Understanding fluorophores

Learn about what makes fluorophores so versatile and how you can take advantage of their incredible properties.

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.

Tandem dyes

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.

Tandem dyes increase your panel size and flexibility

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:

  • Always protect tandem dyes from light as they are highly susceptible to photobleaching.
  • Do not freeze tandem dye antibody conjugates as it might result in denaturation of the donor fluorophore.
  • Minimize sample fixation or permeabilization as much as possible as it reduces their brightness.
  • Each batch requires optimization due to high batch-to-batch variation.
  • Some bleed-through emission from the donor might be observed as FRET efficiency is never 100%.
  • Label cells at 4°C to avoid degradation or decoupling of tandem dyes.

Related resources:

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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