5 1
Ctirad Hofr
Time-Resolved Fluorescence
Fluorescence methods in life sciences
Overview
• Definition of Time-Resolved (TR) fluorescence
• Types of TR fluorescence measurement
• Practical applications of TR fluorescence
5 2
5 3
Steady-state and Time-resolved
fluorescence
• Time-resolved fluorescence is measured using
excitation pulse (pulse length is usually shorter than
the fluorescence decay of the sample) or phasemodulated
excitation light and enables to analyze
the time dependence of the measured parameters.
• Steady-state fluorescence is measured at
excitation by continuous radiation and then we get a
time average value of intensity or fluorescence
polarization.
5 4
Why to measure time-resolved
fluorescence?
• Describes about the decrease in
fluorescence intensity in time
• Measurement is relative, does not depend
on actual intensity value
• Depends on the characteristics of
surroundings
• Informs about dynamics – rotation of
molecules
5 5
0
0.2
0.4
0.6
0.8
1
0 500 1000
time, ps
I(t)
How long the fluorescence lasts?
Random decay to the ground state:
each molecule emits 1 photon
τ=1/e=37%
The population of the excited
molecules by flash (pulse)
5 6
Time course of fluorescence
• The simplest is the exponential decline for
spherical particles
τ
t
eII t
−
= 0)(
0
0.2
0.4
0.6
0.8
1
1.2
-2 0 2 4 6 8 10 12
time (ns)
0I
I
5 7
Fluorescence lifetime τ
• τ is mean time between excitation of the molecule
(i.e. absorption of a photon) and emission of the
light when returning molecules to the ground state
What is the fluorescence intensity
in time τ after photon absorption?
τ
t
eII t
−
= 0)(
Lifetime τ = 2 ns, then in this
time t = τ fluorescence is 37% of the
original intensity in time 0
0
0.2
0.4
0.6
0.8
1
1.2
-2 0 2 4 6 8 10 12
37%
Lifetime 2 ns
37% of intensity in time 0
time (ns)
0
0.0001
0.001
0.01
0.1
1
log I(t)
0
)(
I
I t
5 8
Why to measure fluorescence
lifetime τ ?
• Absolute measurement – lifetime does not depend on
the concentration of the sample
• The lifetime is dependent on the surrounding
environment and can be used to determine its
polarity, pH, temperature, ion concentrations,
presence of quenchers
• Adds another dimension in fluorescence mapping increases
its selectivity
• Enables to measure rotational correlation time of
molecules - rotational mobility
5 9
The main ways of measuring timedependence
of fluorescence
• Pulse method (time-domain) - source of
excitation radiation is a short pulse
• The method of phase-modulated
excitation radiation (frequency domain)
- source of excitation radiation is sinusoidally
modulated light
5 10
Pulse method
• Vzorek je excitován krátkým pulzem
s trváním mnohem kratším než doba
dohasínání τ
• Závislost intenzity fluorescence na čase
(dohasínání) je použita pro výpočet doby
dohasínání τ
5 11
Counting of photons: Time-Correlated
Single Photon Counting TCSPC
time
One emitted photon can be
detected with each pulse of
excitation light.
A typical arrangement is one photon
per 100 excitation pulses.
Time between the excitation pulse
and detected photon is measured.
Histogram which illustrates the
temporal distribution of photons is
created.
Is necessary to have a set of at
least 4000 photons to evaluate a
lifetime curve.
5 12
Sources for TCSPC
Laser diodes
Pulse width FWHM = 70 ps (full width at half of
maximum)
Pulse repetition rate 40 MHz
LED (Light Emitting Diode)
FWHM = 1,4 ns (full width at half of maximum)
Pulse repetition rate 40 MHz
5 13
Detector - photomultiplier
Light Detectors: Photomultiplier Tube (R928 Side-on)
Side window
24 mm2
Photocathode
Fokusing electrode
Electron
multipliers
(dynodes)
Photon
Photoelectron
Anode
Vacuum
10-4 Pa
Provided by HORIBA Jobin Yvon
5 14
Fast detector (MCP PMT)
• It is the most important when
monitoring the time dependence of
the fluorescence
• Currently the fastest one are the
microchannel plate photomultiplier
(MicroChannel Plate
PhotoMultiplier Tube)
• They have a quick response, the
electron travels in a detector less
than 1 ns. In comparison with the
conventional photomultiplier the
response is 10 times faster.
5 15
0
2000
4000
6000
8000
10000
12000
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
TIME, CHANNELS
PHOTONCOUNTS
MCA
S
CFDSYNC
IBH 5000U
TAC rate 1MHz
Coaxial Delay 50 Ns
Sync delay 20 ns
TBX-04
nanoLED
α ≤ 2%
statistical
single photon
events
periodic pulses
Cumulative
histogram
TAC
Time to amplitude
converter
V
Principle of devices with TCSPC
Provided by HORIBA Jobin Yvon
detector
source
sample
Constant fraction
discriminator
5 16
Practical determination of τ
τ
t
eII t
−
= 0)(
37.00
1
0)( ⋅== −
IeII τ
5 17
1
10
100
1000
10000
100000
-10 -5 0 5 10
ČAS, KANÁLY
Photoncounts
1
10
100
1000
10000
100000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
TIME, CHANNELS
PHOTONCOUNTS
δ−4
δ−3
δ−2
δ−1
δ 0
decay after pulse δ
Intensity is a function of time:
I(t)=α exp (-t/τ)
Intensity of the light source
is also a function of time
L(t)
Convolution of fluorescence :
F(t)= I(t) ⊗ L(t)
Composite signal
of lifetime
Principle of composing
(convolution) signals
5 18
Time-resolved fluorescence of
fluorescein
IRF
• Fluorescein in water at pH 9.0
• Excitation 450 nm
• Pulse LED with repetition rate 20 MHz
IRF Instrument Response Function
signal of the source and response
of the instrument
IRF is the curve corresponding to
the shortest possible lifetime,
which can be measured
on the instrument
5 19
More fluorophores in the system:
multi-exponential decay
• αι proportional representation of fluorophore
• τι lifetime of given fluorophore
• The total intensity is the sum of (convolutions)
contributions from each fluorophore
• In the analysis it is necessary to use
deconvolution - signal decomposition on the
contributions of individual fluorophores
)/(exp
...
)(
21)(
21
iit
Nt
tI
eeeI N
ttt
τα
ααα τττ
−=
++=
−−−
5 20
Resolution of composing signals from
two fluorophores (deconvolution)
• In determining
contributions from
individual fluorophores,
sometimes it is not
possible to determine
precisely α and τ, because
their various combinations
can be used for the same
curve fitting
• Various pairs αι and τι can
give very similar shape of
the decay curve
5 21
Time-resolved fluorescence of
human serum albumin HSA
Fluorophore is tryptophan
There is only one tryptophan in the
protein, nevertheless the time
dependence of decay is multi-exponential.
It is caused by different conformations of
the protein in solution.
Tryptophan is found in several different
local environments. Provided by HORIBA Jobin Yvon
5 22
Method of phase-modulated
excitation radiation
1. The sample is excited with sinusoidally modulated light
with high frequency range comparable with reciprocal
value of the lifetime
2. When the sample is excited by the modulated wavelength,
emitted wavelength of fluorescence corresponds to
the modulation frequency of the absorbed excitation light
3. Emission is time-delayed in comparison with the excitation
light, resulting in a phase shift. Phase shift is used to
calculate fluorescence lifetime τ
5 23
Degrees / Time
0 2π (360°)/T
Intensity
-2
-1
0
1
2
Phase shift
Determination of lifetime using method of
phase-modulation
Φ
b
a
B
A
4π (720°)/2T 6π (1080°)/3T
modulation of excitation
b/a
(peak height / middle intensity)
Modulation of emissions
B/A
Relative modulation m
ab
AB
m
/
/
=
Determination of lifetime
from phase shift Φ
Φ=Φ ωτtan 22
1
1
m
m
τω+
=
Determination of lifetime
from modulation m
excitation light
fluorescence emission
5 24
-1.5
-1
-0.5
0
0.5
1
1.5
TIME
AMPLITUDE ( )
( )
M =
φ
0
10
20
30
40
50
60
70
80
90
1 10 100 1000
Frequency, MHz
φ
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
M
Principle of
measurement using
phase modulation
ab
AB
m
/
/
=
%
Provided by HORIBA Jobin Yvon
5 25
Example of calculation
Φ = 68°
Φ=Φ ωτtan ;
1
1
22
m
m
τω+
=
m= 0.4
ω
τ
Φ
=Φ
tan




−= 1
11
22
m
m
ω
τ
f⋅= πω 2
5 26
Dependence of parameters obtained by
method of phase modulation on τ
• The shorter lifetime τ, the
larger shift of intersection
curves to higher frequencies
• For longer lifetime
τ = 10 µs is necessary to
use the modulation
frequencies 10 kHz - 1 MHz
• For shorter lifetime
τ = 100 ps is necessary to
use the modulation
frequencies up to 2 GHz,
thus 10,000-fold higher
5 27
Time-resolved fluorescence of
fluorophores in a different environment
• Flourophores have the
same value τ in the same
environment
• After adding of quencher
the fluorecence lifetime is
reduced for the fluorophore
on the surface τ2
• The result is "bent" curve
• The task of deconvolution is
then to determine τ1, τ2 and
the relative contributions of
fluorophores α1,α2
• Protein with two
fluorophores - tryptophans
21
21)(
ττ
αα
tt
eeI t
−−
+=
5 28
Change in rotational correlation time
during the protein multimerization
• The time dependence
of the polarized
fluorescence can be
used to monitor
changes in the
dynamics of molecules
• Observation of
phosphofructokinase
multimerization
0 10 20 30 40
Čas (ns)
log r (t)
θ = 5 ns
θ = 20 ns
M
T
5 29
Fluorescence LifeTime Imaging Microcsopy
FLIM
• Observation of time dependence of fluorescence in 2D space
• It enables to distinguish an environment in which the
fluorophores are located
• It enables to observe quenching of fluorophores, either due
to environmental influences, quenchers or interactions with
other molecules
5 30
Using FLIM for monitoring protein
interaction
H. Wallrabe and A. Periasamy, Imaging protein molecules using FRET and FLIM microscopy, Current Opinion in Biotechnology, Volume 16, Issue 1, Analytical biotechnology, 2005, Pages 19-27.
Faster decay in
the presence of
quenching protein
Normal
- slow decay
5 31
Interaction or colocalization?
5 32
Example of detailed display of FLIM
Dependence of fluorescence lifetime of NADH is a twoexponential
with a value of 0.4 ns for free and 3 ns for
bound NADH molecule.
5 33
Using of time-resolved fluorescence in
proteomic analysis
Gel stained with
Sypro-Ruby
Intensity Time resolution
The intensity is supplemented with a pseudo-color time resolution to visualize
both parameters at the same time.
Time-resolved fluorescence can help in the analysis of proteins in overlapping
bands. Modified according to materials of Photonic Research Systems Ltd. www.prsbio.com
5 34
Literature
• Lakowicz J.R.: Principles of Fluorescence
Spectroscopy. Third Edition, Springer +
Business Media, New York, 2006.
• Fišar Z.: FLUORESCENČNÍ SPEKTROSKOPIE
V NEUROVĚDÁCH
http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
Graphics from the book Principles of Fluorescence was for the purpose of
this lecture kindly provided by Professor JR Lakowitzem.
Acknowledgment