When the excited electron falls back to the ground state, the fluorochrome emits light of specific wavelengths . This shift makes it possible to separate excitation light from emission light with the use of optical filters. The emitted light can then be measured. The difference in wavelength between the apex of the absorption and emission spectra of a fluorochrome is called the Stokes shift . The signal becomes nonlinear at very high fluorochrome concentrations.
Fluorochromes differ in how much intensity they are capable of producing.
This is important because a dull fluorochrome is a less sensitive probe than a bright fluorochrome. The photobleaching occurs because the excited state of a molecule is generally much more chemically reactive than the ground state. Measuring fluorescence is analogous to measuring radioactivity. In fact, scintillation methods of counting radioactive disintegrations have for many years used a cascade of energy transfers to create emitted light, which is then measured by the recording instrument. The amount of current is proportional to the intensity of the incident light.