Principles and Theory of Fluorescence Spectroscopy

Fluorescence is a type of luminescence caused by photons exciting a molecule, raising it to an electronic excited state. It’s brought about by absorption of photons in the singlet ground state promoted to a singlet-excited state. As the excited molecule returns to ground state, emits a photon of lower energy, which corresponds to a longer wavelength, than the absorbed photon.

Fluorescence spectroscopy analyzes fluorescence from a molecule based on its fluorescent properties. Fluorescence is a type of luminescence caused by photons exciting a molecule, raising it to an electronic excited state.

Fig. 3 shows the Jablonski diagram (Jablonski, 1933), a schematic of the transition of electronic state of a molecule during the fluorescence phenomenon. The left axis shows increasing energy, where a typical fluorescent molecule has an absorbance spectrum. This spectrum shows the energy or wavelengths, where the molecule will absorb light.

In simple terms, fluorescence lifetime of a molecule is the average length of time it spends in the excited state. This depends on the type of molecule and its local environment. Typically, the excited state decays in an exponential manner, as indicated in the equation below. The use of fluorescence lifetime has its advantages over that of an intensity measurement, as it is an "absolute" measurement, rather than the "relative" steady state measurement (which gives a time-averaged signal).

I(t) = I₀ exp^(-t/τ)

τ is the fluorescence lifetime or the time for the intensity to decay to 1/e of its initial value.

If more than one excited state is present, sometimes because the sample under study contains a mixture of fluorescing molecules, and there are different local environments or a molecule undergoes a transformation, giving rise to different excited state species, the decay is expected to be more complex. There can be one exponential decay per excited state present. This can be represented by a sum of exponentials (see below), where α (the pre-exponential factor) is indicative of the relative concentration of each t decay to the observed overall decay.

In order to compare measurements, it is often useful to normalize the pre-exponential factors in some way. If a comparison of the concentration of each fluorescing species is required, then the normalized α may be used. If a comparison of the contribution to steady state spectrum (overall fluorescence emission) is needed, then the fractional or relative amplitude (in %) can be used. The latter is the pre-exponential factor weighted by the lifetime.

At times, it can be just as acceptable to represent a complex decay by an average lifetime. However, it should be noted that this is best done by actually correctly modelling the complex decay, rather than just attempting to fit a single exponential decay to it. In most cases, the use of the amplitude average lifetime is appropriate, however, when considering quenching experiments, it is more correct to employ the intensity average fluorescence lifetime. There are published works going into the details of the relative merits of these averages. (Lakowicz, 2006) (Berezin, 2010)

Phosphorescence is a process where the photon is emitted, not from a singlet-excited state, but from a forbidden triplet state. The time scale of emission is generally in the picosecond to nanosecond range, while phosphorescence typically lasts for fluorescence microseconds, milliseconds, or even longer...minutes or hours. Researchers typically use a pulsed source such as a flash lamp or LED to measure phosphorescence spectra and decays on these longer time scales. Phosphorescence measurements use a longer-lived pulsed source, such as a xenon flash lamp. The timing of the flashing lamp can be used to measure spectra at different phosphorescence lifetimes.

Phosphorescence measurements use a longer-lived pulsed source, such as a xenon flash lamp. The timing of the flashing lamp can be used to measure spectra at different phosphorescence lifetimes.