Effects of fluorophore’s environment on its spectra

Lenka Beranová, Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

S0

S2

S1

Absorption

Flu

ore

scen

ce

kf ~

10

7 – 1

09 s

-1

The fluorescence spectrum

The fluorophore’s spectrum is determined by the spacing of its energy levels and the probabilities of transitions between them (Jablonski diagram).

The fluorophore’s environment influences its lifetime (transitions kinetic constants) and also its spectrum (spacing of levels)

To explain that we need to regard the fluorophore and the molecules surrounding as one quantum system and look at its energy states.

Dipole-dipole interactions are the most important source of the interactions polar solvents have most pronounced effects

S1FC

S0FC

S1Rel

S0

Exc

itatio

n

solvent relaxation

Fluorophore in a polar solvent

Franck-Condon principle: redistribution of electron density caused by an electronic transition happens on a much faster scale than reorientation of nuclei Reorientation of the fluorophore’s dipole moment upon excitation leads to en energetically unfavourable Franck-Condone state from which the system relaxes through reorientation of fluorophore’s solvation envelope to a state of lower energy.Similar situation upon emission of photon from relaxed state

Em

ission

The molecules of the polar solvent are oriented in such a way that their dipole moments compensate for the dipole moment of the fluorophore in order to minimize the total energy of the system fluorophore + solvation envelope

S1FC

S0FC

S1Rel

S0

Exc

itatio

n

solvent relaxation

Fluorophore in a polar solvent

Em

ission

The solvent relaxation introduces an additional red shift to the Stokes shift of the fluorophore spectra of fluorophores in more polar solvents tend to be shifted more to the red

The red shift is the bigger:• the more polar the solvent is, • the bigger the dipole moment of the fluorophore is and • the bigger its change upon excitation is.

S1FC

S0FC

S1Rel

S0

Exc

itatio

n

solvent relaxation

Lifetime vs. solvent relaxation

Em

ission

The time-scale of the solvent relaxation depends on the mobility of fluorophore’s solvation envelope (local viscosity). If it is slower or comparable to the fluorescence lifetime, emission from non-relaxed state contributes largely to the spectrum.

The lower the temperature:• the higher the local viscosity is, • the smaller the red shift of the emission spectrum is.

The centre of mass of the emission spectrum is shifting to red side with advancing relaxation (molecules which have stayed longer in the excited state emit photons of higher wavelength). For a homogeneous sample a

mono-exponential decay of emission spectrum centre of mass can be assumed.

Lifetime vs. solvent relaxation

)/exp()()( 0 SRtt

Assuming a mono-exponential decay of fluorescence intensity (lifetime ), we can write for the centre of mass of the steady-state spectrum:

SR

SRS

tt

ttt

)(

d)/exp(

d)/exp()(

0

0

0

Note that the steady-state spectrum of a fluorophore, whose lifetime is sensitive to the polarity of environment, is an interplay between the effect of

solvent on total red shift and fluorescence lifetime

SSRSSR 0

heptane

water

Increase of solvent polarity leads to larger red-shift

Emission spectra of prodan in different solvents:

E1Fluorescence spectra of ProdanN

C

CH3

O

CH3

H3C

400 440 480 520 560 600

0.2

0.4

0.6

0.8

1.0

100 K

300 K

Decrease of temperature → increase of viscosity → increasing fluorescence contributions of non-relaxed states → blue-shift

Fluorescence spectra of Prodan E1N

C

CH3

O

CH3

H3C

Emission spectra of prodan at different temperatures:

wavelength (nm)

480 520 560 600 6400,0

0,2

0,4

0,6

0,8

1,0

Inte

nsi

ty

wavelength (nm)

Experimental characterization of solvent relaxationThe most comprehensive information is obtained form

Time Resolved Emission Spectra (TRES)

S1FC

S0FC

S1Rel

S0

Exc

itatio

n

solvent relaxation

Em

ission

Fluorescence is excited by short pulses (like in lifetime measurements), photons emitted shortly after excitation pulse come from molecules in

nonrelaxed state (had not enough time to relaxed). The longer after excitation pulse, the more relaxed the molecules are.

The measurement requires spectral and time resolved photon detection – can be achieved by a streak camera combined with imaging spectrograph (2-dimensional detector, one dimension arrival time, other wavelength).

Most often measured indirectly

10 ns0.1 ns

0

0

),(

)(),(),(

dttD

StDtS

Intensity decays (TRES)

D(t,λ)

10 ns

400 nm440 nm470 nm

500 nm

Steady-state emission spectrum S0(λ)

5 ns

2 ns

0.1 ns

Time Resolved Emission Spectra (TRES)

Time-zero estimation

18 20 22 24 26 28 30 32

0.0

0.2

0.4

0.6

0.8

1.0

Ab

sorp

tion

or

Inte

nsi

ty

Wavenumber ( 103 cm-1 )

Spectra of DTMAC 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)coumarin

Measurements:

1. Emission and absorption spectra of the dye in non-polar solvent (hexan,...)

2. Absorption spectrum of the dye in the polar system of interest (liposomes,...)

Data treatment:

3. Calculation of the so called lineshape functions f(), g() from the non-polar reference spectra

4. Finding shift distribution p(δ) by fitting convolution of p(δ) and g() with polar absorption spectrum Ap()

5. Calculation of time-zero spectrum using f(), g(), p(δ)

J. Sykora et al. Chem. Phys. Lipids (2005) 135 213

static (spectral shift)

)()0(

Frank-Condon state

fully relaxed state

the change in position of the centre of mass of the spectrum is proportional to the polarity of the fluorophore’s environment

0 5 10 15 2020000

21000

22000

23000

24000

TR

ES

ce

ntr

e o

f m

ass

(cm

-1)

Time (ns)

TRES and description of the relaxation

is directly proportional to the polarity function F

example:

C1OH: F = 0.71; = 2370 cm-1

C5OH: F = 0.57; = 1830 cm-1

Horng et al., J Phys Chem 1995 99:17311

O ON

CF3

[cm

-1]

F

= [(s-1)/ (s+2)] - [(n2-1)/ (n2+2)]

E2Dependence of spectral shift on fluorophoe’s environment polarity

Coumarin 153

Reflects local viscosity of the fluorophore’s surroundings

)()(

)(t

tC

0

d)( ttCSR

0 5 10 15 200,0

0,2

0,4

0,6

0,8

1,0

C(t

)

time (ns)

TRES and description of the relaxation

Kinetic (correlation function and relaxation time)

)()0()()(

)(

t

tC

Kinetics of the relaxation reflect local viscosity surrounding the fluorophore

R. Richert et al. Chem. Phys. Lett. (1994) 229:302

N

N

Ru(bpy)2(CN)2

N

O

O

H

N

H

H

92 K

170 K

τF = 20 ns

τCT = 4 s

P = 0.25 s

dyes in tetrahydrofuran 90-170 K

}}} Probed by S1S0

fluorescence

Probed by charge-transfer emission

Probed by phosphorescence

E3

TRES and width (FWHM) of the spectra

Width (FWHM) of emission spectra changes during relaxation process. In ideal case (all fluorophores in identical environment) it would decrease monotonically to the width of the fully relaxed spectrum. In real samples a maximum is observed (differences in local environment relaxation not “in phase”).

Together with the time-zero estimation it can be used to estimate how much of the relaxation process is observable in the experiment. Furthermore, more complex dependence suggests fluorophore populations located in distinct environments

0 2 4 6 83000

3500

4000

4500

5000

5500

6000F

WH

M (

cm-1)

time (ns)

relaxation too slow compared to lifetime

relaxation too fast compared to

experimental time resolution

Red-edge excitation spectra

The emission spectra are known to be independent on the excitation wavelength. However, that is not exactly so in polar environments of

sufficient viscosity (SR ≈> )

In the equilibrium state, a small fraction of molecules in the ground state have solvation envelopes like excited molecules in the relaxed state. They can be excited by photons of lower energy R (located at the red edge of the excitation spectrum)

S1FC

S0R*

S1Rel

S0

F

solvent relaxation

R

Red-edge excitation spectra

The effect of excitation wavelength depends on ration SR / (whether the emission spectrum is closer to 0 or ∞ )

S1FC

S0R*

S1Rel

S0

F

solvent relaxation

R

emission spectra excited by F or R

SR <<

SR >>

red-edge excitation spectra can be used to estimate the

characteristic timescale of solvent relaxation SR

Applications of solvent relaxation

Investigation of local polarity and viscosity at specific sites of macromolecules and supramolecular complexes (biomembranes, proteins)

A. Solvent relaxation in biomembranes

polarity amount of clustered water (forming solvation envelopes) viscosity restrictions to its motion – packing of molecules

a) External interface: from sub ps to ns.

b) Headgroup region: pure ns process;

mobility of hydrated functional groups

c) Backbone region: several

ns; water diffusion

bulk water: sub-ps

N

SN

O

O

OOH

O

O-

O

F

F

H+

O

NH

O

N

O

N+

N

O

N

O

OOH

O

O

O

O

OOH

O

OOH

O

Cl-

O

N

SO

O

H

OO

O

P OO

O-

N+

DOPC

16-AP

9-AS2-AS

Patm an

Laurdan

Prodan

ABA-C 15

DTMACC 17DiFU

Dauda

Defined localization of the fluorophoresA

local polarities and viscosities in all regions

backbone headgroup region external interface

: 3750 cm-1 (Prodan); 3000 cm-1 (Patman) Prodan probes larger polarity

τSR: 1.0 ns (Prodan); 1.7 ns (Patman)

Prodan probes lower “micro-viscosity“

Headgroup labels (DOPC - fluid bilayer)A1

N

O

N+

N

O

Cl-

Patman

Prodan

O

O

H

O

O

O

PO

O

O-

N+

DOPC

•Deeper localisation means probing lower polarity and higher “vicosity”

•Significant part within the external interface < 50 ps; partially “bulk” water•Head group labels: “pure” ns SR: bound water to charged and polar groups •Backbone: SR slows down with depth of location: water diffusion

(cm-1) 2100 2750 3000 3750 3100 1700τSR(average) (ns) 3.4 2.1 1.7 1.0 0.5 n.d.

% SR (<50 ps) 75 95 95 95 85 50

Sykora, Kapusta, Fidler, Hof (2002) Langmuir 18 571

O

O-

O

F

F

H+

O

NH

O

N

O

N+

N

O

OO H

O

O

O

O

OO H

Cl-

O

O

H

O

O

O

P O

O

O-

N+

DOPC

9-AS2-AS

Patman

Prodan

ABA-C15

C16

DiFU

O

O

H

O

O

O

P O

O

O-

N+

DOPC

A1 Summary of SR in DOPC vesicles

Large unilamellar vesicles (LUV) = low curvature

Small unilamellar vesicles (SUV) = high curvature

d≈200nm d≈30nm

Membrane curvature and headgroup hydrationA2

Motivation: membrane fusion, vesiculation, formation of new organelles, ... are intermediated via highly deformed bilayer structures.

•degree of hydration remains constant

•relaxation becomes faster with increasing curvature

mobility of the dye microenvironment increased when the bilayer is more bent – different

packing of the bilayer

τSR = 1.2 ns

τSR = 0.9 ns

J. Sýkora et al. Chem. Phys. Lipids (2005) 135 213

A2 Membrane curvature and headgroup hydration

d≈200nm

d≈30nm

B Solvent relaxation in proteins (haloalkan dehalogenases)

Proteins substituting halogens in haloalkans with hydroxyl. Reaction in a tunnel shaped active site

The active site of two mutations is investigated by SR – fluorescently labelled substrate + inhibition of enzymatic activity the fluorophore

stays for a long time in the active site

DbjA DhaA

Jesenská et al. JACS (2009) 131 494

B Solvent relaxation in proteins (haloalkan dehalogenases)

DbjA DhaA

(cm-1) 1300 950SR (ns) 2.8 4.1

% observed 70 90

The difference correlates with molecular modelling – more polar and mobile in wider tunnel mouth. DbjA has higher enzymatic activity

Pure ns dynamics no “bulk” water, water in enzyme active site is structured (like solvation envelope)

Dinitrostilbene in different solvents

Dissolved in:

a)Cyclohexane (nonpolar)

b)Diethyl ether (medium polar)

c)Ethyl acetate (polar)