Fig. 3 is showing 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.
If the incident light is at a wavelength where the molecule will absorb the photon, the molecule is then excited from the electronic ground state to a higher excited state, denoted S2 here.
The electrons then go through internal conversion, affected by vibrational relaxation and heat loss to the environment. A photon is then emitted from the lowest lying singlet excited state in the form of fluorescence.
In conventional fluorescence, photons are emitted at higher wavelengths than the photons which are absorbed. This diagram is extremely important to understand fluorescence. When measuring a fluorescence spectrum, one is typically looking at the intensity at which a molecule emits, the wavelength or energy at which it emits, and also the time which the molecule spends in the excited state. This is the fluorescence lifetime, explained further in detail in coming sections.
Any number of things can affect these observables, including energy transfer to and from other molecules, quenching by other molecules, temperature, pH, local polarity, aggregation or binding. Understanding the mechanisms of these interactions can give one insight into what is being observed with a change in fluorescence spectra or lifetime.
There are two non-radiative deactivation processes that compete with fluorescence: internal conversion from the lowest singlet excited to the ground state and intersystem crossing from the excited singlet state to the triplet state. This last process leads to the phenomenon called phosphorescence, explained in further detail later on.
In simple terms, fluorescence lifetime of a molecule can be thought of as 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 fluorescence emission is generally in the picosecond to nanosecond range, while phosphorescence typically lasts for microseconds, milliseconds, or even longer…minutes or hours. A pulsed source is typically used, 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.