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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.

A Jablonski diagram distinguishes electronic excitation from subsequent relaxation. Photon absorption takes a molecule from the electronic ground state $S_0$ to a vibrationally excited level of $S_1$ or a higher singlet state. Rapid vibrational relaxation and internal conversion usually populate the lowest vibrational levels of $S_1$. Fluorescence then emits a photon on a transition to $S_0$, often ending in one of its excited vibrational levels. Nonradiative relaxation competes with emission; intersystem crossing can also populate triplet states. Energy lost to relaxation and solvent reorganization commonly makes the fluorescence maximum occur at lower photon energy, hence longer wavelength, than the photon absorption maximum: the Stokes shift.

The intrinsic fluorescence quantum yield is the number of photons emitted by fluorescence per absorbed excitation. For a simple excited state with radiative rate $k_r$ and other decay rate $k_{nr}$,A nearby acceptor adds a nonradiative energy-transfer channel, not an emitted photon flying from donor to acceptor. The electric dipole-dipole interaction transfers the donor excitation while returning the donor toward its ground state and exciting the acceptor. The acceptor then relaxes and may fluoresce. The donor yield becomesFor the acceptor, distinguish its intrinsic yield per acceptor excitation, $Q_A=k_{r,A}/(k_{r,A}+k_{nr,A})$, from the number of acceptor photons per donor excitation. Transfer increases the supply of acceptor excitations, giving sensitized emission $E{Q}_A$ per donor excitation in the ideal model. It does not by itself change $Q_A$ if the acceptor's own decay rates remain unchanged. Environmental changes, saturation or additional quenching can change those rates, but increased acceptor intensity alone does not establish an increased intrinsic fluorescence quantum yield.