What is fluorescence?

Fluorescence is a light signal detected when a chemical compound called a fluorophore absorbs energy at a specific wavelength, causing it to become excited. The fluorophore then emits light at a longer wavelength as it relaxes and returns to its ground state.

Fluorescence is a three-stage process, as detailed in Figure 1. In the first step (1), the fluorophore is irradiated with electromagnetic light produced by a laser and passed through optical filters to create a very specific wavelength, matching the signature excitation wavelength for the molecule. The light provides the right amount of energy for an electron in the molecule to jump from its ground state (Gs) to an excited state (Es). In the second step (2), the electron absorbs the light and reaches an excited state.

In the final step (3), the electron undergoes vibrational relaxation to the lowest vibrational state within the excited electronic state, followed by relaxation to its ground state. As the electron returns to the ground state, energy is released in the form of a photon. The amount of energy released and the resulting wavelength of the emitted photon is characteristic of the fluorophore and determines its color and signature emission spectrum. The wavelength of the emitted light is longer than the exciting light because the electron undergoes vibrational relaxation, which emits energy as heat before fluorescence. The difference in wavelength between the exciting light and the emitted light is called the Stokes shift.