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Fluorescence Lifetimes

Martin Hof, Radek Mach

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

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

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.

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).

d n* (t )

= - n* (t ) k

dt

times. Integration gives:

*

(

t

)

=

n

n (0) exp (- kk t )

*

The lifetime

is equal to k

-1

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)

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

molecular environment: excited state reactions, energy transfer,

non-radiative processes:

isolated molecules in gas-phase only internal conversion and

intersystem crossing

molecular environment: excited state reactions, energy transfer,

picoseconds but can

be 8 10 ns bound

to proteins

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

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

transfer

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

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

Time (or pulsed) domain

intensity

a

b

A

B

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

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

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

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 )

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).

single-exponential decay

distinct lifetimes)

I t I 0

ie

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

monochromator /

filter

pulsed

laser

sample

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

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

amplitude increases time jitter

threshold

time

(1-f) I(t-)

- f I(t)

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

ramp by charging a capacitor

voltage

START

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

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

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 )

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

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

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

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

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

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 )

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

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

harmonically modulated LED or laser diode is used. The frequency is typically in the

range of ~10 100 MHz

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

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

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

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

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

FRV 33/119970

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