Quenching of Fluorescence

CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Quenching of Fluorescence
Martin Hof, Radek Macháň
The fluorescence lifetime t = k-1 = (kf + knr)-1 depends on the
environment of the molecule through knr = ki + kx + kET + ….
Fluorescence quantum yield:
QY 
kf
k
t
 f 
t
kf  knr
k
tr
is proportional to fluorescence lifetime.
Addition of another radiationless pathway increases knr and, thus,
decreases t 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
Quenching
A number of processes can lead to a reduction in fluorescence intensity, i.e.,
quenching
These processes can occur during the excited state lifetime – for example collisional
quenching, energy transfer, charge transfer reactions or photochemistry – or they
may occur due to formation of complexes in the ground state
We shall focus our attention on the two quenching processes usually encountered –
namely collisional (dynamic) quenching and static (complex formation) quenching
Collisional Quenching
Collisional quenching occurs when the excited fluorophore experiences contact with
an atom or molecule that can facilitate non-radiative transitions to the ground
state. Common quenchers include O2, I-, Cs+ and acrylamide.
In the simplest case of collisional quenching, the following relation, called the
Stern-Volmer equation, holds:
I0/I = 1 + KSV[Q]
where I0 and I are the fluorescence intensities observed in the absence and
presence, respectively, of quencher, [Q] is the quencher concentration and KSV is
the Stern-Volmer quenching constant
In the simplest case, then, a plot of I0/I versus [Q] should yield a straight line
with a slope equal to KSV. Such a plot, known as a Stern-Volmer plot, is shown
below for the case of fluorescein quenched by iodide ion (I-).
In this case, KSV ~ 8 Lmol-1
1.7
KSV = kq t0 where kq is the
bimolecular quenching rate
constant and t0 is the excited
state lifetime in the absence
of quencher.
1.6
I0F0/I/F
1.5
1.4
1.3
1.2
1.1
1.0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Concentration of I- (M)
In the case of purely collisional quenching, also known as dynamic quenching,:
I0/I = t0/t.
Hence in this case: t0/t = 1 + kq t0[Q]
In the fluorescein/iodide system, t = 4ns and kq ~ 2 x 109 M-1 sec-1
Collisional Quenching
derivation of Stern-Volmer equation:
1
t0 
kf  knr
1
t 
kf  knr  kq [Q]
presence of quencher – additional nonradiative deexcitation channel
t 0 t  1  kqt 0[Q]
quantum yield:
QY  t kf
I0 I  QY0 QY  t 0 t  1  KSV [Q]
Static Quenching
In some cases, the fluorophore can form a stable complex with
another molecule. If this ground-state is non-fluorescent then
we say that the fluorophore has been statically quenched.
In such a case, the dependence of the fluorescence as a
function of the quencher concentration follows the relation:
I0/I = 1 + Ka[Q]
where Ka is the association constant of the complex. Such
cases of quenching via complex formation were first described
by Gregorio Weber.
In the case of static quenching the lifetime of the sample will not be
reduced since those fluorophores which are not complexed – and
hence are able to emit after excitation – will have normal excited
state properties. The fluorescence of the sample is reduced since the
quencher is essentially reducing the number of fluorophores which
can emit.
Static Quenching
In some cases, the fluorophore can form a stable complex with
another molecule. If this ground-state is non-fluorescent then
we say that the fluorophore has been statically quenched.
F+Q
FQ
[FQ]
Ka 
[F][Q]
[FQ] = Ka [F][Q]
I0 [F]tot . [F]  [FQ]


 1  K a[Q]
I
[F]
[F]
If both static and dynamic quenching are occurring in the sample
then the following relation holds:
I0/I = (1 + kq t0[Q]) (1 + Ka[Q])
In such a case then a plot of I0/I versus [Q] will have an upward
curvature due to the [Q]2 term.
I0/I
[Q]
However, since the lifetime is unaffected by the presence of quencher
in cases of pure static quenching, a plot of t0/t versus [Q] would give
a straight line
I0/I
t 0/ t
[Q]
Non-linear Stern-Volmer plots can also occur in the case of purely
collisional quenching if some of the fluorophores are less accessible
than others. Consider the case of multiple tryptophan residues in a
protein – one can easily imagine that some of these residues would
be more accessible to quenchers in the solvent than other.
In the extreme case, a Stern-Volmer plot for a system having
accessible and inaccessible fluorophores could look like this:
I0/I
[Q]
The quenching of LADH intrinsic protein fluorescence by iodide gives, in fact, just such
a plot. LADH is a dimer with 2 tryptophan residues per identical monomer. One residue
is buried in the protein interior and is relatively inaccessible to iodide while the other
tryptophan residue is on the protein’s surface and is more accessible.
E1
350nm
323nm
In this case (from Eftink and Selvidge, Biochemistry 1982, 21:117) the different
emission wavelengths preferentially weigh the buried (323nm) or solvent exposed
(350nm) tryptophan.
aqueous phase
Where are the dyes localized?
O
-
N
+
O
N
+
O
O
O
O
H
O
N
+
+
O
O
H
O
N
P O
P O
O
-
E2
H
O
N
O
O
O
O
O
N
O
Laurdan and Patman are
fluorescent probes which,
thanks to their structure,
incorporate to lipid bilayers
(biological membranes) in well
defined depths
O
hydrophobic interior
O
Laurdan
DOTAP
DOPC
DOPC
Patman
The depth can be determined
by paralax method
Quenchers used for paralax method...
E2
TEMPO-PC
5-DSA
16-DSA
The quenchers used in paralax method are lipid analogues with groups
with unpaired electrons at well defined positions (spin probes) – strong
quenchers
Parallax method ...
Distance from the center of
DOPC bilayer for:
• Patman – 10.45 A
• Laurdan – 11.35 A
E2
Does the fluorophore change its location after addition of
positively charged lipids?
Laurdan
Quencher ...
Patman
Acrylamide
E3
Fluorescense intensity
Emission spectra
without quencher
with quencher
with quencher (dye
more accessible)
Wavelength
+
less accessible dye
for a quencher
more accessible dye
for a quencher
I0/I
+
Stern-Volmer equation for dynamic
quenching:
Io/I= 1+KSV [Q]
Concentration of quencher Q
Positively charged dye (Patman) changes its location in the
presence of positively charged lipids in the outward direction
E3
Self quenching
The intensity of fluorescence is proportional to the concentration of the
fluorophores in a reasonable concentration range.
However, at high concentrations of the fluorophores the proportionality
is no more satisfied, because significant collisional quenching between
the molecules of the fluorophore themselves appears.
Fluorescence intensity of calcein as
a function of fluorophore
concentration
Andersson et al. Eur Biophys J 2007,
36: 621
E4
Self quenching
Typically used to study the formation of pores in vesicles caused by
membrane-active molecules – vesicle leakage assay
vesicles loaded
with 60 mM
calcein
vesicles
Triton X-100
detergent
peptide LAH4
creates pores in
the lipid bilayer,
through which the
dye can leak out
LAH4 peptide
detergent Triton
X-100 micellizes
the vesicles
Vogt and Bechinger, J Biol Chem 1999, 274: 29 115
final calcein
concentration
~ 5 mM
Acknowledgement
The course was inspired by courses of:
Prof. David M. Jameson, Ph.D.
Prof. RNDr. Jaromír Plášek, Csc.
Prof. William Reusch
Financial support from the grant:
FRVŠ 33/119970