Understanding fluorescence spectra
The basic fluorescence properties of a fluorophore—excitation and emission—are often presented in the form of line graphs. These curves describe the likelihood that excitation and emission will occur as a function of wavelength and provide important information about the expected behavior of the irradiated fluorophore. Understanding the information is an important step in understanding the phenomenon of fluorescence.
A fluorophore is excited most efficiently by light of a particular wavelength. This wavelength is the excitation maximum for the fluorophore. Light with a wavelength near the excitation maximum can also cause excitation, as shown by the shaded areas below, but it does so less efficiently.
Excitation range and maximum. A) Excitation spectrum (line) and maximum (arrow) of a fluorophore. B) Shaded areas of the spectrum indicate wavelengths were excitation of the fluorophore is significantly less efficient.
Learn more about excitation range and maximum in Introduction to Fluorescence—The light spectrum and its relationship with fluorescence
Fluorescence emission behaves in a similar way: the fluorescence output of a fluorophore is most likely to occur at a particular wavelength. This wavelength is the emission maximum for that fluorophore. The excited fluorophore can also emit light at wavelengths near the emission maximum, as shown. However, this light will be less intense.
Emission range and maximum. A) Emission spectrum (line) and maximum (arrow) of a fluorophore. B) Shaded areas of the spectrum indicate wavelengths were emission of the fluorophore is significantly less intense.
It is important to remember that although illumination at the excitation maximum of the fluorophore produces the greatest fluorescence output, illumination at lower or higher wavelengths affects only the intensity of the emitted light—the range and overall shape of the emission profile are unchanged. As this animation shows, less efficient excitation can occur at wavelengths near the excitation maximum; however, the intensity of the emitted fluorescence is reduced.
Effect of excitation at different wavelengths on the fluorophore emission. Excitation input (blue curve) and emission output (red curve) are shown at different excitation wavelengths (A-E). A) Excitation at the fluorophore’s excitation maximum results in maximum emission. B-E) Excitation at other suboptimal wavelengths results in decreased emission intensity proportional to the decreased amount of excitation input. The wavelengths at which the fluorophore emit fluorescence do not shift when excited at suboptimal excitation wavelengths; they just decrease the amount of fluorescence emitted by the fluorophores.
Learn more about emission range and maximum in Introduction to Fluorescence—The light spectrum and its relationship with fluorescence
Notice that the emission maximum for the fluorophore is always at a longer wavelength—that is, has lower energy—than the excitation maximum. This difference between the excitation and emission maxima is called the Stokes shift. The magnitude of the Stokes shift is determined by the electronic structure of the fluorophore, and is a characteristic of the fluorophore molecule. So, what causes this energy loss?
The Stokes shift is due to the fact that some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecule's excited state. This energy is dissipated as heat to surrounding solvent molecules as they collide with the excited fluorophore.
Stokes shift schematic. The excitation maximum of the fluorophore is achieved (1) as the energy level of the molecule peaks during the excitation process. As light is emitted from the fluorophore during the fluorescence process energy is lost (2) which results in shift in the emission maximum (3). This process is called the Stokes Shift.
In summary, the excitation and emission spectra of a fluorophore contain important practical information about what wavelengths of light we need to supply and detect, in order to use that fluorophore effectively. In addition, excitation and emission spectra must be examined carefully, when choosing two or more fluorophores to use simultaneously in an experiment, so that the fluorophores can be excited in a manner that will generate distinct emissions.
The complete tutorial is also available on this video.
Plot and compare spectra and check the spectral compatibility of multiple fluorophores.
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