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Fluorophores (or fluorochromes) are commonly used in conjugation with antibodies as detection reagents in applications such as flow cytometry. Fluorophores can absorb and emit light within a range of wavelengths, normally referred to as the absorbance (excitation) and emission spectra.
Figure legend. Top: Excitation and emission fundamentals of fluorophores. 1) The fluorophore absorbs light energy of a specific wavelength. 2) Light absorption results in excitation of the fluorophore's electrons. 3) The fluorophore re-emits the absorbed light energy at a longer wavelength upon the electrons return to their basic state. Bottom: Excitation and emission spectra of FITC.
Click here to learn more about the changes that fluorophores undergo upon excitation.
The design of multi-color panels often requires the use of tandem dyes. They consist of two different fluorophores that are covalently attached. One fluorophore (donor) provides the excitation characteristics, while the other fluorophore (acceptor) provides the emission characteristics. In PE-Cy7, for instance, Phycoerythrin (PE) and Cy7 act as the donor and acceptor fluorophores respectively. Therefore, PE-Cy7 will have the excitation characteristics of PE and the emission characteristics of Cy7.
Figure legend. Top: Excitation and emission fundamentals of tandem dyes. 1) The fluorophore acting as a donor absorbs light energy of a specific wavelength. 2) Upon excitation of the donor, energy is transferred to the acceptor due to their proximity through a phenomenon called Förster or fluorescence resonance energy transfer. 3) The fluorophore acting as an acceptor emits the transferred energy as fluorescent light. Bottom: Excitation and emission spectra of PE-Cy7.
Using tandem dyes, a single laser can excite several fluorophores, which are measured by different detectors. For example, Alexa Fluor® 488, PE, PerCP-Cy5.5 and PE-Cy7 are all excitable with a blue laser (488 nm). However, they will produce green, yellow, purple and infrared emissions respectively.
Figure legend. Excitation and emission spectra of Alexa Fluor® 488, PE, PerCP-Cy5.5, and PE-Cy7 following excitation with the blue laser (488 nm).
Here are some hints and tips on how to handle tandem dyes: