Fluorescence spectra

Fluorescence is generated when electrons in the fluorophore that are brought to a higher energy state, fall back to the ground state (Figure 1). The electrons are brought to excited state s₂, by light of a specific wavelength. The electrons lose some of this energy, for example through heat, and fall back to excited state s₁. Fluorescence is emitted when the electrons lose the remaining energy as they fall back from s₁ to the ground state. The lower energy of the emitted light means this light has a longer wavelength.

Figure 1: The absorption of light of a specific wavelength causes excitation of electrons in a fluorophore into the s₂ excited state. The electrons lose some of the energy, for example through heat loss and fall back into a lower energy, but still excited state called s₁. When the electrons fall back to the ground state, fluorescence is emitted. The lower energy of the emitted light means that it has a longer wavelength.

When using different fluorophores in the same sample in fluorescence microscopy, it is important that the emission spectra of the two fluorophores do not overlap significantly. If they do, it may be impossible to distinguish the signal of the two molecules from one another in the detection. This is known as bleedthrough.