Fluorescence lifetime
Molecular dye probes emit fluorescent light of a particular
wavelength following the absorption of energy of a shorter wavelength.
Depending on the energetics (see Figure 1) of the dye molecule concerned, the
length of time between the absorption of a photon and subsequent fluorescent
emission of another photon can be very short (less than one nanosecond) or
rather long (more than one millisecond). For an individual molecule, this time
interval is the fluorescence lifetime of that molecule during that excitation
and emission. In biochemical assays, a large number of dye molecules are
typically excited and an average fluorescence lifetime is observed. One way to
measure this average fluorescence lifetime is to excite with a very short pulse
(perhaps 200 picoseconds) of light and then measure the rate of light emission
as a function of time. An exponential decay curve is obtained as shown in
Figure 2.
Figure 1 Fluorescence
process
Figure
2 Fluorescence intensity decay of a dye with 5ns lifetime
The
fluorescence lifetime, given the symbol t, is an arbitrary
number equal to the time after which the intensity as dropped to 1/e (1/2.718)
of its initial value. The simple equation relating the intensity(I) to time(t)
is:
It=I0.exp(-t/t)
