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Time Correlated Single Photon Counting
(TCSPC): Examples
Prof. C. Altucci
Corso di Fisica Atomica Molecolare e Spettroscopia a.a. 2014-2015
Laurea Magistrale in Fisica
PhD School in Physics
1
What do we mean by Fluorophore “lifetime”?
Absroptiona dn emission are relative to populations of molecular
species. Generally, single molecule properties are deduced by the
measured ensemble response.
For a population of excited fluorophores we can write the rate
equation:
*
dn()
t
kn*()
t
dt
describing the variation per unit time of the number of molecules in the
excited state at the time t
2
The solution is:
*
()
t

)e
x
p
(-kt)
n
n(0
*
The lifetime  is equal to k -1
The lifetime is then the time needed for the excited molecules to decay at 1/e
(36.8%) of the initial value. t=0 is the end of the excitation pulse.
*
n (t)
t /
e
*
n (0)
3
The decay constant k stands for the sum of the constants related to all
possible decay channels.
k = kf + ki + kx + kET + …= kf + knr
•kf fluorescence decay,
•ki internal conversion decay,
•kx inter-system crossing decay,
•kET energy-transfer decay,
•knr all non-radiative decays
4
Non-radiative processes:
•
Isolated molecules in “gas-phase” undergo only internal conversion or
inter-system crossing
•
In the condensed phase additional mechanisms have to be accounted for
due to interactions with the micro-environment: chemical reactions in
the excited state, energy transfer, …
Coumarine in ethanol
has a 4 ns lifetime
Isoalloxazine in water has
a 4.5 ns lifetime
Lifetime
of
Tryptophan
in
proteins vary from
~0.1 ns to ~8 ns
5
The radiative lifetime r = kf-1 is nearly constant for a given molecule
The overall fluorescence lifetime  = k-1 = (kf + knr)-1 depends on the
micro-environment surrounding through knr.
Quantum yield for fluorescence:


k k
f
QY
 f 

k
k
f
nrk r
It is proportional to fluorescence lifetime
The addition of a further decay path, non-radiative, increases knr and
decreases  and thus QY.
The fluorescnce intensity is proportional to n*(t), I (t) = kf n*(t)
6
The Stokes shift consists in the fluorescence to occur with a photon
energy smaller (larger wavelength) than that of the excitation radiation
The emission spectra or substantially independent on the excitation
wavelength. The excess energy is rapidly dissipated (10-12 s).
The mirror rule.
7
Diffusion times in solution. The excess energy is quickly dissipated (10-12
s): x2=2D.
For example: for oxygen in water D  2.510-5 cm2/s, if the fluorephore
lifetime is 10 ns we obtain x  70 Å which is comparable with the
thickness of a biological membrane or with the linear size of a protein.
Absorption spectroscopy gives info of the ground state structure.
Fluorescence/Emission spectroscopy gives info on the excited state
structure.
8
Let’s focus on TCSPC.
How to measure the fluorescence lifetime?
Time domain
The real fluorescence decay is a convolution with
the excitation pulse profile
IR()
t I()
t
P
()
t
The measured fluorescence decay decadimento di
fluorescenza misurato ism a convolution the overall
system response
Molecules are excited by a short pulse
(ideally a function) at t = 0. The
intensity of the fluorescence decay is
usually measured by the Time
Correlated Single Photon Counting
(TCSPC)
I(t) =ex
p(-t/τ)
It
(
)

I
(
t
)

R
(
t
)
M
R
It
(
)

I
(
t
)P
(
t
)

R
(
t
)

I
(
t
)i
(
t
)
M
R
E
F
The system response function iREF is typically
measured as the response to the “direct” excitation
pulse.
9
Steady-state and time-resolved regimes
While steady-state fluorescence measurements are simple, nanosecond time-resolved measurements
typically require complex and expensive instrumentation. Given the relationship between steady-state and
time-resolved measurements, what is the value of these more complex measurements? It turns out that
much of the molecular information available from fluorescence is lost during the time averaging process.
10
Time domain
I
(
t
)

[

e
x
p
(tτ
-/)
]

i
(
t
)
M
R
E
F
I(t) parameters are usually obtained by a non-linear best-fit combined to a
deconvolution.
The deconvolution is not required when the excitation pulse is much shorter
than the lifetime to measure and/or when we do not need a high accuracy in the
lifetime determination.
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Mono-exponential decay
It
()ex
p
(
t/)
Multi-exponential (at least two distinct
lifetimes)
t
It
ie
i
i
A similar analysis is performed in case of multi-dim. Decay to extract the lifetimes τi and the weights
αi. Increasing the number of parameters in the fitting procedure reults in increasing the risk of
numerical artifacts (more than 3 lifetimes are not recommended).
Alternatively, the Method of Maximum Entropy can be used to analyze distributions of lifetimes.
Esposito R., Altucci C., Velotta R., Analysis of Simulated Fluorescence Intensities Decays by a New Maximum
Entropy Method Algorithm, Journal of Fluorescence, 23, 203-211, 2012
∑ αi
Mean lifetime – the time that a molecules spends in its excited state as
an average over the molecular ensemble
τ m=
i
τi
∑ αi
i 12
Time Correlated Single Photon Counting (TCSPC)
Simple experimental set-up for fluorescence
decay measurements with TCSPC.
Measurement of start-stop times in time-resolved
fluorescence measurement with TCSPC.
Histogram of start-stop times
in time-resolved fluorescence
measurement with TCSPC.
13
Glucose Oxidase
Red: FAD cofactors bound deep inside
the enzyme.
FAD (Flavine Adenine Dinucleotide)
*
The active site where glucose binds
just above the FAD.
This enzyme, like many other proteins, is
covered with carbohydrate chains, shown
in green. It is produced by a mould
species , the Aspergillus Niger
60 x 52 x 37 Å
14
Flavin Adenine Dinucleotide (FAD)
Flavine
“Open” configuration
“Stacked” configuration
Fluorescent site
Adenine
15
Reaction induced by Glucose
Glucose oxidation and FAD reduction
Glucose + GOD (FAD+)  gluconic acid + GOD (FADH2)
GOD (FADH2) + O2  H2O2 + GOD (FAD+)
(1)
(2)
Unlike other enzymes GOD needs an external agent (O2) to complete the cycle
(this allows one to control the reduced FAD concentration)
16
Experimental set-up
GOD
GOD+Glu
Diode laser
l= 404 nm
rep. rate= 40 MHz
<P>= 1 mW
width=80 ps
IRF~120 ps
Bandpass Filter
@520nm
FWHM:10nm
Microscope
objective
Sample
(stirring cell)
17
Effect of Glucose
GOD+GLU 1 mM
A01  32.0  0.310 3
 01  339  4 ps
A02  100.0  0.310 3
 02  3106  21 ps
signal (arb. units)
GOD + GLU 1mM
GOD
100
2
 red
 0.27
10
0
2000
4000
6000
time (ps)
GOD
GOD+GLU
FAD in oxidized form
FAD in reduced form (due to Glucose)
GOD
A01  7.0  0.1  10 3
 01  10.7  1.0 ps
A02  72.0  0.2   10 3
 02  2911  14 ps
2
 red
 0.28
18
Free FAD Lifetime
(conformational effects)
GOD+glu
GOD
The experiment with acid and basic buffers shed light on the FAD conformation in GOD!
19
Estimation of the features for sensors
(preliminary results in sol-gel)
amp2/(amp1+amp2) (arb. units)
Assuming Michaelis-Menten behaviour

S
ES  
S   K M
0
2
4
6
8
10
12
[GLU]mM
14
16
18
20
22
A1
C S 

A1  A2 S   K M
KM  1.7  0.4 mM
C  0.59  0.03
20
21
Another example: 5-Benzyluracil
A Model System for
DNA-Protein cross-link
UV LASER Pulse
@ 258 nm
5-benzyluracil
(5BU)
Dynamics and
Photocyclization of
5-benzyluracil
5,6-benzyluracil
(5,6BU)
take a brick from DNA side (Uracil) and a part from the protein side (benzene)
and study their interaction and dynamics induced by UV light.
Sun, G.; Fecko, C. J.; Nicewonger, R. B.; Webb, W. W.; Begley, T. P., “DNA-protein cross-linking: model systems for
pyrimidine-aromatic amino acid cross-linking.” Org. Lett. 2006, 8, 681–3
22
TCSPC Results
Decay Constants:
τ1= 50 ± 5 ps
Counts
1000
τ2= 1.6 ± 0.2 ns
100
Fitting function:
10
6
7
8
9
10
time (ns)
𝒇 𝒕 = 𝑨𝟏 𝑰𝑹𝑭 𝒕 ⊗ 𝑬𝒙𝒑(−
𝒕
𝒕
) + 𝑨𝟐 𝑰𝑹𝑭 𝒕 ⊗ 𝑬𝒙𝒑(− )
𝝉𝟏
𝝉𝟐
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