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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 fluorophore is a fluorescent molecule or fluorescent part of a molecule. Its usefulness as a label depends on how efficiently it absorbs the chosen excitation light, its fluorescence quantum yield, and its resistance to photobleaching. A large fluorescence quantum yield alone does not guarantee a bright image if few excitation photons are absorbed or collected.

Photobleaching is an irreversible loss of fluorescent capacity caused by illumination. It changes the number of active fluorophores over experimental time; fluorescence lifetime concerns the delay after an individual excitation. Reversible dark states and ordinary return to the ground state are different processes.

In a stipulated population-loss model, let $T$ denote elapsed illumination time and $t$ denote delay after a pulse. If active population declines as $e^{-k_{\mathrm{b}}T}$ while surviving fluorophores retain the same decay rates, then $I(t;T)=A_0{e}^{-k_{\mathrm{b}}T}{e}^{-t/\tau }$. Brightness falls while $\tau$ stays fixed. This example does not assert that all bleaching follows exponential decay: chemical changes and selective loss from a mixture can also change the observed decay shape. The existing FRET photobleaching controls illustrate why deliberate bleaching requires careful interpretation.

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]