1. Effects of fluorophore’s environment on its spectra
Lenka Beranová, Martin Hof, Radek Macháň

2. 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). S2 S1
The fluorophore’s environment influences its lifetime (transitions kinetic constants) and also its spectrum (spacing of levels)
Absorption
Fluorescence
kf ~ 107 – 109 s-1 S0
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

3. Fluorophore in a polar solvent
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
solvent relaxation
S1Rel
Excitation
Emission
S0FC S0
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

4. Fluorophore in a polar solvent
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
S1FC
solvent relaxation
S1Rel
Excitation
Emission
S0FC S0
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.

5. Lifetime vs. solvent relaxation
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.
S1FC
solvent relaxation
S1Rel
Excitation
Emission
S0FC S0
The lower the temperature:
the higher the local viscosity is,
the smaller the red shift of the emission spectrum is.

6. Lifetime vs. solvent relaxation
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.
Assuming a mono-exponential decay of fluorescence intensity (lifetime t), we can write for the centre of mass of the steady-state spectrum:
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 Dn and fluorescence lifetime t

7. Increase of solvent polarity leads to larger red-shift3 O E1
Fluorescence spectra of Prodan
Emission spectra of prodan in different solvents:
heptane
water
Increase of solvent polarity leads to larger red-shift

8. E1 Fluorescence spectra of ProdanH 3 O E1
Fluorescence spectra of Prodan
Emission spectra of prodan at different temperatures:
100 K
300 K
wavelength (nm)
Decrease of temperature → increase of viscosity → increasing fluorescence contributions of non-relaxed states → blue-shift

9. Experimental characterization of solvent relaxation
The most comprehensive information is obtained form Time Resolved Emission Spectra (TRES)
0.1 ns
10 ns
S1FC
S0FC
S1Rel S0
Excitation
solvent relaxation
Emission
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. Steady-state emission
Time Resolved Emission Spectra (TRES)
10 ns
400 nm
440 nm
470 nm
500 nm
Steady-state emission
spectrum S0(λ)
5 ns
2 ns
0.1 ns
Intensity decays (TRES)
D(t,λ)

11. Time-zero estimation 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,...)
Spectra of DTMAC 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)coumarin
Data treatment:
Calculation of the so called lineshape functions f(n), g(n) from the non-polar reference spectra
Finding shift distribution p(δ) by fitting convolution of p(δ) and g(n) with polar absorption spectrum Ap(n)
Calculation of time-zero spectrum using f(n), g(n), p(δ)
J. Sykora et al. Chem. Phys. Lipids (2005)

12. TRES and description of the relaxation
static (spectral shift)
the change in position of the centre of mass of the spectrum is proportional to the polarity of the fluorophore’s environment
fully relaxed state
Frank-Condon state Dn

13. Dependence of spectral shift on fluorophoe’s environment polarity
Coumarin 153
  is directly proportional to the polarity function F
example:
C1OH: F = 0.71;   = 2370 cm-1
C5OH: F = 0.57;   = 1830 cm-1
D [cm-1] F
= [(s-1)/ (s+2)] - [(n2-1)/ (n2+2)]
Horng et al., J Phys Chem :17311

14. TRES and description of the relaxation
Kinetic (correlation function and relaxation time)
Reflects local viscosity of the fluorophore’s surroundings ) ( - = n t C

15. Kinetics of the relaxation reflect local viscosity surrounding the fluorophore
dyes in tetrahydrofuran K }
tP = 0.25 s
Probed by phosphorescence
τCT = 4 s
Ru(bpy)2(CN)2 }
τF = 20 ns
Probed by charge-transfer emission }
Probed by S1S0 fluorescence
92 K
170 K
R. Richert et al. Chem. Phys. Lett. (1994) 229:302

16. 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”).
relaxation too slow compared to lifetime
relaxation too fast compared to experimental time resolution
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

17. 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 (tSR ≈> t)
S1FC
solvent relaxation
S1Rel nF nR
S0R* S0
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 nR (located at the red edge of the excitation spectrum)

18. Red-edge excitation spectra
The effect of excitation wavelength depends on ration tSR /t (whether the emission spectrum is closer to n0 or n∞ )
S1FC
emission spectra excited by nF or nR
solvent relaxation
S1Rel
tSR << t nF nR n
S0R* S0
tSR >> t
red-edge excitation spectra can be used to estimate the characteristic timescale of solvent relaxation tSR n

19. c) Backbone region: several ns; water diffusion
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

20. A Defined localization of the fluorophores external interface backbone
headgroup region
local polarities and viscosities in all regions

21. A1 Headgroup labels (DOPC - fluid bilayer)N O + Cl -
Patman
Prodan H P
DOPC
Dn: 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“

22. A1 Summary of SR in DOPC vesicles- F H + N Cl P
DOPC
9-AS
2-AS
Patman
Prodan
ABA-C 15 C 16
DiFU
Sykora, Kapusta, Fidler, Hof (2002) Langmuir
 (cm-1)
τSR(average) (ns) n.d.
% SR (<50 ps)
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

23. A2 Membrane curvature and headgroup hydration
Motivation: membrane fusion, vesiculation, formation of new organelles, ... are intermediated via highly deformed bilayer structures.
Large unilamellar vesicles (LUV) = low curvature
Small unilamellar vesicles
(SUV) = high curvature
d≈200nm
d≈30nm

24. A2 Membrane curvature and headgroup hydration
d≈30nm
d≈200nm
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)

25. B Solvent relaxation in proteins (haloalkan dehalogenases) DhaA DbjA
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
DhaA
DbjA
Jesenská et al. JACS (2009)

26. B Solvent relaxation in proteins (haloalkan dehalogenases) DhaA DbjA
Dn (cm-1)
tSR (ns)
% observed
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)

27. Dinitrostilbene in different solvents
Dissolved in:
Cyclohexane (nonpolar)
Diethyl ether (medium polar)
Ethyl acetate (polar)