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Fluorescence is photon emission following photon absorption by matter: an excited state releases some of its energy as light. A fluorophore can therefore mark where something is, while its fluorescence emission spectrum and fluorescence lifetime report different aspects of its environment. Fluorescein is one example; FRET exploits a competing route for excitation energy rather than requiring a second emitted photon.

The usual molecular sequence is excitation, relaxation, then emission, as in the Jablonski diagram. The emitted photon commonly has lower energy and longer wavelength, giving a Stokes shift; the exceptions are discussed below.^[ref]

A Jablonski diagram arranges molecular energy eigenstates vertically and draws arrows for changes between them. In the usual closed-shell molecular example, $S_0$ is the electronic ground state, $S_1$ and $S_2$ are electronic excited states with singlet state spin, and $T_1$ has triplet state spin. Each electronic state also has levels of molecular vibration. The diagram records possible processes, not molecular paths in space.^[ref]

Photon absorption can populate an upper level of molecular vibration in $S_1$. Relaxation removes excess energy without photon emission; fluorescence commonly starts from relaxed $S_1$.^[ref] The drawing also includes relaxation from a higher electronic excited state and a competing spin-changing route to a triplet state.

The Stokes shift compares the lowest absorption-band maximum with the fluorescence emission spectrum maximum. Relaxation often makes $\Delta \lambda >0$. This comparison differs from using an arbitrary excitation wavelength. A spectral-density maximum can move when changing from per unit wavelength to per unit spectroscopic wavenumber; specify the spectral convention.^[ref]

An original illustrative energy budget makes the distinction concrete. Excitation at $450\mathrm{nm}$ and emission at $520\mathrm{nm}$ correspond to about $2.76$ and $2.38\mathrm{eV}$ per photon, using $E=hc/\lambda$ with Planck constant $h$ and speed of light $c$ in vacuum. The difference, about $0.37\mathrm{eV}$, must go elsewhere in the complete energy balance. These chosen wavelengths are not measured band maxima for a particular fluorophore.

Anti-Stokes emission has shorter wavelength than excitation. Additional energy, for example from a thermally populated level of molecular vibration or multiple excitation photons, makes it possible. The one-photon, initially relaxed drawing does not cover these cases.^[ref]