The basic principle of fluorescence can be illustrated using a Jablonski diagram (Figure 1 (a)). A fluorescent molecule can be excited to a higher vibrational energy state (S1vib in Figure 1 (a)) with a light source. This excitation is most efficient at the optimal excitation wavelength of the fluorophore, which most often also corresponds to the absorption maximum. After this process of photon absorption, which happens almost instantly, the molecule quickly relaxes without radiation to the lowest vibrational energy level of S1. This state has a much longer lifetime (~ ns) as compared to a vibrational state (~ ps), which leads to the following transition to originate only from this state. During the next transition, the molecule falls back into the ground state (S0) either by non-radiative decay or by spontaneous emission. In the first case energy is released in the form of heat, in the second by releasing energy in the form of a photon. Since energy has been lost during the process due to the vibrational relaxation step, the emitted fluorescent photon has a lower energy than the excitation photon which leads to a red shift of the emission profile relative to the excitation profile. Quantum mechanically the exact transition leading to fluorescence are not clearly defined, but is described by a transition probability. Together with various inhomogeneous and homogeneous broadening effects, the emission profile will have a broad continuous appearance and typically resembles a shifted mirrored version of the absorption profile (Figure 1 (b)).

Figure 1(a) Jablonski diagram illustrating the energy state transitions leading to {fluorescence} and non-radiative decay to the ground state. (b) Excitation and the red shifted emission profile of Alexa 488. Here it is clearly visible that the emission profile is red shifted and is similar to a mirrored version of the excitation profile.