FluorescenceLifetimes培训课件.ppt

What is meant by the “lifetime” of a fluorophore???
Absorption and emission processes are almost always studied on populations of molecules and the properties of the supposed typical members of the population are deduced from the macroscopic properties of the process.
If excitation is switched off at t = 0, the last equation, takes the form:
d n* (t) dt
=
- n* (t)
k
Hale Waihona Puke and describes the decrease in excited molecules at all further times. Integration gives:
n* (t) = n* (0) exp (- kk t)
The lifetime is equal to k -1
If a population of fluorophores are excited, the lifetime is the time it takes for the number of excited molecules to decay to 1/e or 36.8% of the original population according to:
S1
Internal conversion ki ~ 1012 s-1
Radiationless decay knd > 1010 s-1
Inter-system crossing kx ~ 104 – 1012 s-1
kx ~ 10-1 – 105 s-1
T1
Absorption
Fluorescence kf ~ 107 – 109 s-1
In general, the behavior of an excited population of fluorophores is described by a familiar rate equation:
d n* (t) dt
=
- k n*(t) f (t)
where n* is the number of excited elements at time t, k is the rate constant of all deexcitation processes and f(t) is an arbitrary function of the time, describing the time course of the excitation. The dimensions of k are s-1 (transitions per molecule per unit time).
Fluorescence Lifetimes Martin Hof, Radek Macháň
The Jablonski Diagram
The life history of an excited state electron in a luminescent probe
S2
ki ~ 106 1012 s-1
ANS in water is ~100 picoseconds but can be 8 – 10 ns bound to proteins
molecular environment: excited state reactions, energy transfer,…
non-radiative processes: • isolated molecules in “gas-phase” only internal conversion and
non-radiative processes: • isolated molecules in “gas-phase” only internal conversion and
intersystem crossing • in condensed phase additional pathways due to interaction with
n*(t ) n*(0)

et /
The deexcitation rate k is the sum of the rates of all possible deexcitation pathways:
k = kf + ki + kx + kET + …= kf + knr
kf is the rate of fluorescence, ki the rate of internal conversion and vibrational relaxation, kx the rate of intersystem crossing, kET the rate of inter-molecular energy transfer and knr is the sum of rates of radiationless deexcitation pathways.
Phosphorescence kph < 106 s-1
S0
• Fluorescence is observed if kf ~> ki + kx
• The time a molecule spends in the excited state is determined by the sum of the kinetic constants of all deexcitation processes
intersystem crossing
• in condensed phase additional pathways due to interaction with molecular environment: excited state reactions, energy transfer,…