When light is absorbed by a fluorochrome, its electrons become excited and move from a resting state (1) to a maximal energy level called the ‘excited electronic singlet state’ (2). The amount of energy required will differ for each fluorochrome and is depicted in Figure 6 as Eexcitation. This state only lasts for 1–10 nanoseconds because the fluorochrome undergoes internal conformational change and, in doing so, releases some of the absorbed energy as heat. The electrons subsequently fall to a lower, more stable, energy level called the ‘relaxed electronic singlet state’ (3). As electrons steadily move back from here to their ground state they release the remaining energy (Eemission) as fluorescence (4).
As Eemission contains less energy than was originally put into the fluorochrome it appears as a different color of light to Eexcitation. Therefore, the emission wavelength of any fluorochrome will always be longer than its excitation wavelength. The difference between Eexcitation and Eemission is called Stokes Shift and this wavelength value essentially determines how good a fluorochrome is for fluorescence studies. After all, it is imperative that the light produced by emission can be distinguished from the light used for excitation. This difference is easier to detect when fluorescent molecules have a large Stokes Shift.
FIGURE 6: Stokes Shift