What are fluorophores?

Fluorophores or fluorochromes are photoreactive chemicals that absorb and emit energy in a predictable fashion. They re-emit light when excited, making them useful tags for identifying and characterizing cells or molecules in a mixture, visualizing proteins, or quantifying tagged compounds.¹

Fluorophores absorb light energy of a specific wavelength and re-emit it at a longer wavelength. The wavelengths at which the fluorophore absorbs and emits light are the fluorophore's excitation and emission (absorbance) spectra, which collectively make up the fluorescence spectra (Figure 2). Fluorophores differ in the intensity at which they emit light. Their brightness depends on their ability to absorb light and the efficiency of converting the absorbed light into emitted light.

There are many commercially available fluorescent dyes that absorb and emit light at specific wavelengths across the entire visible spectrum, including the infrared region.

Instruments requiring the use of fluorescent dyes, such as fluorescence microscopes and flow cytometers, are equipped with lasers that produce light at a particular wavelength to excite fluorophores, and sensors that detect various light wavelengths. Some combine multiple lasers and/or optical filters for multiplexed analyses. Light emitted by the fluorophores in the sample is then detected by sensors, providing the fluorescence read-out signal.

Beyond basic fluorescence, several more complicated fluorescence techniques are used in research. The most common are time-resolved fluorescence (TRF) and fluorescence resonance energy transfer (FRET), which are explained in more detail below