In recent years fluorescence spectroscopy has become a very important tool in biology, pharmacy and medicine. Numerous bio-molecules can be observed, identified and traced by labeling with a fluorescent dye. For a long time it has been common to make cells and organelles visible by staining with dyes. Nowadays, making use of fluorescence, even the motion of single dye-labeled molecules can be observed in living cells.
Characterization of Fluorescence
On absorption of a photon a dye molecule is excited to a meta-stable state. In most dyes the absorbed energy is dissipated very fast and converted into heat. Only in relatively few dyes these non-radiative transitions are sufficiently slow so that the radiative transition from excited to ground state can successfully compete: the dye is deactivated by emitting a photon (it fluoresces).
A fluorescent dye can be characterized by the spectral properties (excitation and emission spectrum), the fluorescence quantum yield (ηfl), and the fluorescence decay time (τfl). The fluorescence of a dye is independent of the wavelength of excitation.
The spectral properties of a dye are determined by the molecular structure. Absorption in the visible spectral range (400 - 700 nm) requires a corresponding energy gap between the ground and excited state. The striking feature of dye structures is a conjugated pi-electron system.
In most cases the fluorescence spectrum is nearly a mirror image of the long-wavelength absorption band. The fluorescence is typically shifted by 25 - 40 nm to the red relative to the main absorption band. This so-called Stokes-Shift is caused mainly by reorientation of the solvent molecules surrounding the dye. The spectral properties are often used to distinguish and identify different dyes in a sample.
Fluorescence Quantum Yield (ηfl)
One of the most important properties of a dye is the fluorescence quantum yield (ηfl). The quantum yield is defined as the quotient of the number of emitted (nfl) and the number of absorbed photons (nabs).
ηfl = nfl / nabs
It should be noted that the quantum yield can never exceed 100%.
Obviously a high quantum yield is beneficial if a dye is to be observed via fluorescence.
Fluorescence Decay Time (τfl)
The emission of a photon is a statistical process. Therefore the time an excited molecule remains in the excited state is also a statistical quantity. However, in an ensemble of identical molecules the observed decay statistics is well-defined. In the simplest case the number of molecules in the excited state decreases exponentially after exciting an ensemble by a short pulse. The time interval, after which the number of molecules (n1) in the excited state has decreased to 1/e (about 37%), is called fluorescence decay time (τfl).
n1(t) = n1(0) exp(- t / τfl)
The fluorescence decay time is an important property of dye molecules and can be used to identify a dye. Typical values for τFl are in the range of nanoseconds.
The fluorescence decay time as well as the fluorescence quantum yield is not a fixed quantity. They depend on the environment, e.g. solvent, temperature and other factors. Furthermore, the decay time and quantum yield are not independent quantities. They are linked by the relation:
τfl = τ0 × ηfl
τ0 is the so-called natural decay time, where the quantum yield is assumed to be 100% (complete absence of non-radiative deactivation).
Therefore a change in the decay time can be used to determine a change in the local environment of the dye molecule. Numerous experiments have been carried out to study quenching effects on dye molecules. This phenomenon can be used especially to develop switchable dyes or intelligent probes (e.g. the GenePins).