Molecular Probes Tutorial Series- Anatomy of Fluorescence Spectra

This video describes the principle behind fluorescence spectra and how they can be used to determine properties of a fluorescent molecule. Learn more: www.lifetechnologies.com/us/en...
AUDIO TRANSCRIPT
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, but it does so less efficiently.
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.
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.
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.
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.

[@alextobias257]

Yes! I was wondering if the excitation wavelength affected the shape or maximum of the emission curve. Thank you for clarifying that it doesn't - it only affects the intensity.

[@SugarShawty]

Thank you for the great explanation!

[@MUSLIMSHAVESOLES]

This video saved me when I did not know a thing about fluorescence spec and I had a 15 page paper on it due the next day

[@thermofisher]

We've all been there (though in my particular case it was an exam on absorption and emission spectra). Happy to have helped!

[@Edwardyugi1]

Very helpful!

[@teneoaristocrata3270]

Very helpful. Thanks

[@eligirl100]

Great explanation

[@willians.bernardes7478]

Great.

[@miasoraya9565]

Great explanation! But Excitation and Emission maximum for some fluorescent molecules (for Example Quinine) can differ depending on the pH value of the used solvent. Why is that?

[@thermofisher]

Thank you for your question.
Fluorescent molecules that exhibit pH-dependent spectral changes have at least one atom that is subject to protonation/deprotonation. The protonation state (varies with pH) can affect the π-electron configuration, and as a result the spectral properties, of the fluorescent molecule.
For additional technical questions, please contact us at thermofisher.com/askaquestion. Thank you!

[@abdullahhussainjabri9549]

@@thermofisher In this case, what we can do to fix the Stokes shift caused by pH ?

[@capecoaster69]

Neat

[@kieutiensy]

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