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Fluorescence Lifetimes
Martin Hof, Radek Mach
S2
Radiationless decay
knd > 1010 s-1
ki ~ 106
-1012 s-1
Inter-system crossing
kx ~ 104 1012 s-1
S1
Fluorescence
kf ~ 107 109 s-1
T1
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
d n* (t )
*
= - k n (t ) f (t )
dt
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).
The lifetime
is equal to k
-1
n*(t )
t /
e
n*(0)
non-radiative processes:
isolated molecules in gas-phase only internal conversion and
intersystem crossing
non-radiative processes:
isolated molecules in gas-phase only internal conversion and
intersystem crossing
The lifetime of
tryptophan in
proteins ranges from
~0.1 ns up to ~8 ns
QY
kf
k
kf knr
k
r
intensity
a
b
A
B
I (t ) = I (0) exp (- t / )
time
1
= tan
1 1
m =
1
2
m
IR (t ) I(t ) P(t )
The measured fluorescence decay is a convolution of the real decay with the response
of the detection
IM (t ) IR (t ) R(t )
single-exponential decay
I t I 0
ie
distributions of lifetimes.
2
Mean lifetime an average time a
molecule spends in the excited state
t I(t ) dt
0
I(t )dt
0
i
i
pulsed
laser
sample
START
TAC
monochromator /
filter
detector
STOP
discriminator
multichannel analyzer
generates an array of numbers of detected photons within short time
intervals photon arrival histogram
Discriminator
eliminates noise (dark counts of the photodetector) and generates pulses which are
independent of the actual shape and amplitude of the detector pulse (which is
generated when a photon hits the detector)
Leading edge discriminator
voltage
threshold
time
- f I(t)
10 V
voltage
START
STOP
50 ps
the charging is stopped by a pulse from the detector (photon arrival) and the
reached voltage is stored by the multichannel analyzer.
if no photon is detected TAC is reset when reaching the maximum voltage
TACs are usually operated in reverse mode:
the charging is triggered by photon arrival and stopped by the excitation
pulse
the capacitor is charged in those excitation cycles when a photon is detected
sample
monochromator /
filter
voltage
reference pulse
pulsed
laser
STOP
detector
TAC
time to
amplitude
convertor
START
discriminator
value of voltage
reached
multichannel analyzer
generates an array of numbers of detected photons within short time intervals
photon arrival histogram
TCSPC - Artefacts
If more photons arrive within a single time interval (ti + t) after excitation, only a
single count is registered the discriminator does not take into account the size of
the pulse from the detector once it is larger than the discrimination level
The average number of photons wi reaching the detector with each interval (ti + t)
should be less then one
TCSPC - Theory
Consider that within one excitation cycle in the time interval (ti + t) after excitation
(which corresponds to the i-th channel of the multichannel analyzer) on average wi
photons reach the detector.
The probability of z photons reaching the detector in that interval is given by Poisson
z
distribution:
w
pi ( z )
Specifically:
z!
exp(wi )
pi (1) wi exp(wi )
pi (0) exp(wi )
Ni NE pi (1) pi ( z 1)
Low intensities are used in TCSPC, therefore wi << 1 and:
pi (1) wi
pi ( z 1) wi
Ni N E w i w i N E w i w i
The number of counts in the i-th interval is indeed proportional to the intensity in the
interval (ti + t).
TCSPC - Theory
TAC however detects only one photon in each excitation cycle
The actual number of counts NSi stored in the i-th channel of the multichannel
analyzer is lower than Ni.
NSi Ni
1
1
NE
i 1
N
j 1
Here are pulse decay data on anthracene in cyclohexane taken on an IBH 5000U
Time-correlated single photon counting instrument equipped with a LED short
pulse diode excitation source.
= 4.1ns
2 = 1.023
56ps/ch
photon
photoelectron
photocathode
bB
m=
aA
intensity
a
b
A
1
= tan
1 1
m =
1
2
m
B
time
The frequency domain measurement does not provide a direct information on the
shape of the fluorescence decay
The equality of and m indicates single-exponential decay. If they are not equal,
more general expressions have to be used.
High excitation intensity can be applied to shorten the measurement time
d n* (t )
= - k n* (t ) f (t )
dt
considering the harmonic excitation:
f (t ) = A a sin(t )
sin
= tan
cos
bB
= m
a A
1
1 2 2
I(t ) cos(t ) dt
0
I(t ) dt
1
i i
i i
i 1 2 2 m cos
i
i i
i i
i 1 2 2 m sin
i
I(t ) sin(t ) dt
0
I(t ) dt
D
A
I
C
Rho
Weak fluorescence
E1
S
V
Rho
Rho
Strong fluorescence
Lifetime data for two rhodamine isomers (5 and 6) linked to the peptide
D
A
I
C
Rho
Weak fluorescence
S
V
Rho
Rho
Strong fluorescence
E1
E2
Hydrophobicity sensing with lifetime sensitive dyes
exc = 467 nm
100, 1.3 N.A. oil
immersion
300 300 pixels
Fluorescence lifetime
Lifetime distribution
5x104
Frequency [cps]
Fluorescence intensity
4x104
3x104
4
2x10
1x10
6
8
10
Lifetime [ns]
12
14
Acknowledgement
The course was inspired by courses of:
Prof. David M. Jameson, Ph.D.
Prof. RNDr. Jaromr Plek, Csc.
Prof. William Reusch