Quenching of Fluorescence

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

The fluorescence lifetime = k-1 = (kf + knr)-1 depends on the environment of the molecule through knr = ki + kx + kET + ….

Fluorescence quantum yield:

is proportional to fluorescence lifetime.

r

f

nrf

f

kk

kkk

QY

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

QuenchingA 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 QuenchingCollisional 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-).

Concentration of I- (M)

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

F0/

F

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

In the case of purely collisional quenching, also known as dynamic quenching,:

I0/I = 0/.

Hence in this case: 0/ = 1 + kq 0[Q]

In the fluorescein/iodide system, = 4ns and kq ~ 2 x 109 M-1 sec-1

In this case, KSV ~ 8 Lmol-1

KSV = kq 0 where kq is the bimolecular quenching rate constant and 0 is the excited state lifetime in the absence of quencher.I 0

/I

Collisional Quenching

derivation of Stern-Volmer equation:

nrf kk

10

]Q[1

qnrf kkk

presence of quencher – additional nonradiative deexcitation channel

]Q[1 00 qk

fkQY quantum yield:

]Q[1000 SVKQYQYII

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 [F][Q]]FQ[

aK

]Q[1]F[

]FQ[]F[[F]]F[ .0

atot K

II

[FQ] = Ka [F][Q]

I0/I

[Q]

If both static and dynamic quenching are occurring in the sample then the following relation holds:

I0/I = (1 + kq 0[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.

However, since the lifetime is unaffected by the presence of quencher in cases of pure static quenching, a plot of 0/ versus [Q] would give a straight line

I0/I

[Q]

0/

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.

I0/I

[Q]

In the extreme case, a Stern-Volmer plot for a system having accessible and inaccessible fluorophores could look like this:

323nm

350nm

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.

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

O

O

H

O

O

O

P O

O

O- N

+

O

O

H

O

O

N+

N

O

N+

N

O

O

O

H

O

O

O

P O

O

O- N

+

DOPCDOTAP

Patman

Laurdan

DOPC

Where are the dyes localized?Where are the dyes localized?

Laurdan and Patman are fluorescent probes which, thanks to their structure, incorporate to lipid bilayers (biological membranes) in well defined depths

aqueous

phase

hydro

phobic

inte

rior

The depth can be determined by paralax method

E2

Quenchers used for paralax method...Quenchers used for paralax method...

TEMPO-PC

16-DSA5-DSA

The quenchers used in paralax method are lipid analogues with groups with unpaired electrons at well defined positions (spin probes) – strong quenchers

E2

Parallax method ...Parallax method ...Distance from the center ofDistance from the center of

DOPC bilayer for:DOPC bilayer for:• Patman – Patman – 10.45 A10.45 A• Laurdan – Laurdan – 11.35 A11.35 A

E2

Laurdan Patman

Quencher ...Quencher ... Acrylamide

Does the fluorophore change its location after addition of positively charged lipids?

E3

Stern-Volmer equation for dynamic quenching:

Io/I= 1+KSV [Q]

Flu

ores

cens

e in

tens

ity

Wavelength

Emission spectra without quencher with quencher with quencher (dye

more accessible)

E3

Positively charged dye (Patman) changes its location in the presence of positively charged lipids in the outward direction

+

+

Self quenching

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.

The intensity of fluorescence is proportional to the concentration of the fluorophores in a reasonable concentration range.

Fluorescence intensity of calcein as a function of fluorophore concentration

Andersson et al. Eur Biophys J 2007, 36: 621

Self quenching

Typically used to study the formation of pores in vesicles caused by membrane-active molecules – vesicle leakage assay

E4

LAH4 peptide

Triton X-100 detergent

vesicles

vesicles loaded with 60 mM calcein

peptide LAH4 creates pores in the lipid bilayer, through which the dye can leak out

detergent Triton X-100 micellizes the vesicles

final calcein concentration ~ 5 M

Vogt and Bechinger, J Biol Chem 1999, 274: 29 115

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