression, or rare events. While there are several resources and charts online with varying selections of fluorochrome brightness. With the continued development of new fluorochromes, it is critical to stay up on the trends. The polymer dyes are reported to aggregate when more than one is used in staining, so the use of the recommended staining buffer is an important consideration when using them in your panels. If one fluorochrome doesn’t work, consider why it may have failed and look for alternatives. Brighter fluorochromes should be reserved for critical markers, be they of low or unknown expression, or on rare events.
Fluorochromes selection must have considerations including brightness of the dyes, instrument configuration and staining protocol. With the continued development of new fluorochromes, it is critical to stay up on the trends and available options across multiple suppliers to ensure researchers are making the best decisions while not compromising the quality of their data.
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