Photochemistry and Photobiology, 1998, 67(5): 475-486
The Correct Use of “Average” Fluorescence Parameters
Alain Sillen and Yves Engelborghs*
Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit Leuven, Leuven, Belgium
Received 11 August 1997; accepted 16 January 1998
ABSTRACT
When more than one fluorophore is present or when one
fluorophore displays a multiple exponential decay “av-
erage” fluorescence parameters are derived, which can
be combined with “average” lifetimes for further inter-
pretation. However, two kinds of average lifetimes are
used in this context: the intensity and the amplitude av-
erage lifetime. In this paper the different average param-
eters are carefully analyzed and their “best” combina-
tions are derived. These average parameters are ana-
lyzed in the context of external and internal dynamic and
static quenching, Foster energy transfer and the calcu-
lation of the radiative rate constant. The use of the am-
plitude average lifetime for the analysis of multiple fluo-
rophore-containing systems and the detection of inter-
actions is discussed.
INTRODUCTION
Fluorescence measurements are important tools for the anal-
ysis of biological systems as shown by an increasing number
of applications. The correct use of the fluorescence param-
eters is essential for the understanding of their relevance for
the biological system under investigation. The theory of fluo-
rescence parameters is well developed for the monoexpo-
nential decay of fluorescence. In this case simple equations
have been derived that describe situations as dynamic and
static quenching, fluorescence energy transfer and its dis-
tance dependency and radiative and nonradiative processes.
However, in biological systems the situation is usually more
complicated. The fluorescence decay is almost always mul-
tiexponential, due to heterogeneity of the fluorophores or of
their environment. Very often the fluorescence of heteroge-
neous systems is analyzed in the same way as a simple sys-
tem, At a first glance this seems to be justified because the
complex fluorescence behavior of the system can be de-
scribed by an average fluorescence lifetime. However, this
is not always true. Moreover, two different types of average
lifetimes can be calculated (the amplitude and the intensity
average lifetime).
In this manuscript we analyze the meaning of the different
*Author to whom correspondence should be addressed at: Labora-
tory of Chemical and Biological Dynamics, Katholieke Univer-
siteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium.
Fax: 32-16-327982; e-mail: yves.engelborghs@fys.kuleuven.ac.be
0 1998 American Society for Photobiology 0031-8655/98 $5.00+0.00
475
parameters obtained by using these average lifetimes, and
we indicate which lifetime is to be used dependent on the
situation to be analyzed.
When studying dynamic quenching, using an externally
added quencher, we show that the use of the amplitude av-
erage lifetime leads to a linear Stern-Volmer plot at low
quencher concentrations, and the average collisional quench-
ing constant (k,) reflects closely the kq of the major com-
ponent.
A decrease of the amplitude average lifetime upon
quenching is usually attributed to dynamic quenching. How-
ever, for a heterogeneous system, the amplitude average life-
time can also be changed by static quenching. This is the
case when the static quenching is selective for some of the
components of the heterogeneous system and changes the
amplitude ratio. This situation can only be identified by the
inspection of the individual lifetimes and amplitude frac-
tions. In analyzing heterogeneous fluorescence in the ab-
sence of lifetime data one has to be aware of this.
For the calculation of the average radiative rate constant
(kr) only the amplitude average lifetime can be used. It
should be noted, however, that in a heterogeneous system
the value of k, can be underestimated by the presence of
static quenching.
The amplitude average lifetime is also a very useful tool
in the analysis of interactions between fluorophores, e.g. the
different tryptophan residues within a protein. The amplitude
average lifetime of a multitryptophan protein is identical to
the amplitude average lifetime calculated from the lifetime
data of the individual tryptophan residues, provided no en-
ergy transfer occurs. If energy transfer does occur between
a pair of tryptophan residues, this pair can be identified.
DEFINITIONS
Multiexponential fluorescence decay F(t,X) is described as a function
of time (t) and wavelength (A) by the following equation:
F(t,X) = I(X)z a,(k)exp(-t/T,) (1)
where I(X) is the total amplitude, a,(X) are the amplitude fractions
and T , are the fluorescence lifetimes. Fluorescence lifetime analysis
gives only the amplitude fractions, because I(h) is normalized.
On measuring fluorescence lifetimes it is possible to calculate
different average lifetimes, the two most commonly used are the
intensity average lifetime and the amplitude average lifetime.
Intensity average lifetime. Because the fluorescence intensity con-
tribution of a component is proportional to the product a?, the in-
z
tensity average lifetime is defined as ( T ) ~= a,T,%a,T,. The intensity
average lifetime is the average amount of time a fluorophore spends
in the excited state (1). This average is given by
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476 Alain Sillen and Yves Engelborghs

Table 1. Dynamic and static quenching parameters

7 a Kq K,

Fig. 1 5 0.7 3 0
1 0.3 0.5 0
Fig. 2 5 0.7 3 0
1 0.3 0.5 0
Fig. 3 1 0.6 4 3
0.2 0.4 0.5 1

is the preferred parameter to be used. Only for the calcula-

-
--
c a,exp(-t/T,) dt c a,7,‘
(T)f = ~ (2)
The intensity average lifetime can also be defined as the
average lifetime of a collection of different excited-state
populations, where the lifetime of each population is weight-
ed by the relative contribution of that population to the total
fluorescence.
Amplitude average lifetime. The amplitude average life-
time is defined as ( T ) ~= 2 a,Tlfi a, and because 2 a, = 1,
this equals 2 a,T,. This is the lifetime a fluorophore would
have if it had the same steady-state fluorescence as the fluo-
rophore with several lifetimes (2,3). The weight factor for
the calculation of this average is the amplitude fraction a,.
The same definitions of average lifetimes apply when the
time-dependent fluorescence is described by a lifetime dis-
tribution (e.g. a discrete distribution as given by maximum
entropy analysis or an analytical distribution by fitting to a
gauss equation). The problem with the average lifetimes is
that it is not always clear which one is to be used and in the
literature they are both used in diverse conditions. Therefore
six examples are worked out to investigate the exact mean-
ing of the average lifetimes in the appropriate circumstances.
In most cases it turns out that the amplitude average lifetime
18
16 4 are measured, then an average K,, is obtained. The question
tion of the average collisional quenching constant the inten-
sity average lifetime has to be used. Our analysis also shows
how in heterogeneous systems a distinction between dynam-
ic and static quenching can be made.
RESULTS AND DISCUSSION
1. Analysis of dynamic fluorescence quenching by the
Stern-Volmer equation
Because dynamic fluorescence quenching is a process par-
allel to the normal way of decay, the rate constants of the
two processes have to be added and k = ko + kq[Q] where
k is the rate constant of decay in the presence of quencher,
ko the constant in the absence of quencher and k, is the
collisional quenching constant, [Q] is the concentration of
quencher. This leads immediately to the Stern-Volmer equa-
tion for dynamic quenching for one lifetime:
(3)
where Fo (F) is the steady-state fluorescence intensity in the
absence (presence) of quencher, T~ (7) is the fluorescence life-
time without quencher (with quencher), the product T&, is the
Stern-Volmer constant K,,, K,, is thus the ratio of kq/k,,.
If the time dependence of the fluorescence intensity has
to be described by several lifetimes, it is possible that kq
differs for each lifetime component, even if they are derived
from a single fluorophore (4).If only steady-state intensities
is, what is the meaning of this average K,, obtained by
steady-state measurements. To investigate this we made the
following theoretical analysis. The average lifetime that is
proportional to the steady-state intensity is the amplitude av-
erage lifetime (3) (no static quenching). Therefore, for het-
erogeneous systems the ratio of Eq. 3 can be redefined as

where I is the total amplitude, a is the amplitude fraction

and subscript 0 indicates absence of quencher (in absence of
static quenching = I). For a simulation we take the life-
times, amplitude fractions and k, values in Table 1 and plot
I I I I T d T and (?o)/(T) as a function of quencher concentration (Fig.
00 02 04 06 08 10 1). In Fig. 1 the dotted lines deviate from linearity meaning
[Ql that the “average kq” obtained by plotting the amplitude
Figure 1. Stern-Volmer plots without static quenching of T, ( T ) ~ ,
average lifetime or the intensity average lifetime is nonlin-
the amplitude average lifetime and ( T ) ~ the
, intensity average lifetime early dependent on the quencher concentration, when the
(see Table 1). Key to plot: - T ~ .- - T ~ .,. . ( T ) , and -.-. ( T ) ~ . system follows the simple collisional quenching model,

which is not always the case due to transient effects (5). The
equation for quenching of populations of fluorophores where
one fluorophore is totally inaccessible for the quencher is
rewritten and adapted from Lehrer (6) for the general case
of different fluorophores with different accessibilities:
(70)a= (70)a
(5)
(?)a a01701
? 1 + kql~OSQ1
This gives a deviation from a straight line in the Stern-
Volmer plot. However Eq. 5 is not in the Stem-Volmer form
y = 1 + f([Q]), so it does not help to understand what
information can be obtained from the Stem-Volmer plot for
the heterogeneous case.
To obtain the equations of the dotted lines of Fig. 1 the
different average lifetimes are calculated. (Derivation: see
Appendix 1) The equation using the ratio of amplitude av-
erage lifetimes and by introducing intensity fractions f, =
a,TIa a,T, turns out to be
and can be written as
(7)
where ( )fq means an average Ksv weighted with the intensity
fraction in the presence of quencher.
Note that this intensity average Stern-Volmer quenching
“constant” is a function of [Q]. In case the intensity average
lifetime is used, instead of the amplitude average lifetime,
to characterize the quenching (4,7),the equation turns out to
be much more complex and very difficult to interpret (see
Appendix 1).
Collisional quenching constant. For a single lifetime, the
Stern-Volmer constant Ksv can be used to calculate a bi-
molecular quenching constant kq. For heterogeneous sys-
tems, some authors (8) derive an apparent bimolecular
quenching rate constant (kJm from the steady-state Stern-
Volmer constant by dividing Ksv by the intensity average
lifetime (To)fo obtained in the absence of quencher:
To investigate the quality of this (k& we plot (kq)m as a
function of quencher concentration, (k,), being calculated on
the basis of the (Ksv)fqusing Eq.7:
and this is compared with the (kq)fqsuggested by Eq. 6:
Fig. 2.
are plotted in
These quenching constants and (KSV)fq/(~)a
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Photochemistry and Photobiology, 1998, 67(5) 477
0 0 0 2 0 4 0 6 0 8 1 0
I0 I
Figure 2. Plot of the apparent quenching constants as a function of
the concentration quencher. Key to plot: - 7 , . - - T ~ ., . . KSv/(&,.
-.-. Ksv/(~)aand Ksvl(~)f.
The (k& “constant” varies from 2.9 to 1.7 while the
(kJfq only varies from 2.9 to 2.6 in changing the quencher
concentration from 0 to 1 M. However to use the (kq)fq de-
rived from Eq. 6 it is necessary to know all r values at
different quencher concentrations that of course allows the
determination of the individual kT If this information is not
available one can measure (Ksv)fqnear [Q] = 0; there Eq.
10 has nearly the same value as Eq. 9, so a good value of
(kJm can be obtained. For the calculation of the average
collisional quencher constant the use of the intensity average
lifetime is clearly justified.
2. Analysis of fluorescence quenching in the presence of
a static component
Static quenching is the loss of fluorescence intensity due to
the inability of a fluorophore to emit light. This can occur
upon the formation of a nonfluorescent complex before ex-
citation. In case of static quenching a simple equation is
derived for one lifetime (1):
F O E= (1 + KSV[Ql)(l + Ks [QI). (1 1)
In this equation K, is the binding constant of the quencher
to the ground state. Diagnostic for such a situation is the
difference between the ratios: F,JF and rdr.
When multiple lifetimes are present the analysis is much
more complex. The derivation of the equations is done in
Appendix 2 and the final result is
(12 = 74)
with yI = aoi - q the difference in amplitude fraction due
to static quenching. This difference can only be obtained by
measuring the amplitude fraction of the individual lifetimes
in the presence of quencher. Equation 74 is a modified form
of the Stern-Volmer equation. However in the case [Q] 0
the correction term containing y can be neglected in com-
-

478 Alain Sillen and Yves Engelborghs
12
14 I possible to obtain an average k, by dividing the quantum
I I I I
0.0 0.2 0.4 0.6 0.8
[Ql
Figure 3. Stern-Volmer plots with static quenching of T, ( T ) ~ , the
amplitude average lifetime and ( T ) ~ , the intensity average lifetime
(see Table 1). Key to plot: - T , , = T ~ ., . . ( T ) ~ , -.-. ( T ) ~ , 0 Fa, the
steady-state fluorescence calculated with the amplitude average life-
time and A Ff, the steady-state fluorescence calculated with the in-
tensity average lifetime.
parison with (Ksv)f [Q] and a, = a,, so that Eq. 11 is ob-
tained. But at higher concentrations of quencher the analysis
is not straightforward.
Examples. In order to investigate further Eqs. 6, 7 and 74
a simulation is performed. Different amplitude fractions are
combined with different lifetimes (Table 1) and the behavior
of (70)=/(7), and (70)f/(7)f is obtained as a function of [Q], first
with dynamic quenching only (Fig. 1) and subsequently for
dynamic and static quenching (Fig. 3).
Figures 1 and 3 show at low [Q] that the ratio of the
average lifetimes follows the lifetime with the largest am-
plitude fraction. The curve of the intensity average lifetime
has the tendency to have a larger curvature than the ampli-
tude average lifetime and the deviation from linearity starts
at lower quencher concentrations for the intensity average
lifetime than for the amplitude average lifetime.
3. Average radiative rate constant
For a single lifetime the radiative rate constant (k,) is ob-
tained by dividing the quantum yield by the lifetime if there
is no static quenching component. If a static quenching com-
ponent is present the measured quantum yield has to be cor-
rected for static quenching to obtain the real quantum yield.
The same question arises here as in the previous case: when
there are multiple lifetimes, what average lifetime is the best
to use? The additional problem here is that average lifetimes
are wavelength dependent and the radiative rate constant is
wavelength independent. Therefore dividing by an average
lifetime will give a wavelength-dependent rate constant (9).
To solve this problem a new amplitude fraction a is defined
that is independent of wavelength.
/ Ii(X) dh
-
/ &(A) dh
a,=
9
J
I=I
Il(h) dh - I
J
I(h) dh
(13 = 81)
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where I(X) is the total amplitude, A is the wavelength, n is
the number of lifetimes and 1 is the counter. With this it is
yield by the amplitude average lifetime. For the derivation
see Appendix 3, the result is
(14 = 8 5 )
This (k,) is the absorption weighted average k,. Using the
intensity average lifetime yields a much more complicated
formula (Appendix 3).
4. Comparison of a system with multiple fluorophores
1 .o
and multiple lifetimes with a system containing one
fluorophore with multiple lifetimes
When comparing the lifetimes of a heterogeneous system
with multiple fluorophores with its components, e.g. a mul-
titryptophan protein with its single tryptophan-containing
mutants, the situation can become very complicated. In some
cases the single tryptophan-containing mutants display the
same classes of lifetimes as the wild type. This is the case
for colicin (4). The question that can be asked is whether
additivity can be used to explain the lifetime data of the
wild-type protein. In this context we found it simplifying to
use an average lifetime (4). Salient features are more appar-
ent in the average lifetime than in the full details of all the
lifetimes and fractions. The question again arises which av-
erage is the best one to be used? Here there are m fluoro-
phores (1, . . . j, . . . m) that all have n lifetimes (1, . . . i,
. . . n). The ith observed lifetime is an average of the ith
lifetime of each fluorophore, appropriately weighted (see
Appendix 4). The amplitude fraction in these averages is
weighted by the molar absorption coefficient (E) divided by
the average intrinsic lifetime (7J (this is equal to the inverse
of the average radiative rate constant; see Appendix 4).
The equation of the intensity average lifetime is
(15 = 102)
The equation of the amplitude average lifetime is
L
J
Supposing that all the tryptophans have the same E, Eqs. 102
and 104 simplify to the simpler forms without E. For mul-
titryptophan-containing proteins, the values obtained by us-
ing Eq. 102 will be approximately the same as Eq. 104 be-
cause both equations are used to calculate an average of
lifetimes that are very similar. By calculating these average
lifetimes obtained from individual tryptophans and comparing
with wild type we could clearly demonstrate the presence of
energy transfer between a particular tryptophan pair (4).

5. Energy transfer calculations in case of multiple
lifetimes (single donor, single acceptor)
According to Foster's classic theory (lo), the rate constant
of energy transfer (kT) between two fluorophores, donor D
and acceptor A, can be calculated by
where T" is the lifetime of the donor in the absence of the
acceptor, R is the distance between the donor and the ac-
ceptor and & is the distance at which 50% energy transfer
occurs, which depends on the overlap of the emission spec-
trum of the donor and the absorption spectrum of the accep-
tor, the orientation factor, the quantum yield of the donor
and the refractive index of the medium. The efficiency of
radiationless energy transfer, E, can also be calculated by
where T~~ is the lifetime of the donor in the presence of the
acceptor. When donor and acceptor display multiple life-
times, according to Wu and Brand (1 1) the average lifetime
to be used in Eq. 18 is the amplitude average lifetime. Thus
analogously to Appendix 1 this is
This is the intensity average efficiency. The average rate
constant for energy transfer can be calculated according to:
The efficiency of radiationless energy transfer can also be
calculated by measuring the acceptor lifetimes instead of the
donor lifetimes; this has, e.g. been done in the case of for-
ward and reverse energy transfer (12,13).
If the donor and acceptor have only one lifetime (in each
others absence), the efficiency of energy transfer in each
others presence is (Appendix 5)
In this equation the amplitude average lifetime of the accep-
tor in the presence of the donor ( ( T ) ~ is
~ )used because (due
to the presence of the donor) the acceptor displays a mul-
tiexponential decay, while in the absence of the donor it has
only one lifetime (TA).
If the donor and the acceptor have multiple lifetimes, then
applying Eq. 129 with the amplitude average lifetime for the
acceptor lifetime will yield an average energy transfer effi-
ciency (e.g. for two lifetimes):
where aDis the amplitude fraction of the donor and a* is the
amplitude fraction of the acceptor, which can be rewritten
as:
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Photochemistry and Photobiology, 1998, 67(5) 479
= ap(El) + a:(E,) = (E)A. (23 = 134)
This average energy transfer efficiency is different from the
one calculated with the donor lifetimes. Only when the do-
nor and the acceptor have one lifetime are all the calculated
efficiencies the same. A simplification can be introduced
when R, is the same for every kT. Then (R) can be calculated
with the efficiency obtained from the donor lifetimes:
(24)
or with the efficiency for the acceptor lifetimes:
and both should be equal.
6. Analysis of the nature of quenching upon a
conformational change of a protein or a mutation
When the fluorescence of a protein decreases as a conse-
quence of a conformational change, or as a consequence of
the introduction of a mutation, the question arises whether
this phenomenon is due to a static or a dynamic type of
quenching. Even the static quenching may be due to a dy-
namic quenching on a very short timescale as described by
Webber (3). To answer this question we compare the ratios
of the intensities and the ratios of the amplitude average life-
times of the two conformations (Derivation see Appendix 6).
In order to calculate the fraction of static and dynamic
quenching we can use the following equations. The reduc-
tion factor of the total amplitude due to static quenching
~ 3 (sq,)) is
2-
[l - = sqa
where subscript 0 indicates again the absence of quenching.
This fraction is calculated by taking the ratio of the total
amplitudes. The intensity (I) can be calculated according Eq.
135. The reduction factor of intensity due to static quenching
(sqf) is
This is calculated taking into account the amplitude average
lifetime and the fact that lifetimes are not affected by static
quenching.
The reduction factor of intensity due to dynamic quench-
ing, not taking into account static quenching (dqa) is
(1 - &) = dq,.
This is calculated by the ratio of the amplitude average life-
time taking into account that amplitude fractions are not af-
fected by dynamic quenching.
A second reduction factor due to dynamic quenching is
defined taking into account static quenching (dqf):
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480 Alain Sillen and Yves Engelborghs

APPENDIX 1: ANALYSIS OF DYNAMIC

If we interpret the different lifetimes as belonging to differ-
ent species i.e. [SAEMS]? = [DAS] then we can calculate
the fraction of static and dynamic quenching of every spe-
cies. The equation for static quenching of each species be-
comes
1
QUENCHING OF AVERAGE LIFETIME
COMPONENTS
Analysis using the amplitude average lifetime
For simplification of the equations kq [Q] is abbreviated by
k; in these appendices. The Stern-Volmer equation for one
lifetime is (Eq. 3):

I (lai> I (34)
Il--l= Sqai
L (10aoi)J by multiplying with a,l.~l.~ol,
this can be rewritten as
and the equation for dynamic quenching: a01701= a0171 + a0171701 kii. (35)
Writing a similar equation for a second lifetime 72 with
summing and taking into account that in the absence of static
quenching a, = a leads to
It is interesting to calculate the total intensity loss due to (70)a = (7)a + a,l71701k;, + a0272702 k;27 (36)
quenching. First the intensity loss by static quenching AFsq
which can be rewritten in the ratio form
is calculated:
- - -
(70)a + aO171'olk;, + a02'2T02kb2
(37)
AFsq = Fosqf (32)
(7)a a0171 + a0272
If AFsqi s known, it is possible to calculate the intensity loss and be generalized to
due to dynamic quenching AFdq:

In order to calculate these losses for the different species Eq. where means the average weighted with the intensity
32 can be used if F, is replaced by Foi(=fiFo). To calculate fraction, or
the intensity loss due to dynamic quenching it is important to
1 -
calculate the intensity fraction of the species already corrected _ --1 + -.IT0k;)f (39)
for static quenching: fi = ( ~ T ~ ~ ) /4( X ~and
~ then
~ use
) Eq. 33. (7)a (70)a (70)a

Note: (1) All the considerations above are valid if there
is no shift in the fluorescence spectrum and if there is no
change in k,; if there is a shift it is best to use F, mar for the
calculations. (2) If the quenching of the species is calculated
it is necessary that F, max of that species is used (see DAS).
If Q/Qo # F/Fo this is an indication that it is better to use
FA,,,. (3) It is also possible to use the quantum yield to
calculate the quenching. The apparent change in k, is then
due to static quenching.
Analysis of dynamic quenching of a set of different
lifetimes in the presence of static quenching. If there is a
contribution of static quenching, then a, is not equal to a,
therefore a correction, y (dependent on [Q]) is introduced so
that a,, = a y. Applying this to Eq. 36 yields
(70)a = (a171+ W z ) + (7'171+ Y272) f a0171701k;i+ a0272702k;2
(40)
or

CONCLUSION (7o)a = (7)a + (~171 + ~272)+ a o l ~ l ~ ~ l + (41)

k ;a0~72702kh~
,
which can be written as
In all cases we have studied we found that it is better to use
the amplitude average lifetime, except for the calculation of (70)a
- - - + ( Y I ~ I+ 7'272) + ~ O I ~ I ~ O a0272702k;~
I ~ ~ I
(42)
(kq) from the Stern-Volmer constant. The amplitude average (4, a171 + a272 a171 + a272
lifetime is also to be used when studying conformational
or
changes, e.g. protein denaturation (14). This is because the
amplitude average lifetime is proportional to the fluores- 1 -
_ --l + (?IT1 + Y272)
cence intensity and this is what is directly measured in (4, (TO)~ (alT1 + a272)(a01701 + a027021
steady-state fluorescence measurements. The amplitude av-
erage lifetime can also be used to diagnose energy transfer
in multiple fluorophore systems.
which after switching of the last two terms can be general-
Acknowledgement-The authors thank R. Vos for useful discussions
about the additivity of fluorescence lifetimes. ized to

?Abbreviations: DAS, decay-associated spectra; SAEMS, species-

associated emission spectra.
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Photochemistry and Photobiology, 1998, 67(5) 481

Alternatively Eq. 41 can be rewritten by introducing a. = a We multiply with the amplitude fraction a,, and write an
+ y in the last term as analogous equation for a second lifetime ( T ~ ~ sum
) , up and
divide by C c~,,T~:
_1 -- - 1 +-(T0k;)f [C Yi(Ti +
TiT~ikki)]
(45)
(T)a (70)a (T0)a (T)a(TO)a

and by introducing Eq. 48

In the absence of static quenching, when y = 0 Eq. 44 and

Eq. 46 can be simplified and combined to

(47)

For the calculation of y see further the section about static

quenching.

Analysis using the intensity average lifetime

In order to calculate the intensity average lifetime we start
rewriting Eq. 34:
701 = 71 + TlTo1k;l (48)
and square
Eq. 51 can be written as (with a, = a, in the absence of
7'81 = 7: f 2T:'Tol kil + T:T81ki?. (49) static quenching):

that can be generalized to Analysis of dynamic quenching of a set of different

lifetimes in the presence of static quenching
(54)
In presence of static quenching y is introduced in Eq. 53 so
and rewritten as that a, = a +y.

and finally because T = 1/(1/~0+ k;)

1

1 1 generalizing leads to

(56)
finally resulting in
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482 Alain Sillen and Yves Engelborghs

- -
(70)a
- 1 + M,,)f[QI. (68)
(T)a

If there is static quenching the situation becomes more com-

(C (7, - (T)f)YIT,) plex. Rearranging Eq. 62 leads to
(59)
+ (xaol~ol)(~)f(~o)f
. 10,= 1, + 1,KJQI
APPENDIX 2: ANALYSIS OF STATIC and summation over every i
QUENCHING OF AVERAGE LIFETIME C 101 = C (1, + IlKS,[Ql)
COMPONENTS
or
Calculation of y
To calculate y as function of [Q] we start with the Stern-
10 = 1 + c I,Ks,[Q1 = I +1 a,K,,[QI
Volmer equation for static quenching: or

_
Fo -- 1 + Ks[QJ. (60) I"I = 1 + (C aiKsi)[Q].
F
In this equation Fo and F are the fluorescence intensities in Using the general form of Eq. 4:
the absence and the presence of quencher, respectively. Ap-
Fo = _
- _
Io(T0)
plying Eq. 60 to each component of a heterogeneous system
gives F 1 (4
and substituting Eq. 46 and 72 yields
f

In this equation &, and I are the fluorescence amplitudes in

the absence and the presence of quencher, respectively.
(74)
In the absence of dynamic quenching T , = T ~ therefore
~ .
Eq. 61 can be rearranged as
APPENDIX 3: ANALYSIS OF THE
CALCULATION OF THE RADIATIVE RATE
CONSTANT FOR A SET OF DIFFERENT
and because Ii = a,[, further to LIFETIMES
We start with the equation from the method of Parker and
Rees (15) for the calculation of the quantum yield.
substituting I = Z, [Iol/(l + K,,[Q])] (see Eq.62) this leads to
I F X X ) dh
Qr (75)
Qf = Af F,(X) dX

or where Q is the quantum yield, F' is the fluorescence intensity

and A is the absorbance at the excitation wavelength, sub-
script f refers to the fluorophore and subscript r refers to
reference. We define r as

= 1 F,(h) dX
Q,

where r is a correction factor that contains all instrument-

dependent parameters.
Applying Lambert-Beer's law:
Steady-state fluorescence
Starting with Eq. 3 and introducing lifetimes gives

Qf =
I F;(X)r dh

Ecb
(77)

In this equation E is the molar absorption coefficient, c is the

If there is no static quenching I. = I and Eq. 38 is obtained concentration of fluorophore and b is the optical pathlength.
under the form: Substitution of F' r with F, the corrected fluorescence, yields
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Photochemistry and Photobiology, 1998, 67(5) 483

Eicibkri= ai h ( h ) dh (87)
or
(78)
= Ecb '

If there is only one lifetime, then Q/T = k,, and because

F(A)/T = I(X): Pik, = a1
1 I(A) dA
= ai(kr). (88)
Ecb
The equivalent calculation using the intensity average life-
time yields the following relation:

In this equation, I is the intensity amplitude at wavelength A.

If there are more lifetimes, Eq. 79 can be applied to each
component:

APPENDIX 4: COMPARISON OF A SYSTEM

WITH MULTIPLE FLUOROPHORES AND
MULTIPLE LIFETIMES WITH SYSTEMS
Equation 80 is useful for the calculation of an average k,. CONTAINING ONE FLUOROPHORE WITH
Therefore we define a, a wavelength-independent ampli- MULTlPLE LIFETlMES
tude fraction: Here m is the number of fluorophores and is counted by j

a. =
I Ii(A) dh
I-
-
-
I Ii(X) dh
and k, n is the number of lifetimes and is counted by i and
1. First we start with the evolution of the fluorescence inten-
sity with time (t):
i=n
FJt, A ) = 2 I,,(h)exp(-t/.r,).
,=I
(90)
Rewriting Eq. 80 and summing over every i gives
From this the quantum yield can be calculated:
2 E,c,bQ, = 2 T,CX, II(h) dh
or with PI the absorption fraction P, = (E,c,b)/(Ecb)
Ecb 2 P,Q, = 2 ~ ph(A) , dh.
Thus the quantum yield measured according to Eq.
Q,N, = [[(b ,=I
I,(h)exp(-t/T,)

where N is the number of photons absorbed. Further,

1
dt dh (91)

J
Q = 2 PiQi =
Ecb Recalling the definition of a:
and here an average k, can be calculated by dividing by the
amplitude average lifetime: QjN, = (2 (d a i j ~ i j ) I,@) dh (93)

or

To understand the meaning of this average k, consider the

i=l
following relation:
The number of photons absorbed by one fluorophore (N,)
relates to the total number of photons absorbed (N) by
N
NJ= e J k = m (95)
c
k= I
Ek

combining Eqs. 94, 95 and 85 yield

Thus the average k, of Eq. 85 is the sum of the absorption-

weighted radiative rate constants.
k=l k= I
It is possible to calculate Pi kri. We start with Eq. 80 and
rewrite: Recalling the definition of (Y
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484 Alain Sillen and Yves Engelborghs

FQ is therefore the fluorescence intensity corrected for in-

(97) strumental factors and normalized with respect to the ab-
sorption. From here the derivation is analogous to the pre-
vious one if Q is replaced by FQ, a by a and no integration
or over the wavelength is done. Then Eq. 104 becomes
-
k=m

Defining aI, and applying Eq. 98:

k=m
c
I?‘J k-1 ek
For the sake of completeness the average
k=m k=m
cy is

c Iik

g k= I
kTIil i=n k=m 07)
i=l k=l i=l k = l (Tr)k

The intensity average lifetime

APPENDIX 5: ENERGY TRANSFER, THE
ACCEPTOR LIFETIMES
definition of the intensity fraction and substituting Eq. 99: To investigate a system of energy transfer with different life-
times, a model system with one donor with two lifetimes
and one acceptor with two lifetimes is examined. Energy
transfer is possible between the species responsible for the
first lifetime of the donor (D1) and the species responsible
for the first lifetime of the acceptor (Al) with transfer con-
stant kT1and A2 with transfer constant kT2.For the second
lifetime of the donor (D2) the situation is analogous with
transfer constants kT3and kT4.
L The differential equations describing the decay of D1 and
k=l (Tr)k D2 are
Substituting Eq. 101 in Eq. 100 yields

and

Amplitude average lifetime

with kD the fluorescence decay constant of the donor.
The solutions of these equations are

Substituting Eq. 99 in Eq. 103 yields [Dl1 = [DlIoexP[-(k~i TI k~dt] (110)

and
CD21 = [D210exp[-(kD2 kT3 + kT4)tl. (l 1>
The differential equations describing the decay of A1 and
A2 are
Equations 102 or 104 are better not used when the max-
ima of the DAS spectra of the lifetimes are shifted to other
wavelengths. If there is a large shift the average lifetime
becomes wavelength dependent and it is necessary to use and
the following equations.
To derive these equations, the intensity P at a certain
wavelength is defined as
FXNr
FQ= - The solutions of these equations with [All = [All, at t =
(105)
Ecb ’ 0 are
 17511097, 1998, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1751-1097.1998.tb09082.x by National Taiwan University, Wiley Online Library on [21/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Photochemistry and Photobiology, 1998, 67(5) 485

where I$ and 1: are the intensity amplitudes of the acceptor

and the donor respectively. %A and qDare the amplitude frac-
tions of the acceptor and the donor respectively. qA.%D, I&
and IF can be calculated out of the lifetime measurements
of the acceptor and the donor and the steady-state measure-
ments of the acceptor and the donor. Thus the intensity de-
cay of the acceptor in the presence of donor can be fitted
with Eq. 118 that contains only six unknowns, namely the
two lifetimes of the donor in presence of the acceptor and
the four 6 values.

Calculation of energy transfer efficiency

The calculation of the efficiency of energy transfer for one
lifetime starts with the calculation of the average lifetime of
exp[-(kD2 + kT3 + kT4)tl (1 15) the acceptor in presence of the donor.
with

=
kT1[D110 + kT3 rD210
(1 16)
( ~ A -
I ~ D -I TI - ~ T Z ) ( ~ A -
I koz - k ~ -
3 k~4)
or
kn ID110 + ~T~[D~Io
s2 = (1 17)
(kA2 - kDI - kT1 - (kA2 - kD2 - kT3 - kT4)'
These equations show that the acceptor has four lifetimes if
energy transfer occurs, the lifetimes of the acceptor in ab- or
sence of the donor and the lifetimes of the donor in the
presence of acceptor. Summation of [All and [A21 shows
this more clearly:
[A1 = ([All0 - ~ I ) ~ x P ( - ~+A([A210
I ~ ) - We~p(-kAzt) The efficiency of energy transfer is therefore

+ 63[D110exp[-(kD1 + kT, + h2)tI

+ 64[D210exp[(-kD2 + kT3 + kT4)tI (118)
A similar expression can be calculated for two lifetimes:
with

This can be worked out as

(120) (131)
The acceptor fluorescence intensity decays the same way as and divided by the average lifetime of the acceptor in the
described by Eq. 118: absence of the donor:
I(t) = a,exp(-t/.r,) + a,exp(--t/?,) (kT17A1 + kT27A2) (kT3TA1 +
+ a,exp(-th,) + a4exp(-th4) (121) (- - I)$ = a? a.A

kD1
;' I + a$TA2
+ kTI + kT2
+ a: a?7AI

kD2
kT47A2)

+ a$TA2
+ kT3 + kT4
with
(132)
and generalized to

(123)

Finally an average efficiency can be obtained:


486 Alain Sillen and Yves Engelborghs
APPENDIX 6: DIAGNOSIS OF THE NATURE
OF QUENCHING UPON A
CONFORMATIONAL CHANGE OF A
PROTEIN OR A MUTATION
The total fluorescence intensity F is
F = I i ~=i (2 aiTi)I = (T)~I.
(135)
If we compare quenched with not quenched (subscript 0) we
can write:
Therefore if the following relation holds:
then I = and this implies absence of static quenching. In
this case the amplitude can be used to identify the micro-
states (16).
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