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CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Fluorescence Lifetimes
Martin Hof, Radek Mach

The Jablonski Diagram


The life history of an excited state electron in a luminescent probe
Internal conversion
ki ~ 1012 s-1

S2

Radiationless decay
knd > 1010 s-1

ki ~ 106
-1012 s-1

Inter-system crossing
kx ~ 104 1012 s-1

S1

kx ~ 10-1 105 s-1


Absorption

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

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.

In general, the behavior of an excited population of


fluorophores is described by a familiar rate equation:

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).

If excitation is switched off at t = 0, the last equation, takes the form:


d n* (t )
= - n* (t ) k
dt

and describes the decrease in excited molecules at all further


times. Integration gives:
*
(
t
)
=
n
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:

n*(t )
t /

e
n*(0)

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.

non-radiative processes:
isolated molecules in gas-phase only internal conversion and
intersystem crossing

in condensed phase additional pathways due to interaction with


molecular environment: excited state reactions, energy transfer,

non-radiative processes:
isolated molecules in gas-phase only internal conversion and
intersystem crossing

in condensed phase additional pathways due to interaction with


molecular environment: excited state reactions, energy transfer,

ANS in water is ~100


picoseconds but can
be 8 10 ns bound
to proteins

Ethidium bromide is 1.8 ns


in water, 22 ns bound to
DNA and 27ns bound to
tRNA

The lifetime of
tryptophan in
proteins ranges from
~0.1 ns up to ~8 ns

Note: fluorescence lifetime tends to be shorter in more polar environment, because


larger dipole moments of surrounding molecules can increase the efficiency of energy
transfer

The radiation lifetime r = kf-1 is practically a constant for a given molecule


The fluorescence lifetime = k-1 = (kf + knr)-1 depends on the
environment of the molecule through knr.
Fluorescence quantum yield:

QY

kf
k

kf knr
k
r

is proportional to fluorescence lifetime.


Addition of another radiationless pathway increases knr and, thus,
decreases and QY.
However, the measurement of fluorescence lifetime is more robust than
measurement of fluorescence intensity (from which the QY is
determined), because it depends on the intensity of excitation nor on
the concentration of the fluorophores.
The fluorescence intensity I (t) = kf n*(t) is proportional to n*(t) and
vice versa

How to measure fluorescence lifetime ???


Time (or pulsed) domain

Frequency (or harmonic) domain

intensity

a
b
A
B

Molecules are excited by a very


short pulse (close to a -pulse)
at t = 0 and the decay of
florescence intensity is
measured. Usually by Time
Correlated Single Photon
Counting (TCSPC)

I (t ) = I (0) exp (- t / )

time

Excitation light is harmonically


modulated with circular
frequency and so is the
emission. Fluorescence lifetime
can be deduced from the phase
shift and modulation m.

1
= tan

1 1
m =
1
2
m

Time (or pulsed) domain


Ideal single-exponential decay of fluorescence intensity (excited by a -pulse at t = 0)

I(t ) I(0) exp(t / )


The real fluorescence decay is a convolution with the profile of the excitation pulse

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 )

IM (t ) I(t ) P(t ) R(t ) I(t ) iREF (t )


The instrument response function iREF is typically measured as a response of the
instrument to scattered excitation pulse.
The parameters of I(t) (the lifetime ) are usually obtained by nonlinear fitting
combined with a deconvolution procedure.
The deconvolution is not necessary when the excitation pulse is very short
compared to the lifetime (fs-lasers) and/or high precision of lifetime determination
is not required. A part of the measured decay closest to the excitation pulse is
then excluded from the analysis (tail fitting).

Time (or pulsed) domain

single-exponential decay

multi-exponential decay (at least two


distinct lifetimes)

I t I 0

I(t ) I(0) exp(t / )

ie

An analogous analysis is performed in the case of multi-exponential decay to extract


lifetimes i and fractions i. An increase in the number of fitted parameters represents
increases the risk of artefacts (more than 3 lifetimes not recommended)
Alternatively maximum entropy method can be used allows analysis of continuous

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

Time correlated single photon counting (TCSPC)


monochromator /
filter

pulsed
laser

sample

trigger pulse from a reference


detector and discriminator or
from the pulse generator which
drives the laser pulses

START

TAC

monochromator /
filter
detector

STOP

discriminator

multichannel analyzer
generates an array of numbers of detected photons within short time
intervals photon arrival histogram

detector: multichannel plate photomultiplier tube (MCP PMT), avalanche


photo diode (APD)

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

the pulse timing depends on its


amplitude increases time jitter

threshold

time

Constant fraction discriminator


(1-f) I(t-)

- f I(t)

the signal is divided to two branches, the


signal in one branch is inverted and in
the other delayed and then they are
added together
the zero point used for timing
independent of amplitude

10 V

Time to Amplitude Converter (TAC)

TAC generates a linear voltage


ramp by charging a capacitor

voltage

TAC is the limiting step in TCSPC

START

the charging is started by a trigger


pulse (synchronized with the excitation
pulse)
time

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

Time correlated single photon counting (TCSPC)


monochromator /
filter

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

TAC however detects only one photon in each excitation cycle


The average number of photons reaching the detector in each excitation cycle 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 )

pi ( z 1) 1 pi (0) pi (1) 1 (1 wi ) exp(wi )


After many (NE) excitation cycles, Ni counts will be detected in the i-th interval

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

That is called the pile-up effect


To prevent the need for corrections of the measured decays for pile-up effect very low
intensities are used to make the effect negligible. The intensities are usually adjusted
to ensure that Ni is approximately 1% of NE, that means that a photon is detected
only in 1% of excitation cycles.
High repetition rates of excitation pulses are used to decrease the time necessary for
measurement. However, the fluorescence intensity has to decay completely between
the pulses repetition rates usually 1 10 MHz.
Note: an advantage of TCSPC is the known statistical distribution of noise
(Poisson distribution) and it can be included in the data analysis.

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

Time domain An alternative detection method


The decay of fluorescence can be also recorded with high temporal resolution using a
streak camera (analogous to an oscilloscope)
voltage sweep
phosphor screen

photon

photoelectron

photocathode

Modern streak cameras have time resolution superior to photomulpliers. Parallel


detection in all channels intensity is not limited by pile-up effect.

Frequency (harmonic) domain

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

Frequency (harmonic) domain - derivation


derivation of equations for a single-exponential decay:

d n* (t )
= - k n* (t ) f (t )
dt
considering the harmonic excitation:

f (t ) = A a sin(t )

we assume a solution in the form: n * (t ) = B b sin(t )

b cos(t ) = 1 B b sin(t ) A a sin(t )


to ensure that the equation is solved for all values of t, we search for such values
of phase shift and modulation m that satisfy the equality of terms containing t,
terms containing cos( t) and terms containing sin( t) on both sides of the
equation.

sin
= tan
cos

bB
= m
a A

1
1 2 2

Frequency (harmonic) domain general expressions


An integral transform of the fluorescence decay I(t) gives:

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

The excitation intensity is harmonically modulated by a Pockels cell or a


harmonically modulated LED or laser diode is used. The frequency is typically in the
range of ~10 100 MHz

An example of the use of lifetime data is given by a study of a rhodamine labeled


peptide which can be cleaved by a protease (from Blackman et al. (2002)
Biochemistry 41:12244)
N

D
A

I
C

Rho

Weak fluorescence

E1

S
V

Rho

Rho

Strong fluorescence

In the intact peptide the rhodamine


molecules form a ground-state dimer
with a low quantum yield (green curve).
Upon cleavage of the peptide the
rhodamine dimer breaks apart and the
fluorescence is greatly enhanced (blue
curve).
Lifetime data allow us to better
understand the photophysics of this
system

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

As the lifetime data


indicate, before protease
treatment the rhodamine
lifetime was biexponential
with 95% of the intensity
due to a long component
and 5% due to a short
component. Hence one
can argue that the intact
peptide exists in an
equilibrium between open
(unquenched) and closed
(quenched) forms.

E2
Hydrophobicity sensing with lifetime sensitive dyes
exc = 467 nm
100, 1.3 N.A. oil
immersion
300 300 pixels

fluorescence lifetime image of a part of a membrane of


a living hepatocyte cell stained with the dye NBD (7nitrobenz-2-oxa-1,3-diazole) lifetime is depending
on the hydrophobicity of the environment

Fluorescence lifetime

Lifetime distribution

5x104

Frequency [cps]

Fluorescence intensity

acquisition time: 2 ms/pixel

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

Financial support from the grant:


FRV 33/119970