Basics of fluorescence

Fluorescence is characterized by its spectrum (excitation and emission wavelengths) and its intensity.

Spectrum

It is possible to measure two types of fluorescence spectra, excitation and emission spectra.

The excitation spectrum is measured by fixing the fluorescence emission wavelength and scanning the excitation wavelength. Maximum excitation wavelength can then be determined. It is similar to the absorption spectrum for a single molecule in solution.

The emission spectrum is measured by fixing the excitation wavelength and scanning the fluorescence emission wavelength.

According to the Jablonski diagram:

• The emission spectrum (S₁-S₀) is the opposite image of the excitation spectrum (S₀-S₁)
• The fluorescence wavelength does not depend on the excitation wavelength
• The fluorescence wavelength is slightly longer than the absorption wavelength, since there has been a loss of energy through vibrational de-excitation (the basis of the Raman effect). This phenomenon is called the Stokes shift.

Intensity

Fluorescence intensity is the quantity of photons emitted per unit of time and per unit of volume. It depends on the sample concentration, as well as the excitation wavelength and quantum yield.

Molecules showing π → π* transitions have a high quantum yield. These are highly fluorescent molecules. By increasing the conjugation of the π electrons, the fluorescence yield and the emission wavelength increase.

Fluorescence is a very sensitive detection method. As fluorescence intensity is measured over a background noise, it is possible to measure very weak signals. Fluorescence can be 1,000 times more sensitive than absorption which is the comparison between two similar and very intense signals (incident intensity/transmitted intensity).