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Effects of fluorophores environment on its spectra Lenka Beranová, Martin Hof, Radek Macháň.

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Presentation on theme: "Effects of fluorophores environment on its spectra Lenka Beranová, Martin Hof, Radek Macháň."— Presentation transcript:

1 Effects of fluorophores environment on its spectra Lenka Beranová, Martin Hof, Radek Macháň

2 S0S0 S2S2 S1S1 Absorption Fluorescence k f ~ 10 7 – 10 9 s -1 The fluorescence spectrum The fluorophores spectrum is determined by the spacing of its energy levels and the probabilities of transitions between them (Jablonski diagram). The fluorophores 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

3 S 1 FC S 0 FC S 1 Rel S0S0 Excitation 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 fluorophores dipole moment upon excitation leads to en energetically unfavourable Franck-Condone state from which the system relaxes through reorientation of fluorophores solvation envelope to a state of lower energy. Similar situation upon emission of photon from relaxed state Emission 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

4 S 1 FC S 0 FC S 1 Rel S0S0 Excitation solvent relaxation Fluorophore in a polar solvent Emission 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.

5 S 1 FC S 0 FC S 1 Rel S0S0 Excitation solvent relaxation Lifetime vs. solvent relaxation Emission The time-scale of the solvent relaxation depends on the mobility of fluorophores 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.

6 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 Assuming a mono-exponential decay of fluorescence intensity (lifetime ), 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 and fluorescence lifetime

7 heptane water Increase of solvent polarity leads to larger red-shift Emission spectra of prodan in different solvents: E1 Fluorescence spectra of Prodan N C CH 3 O CH 3 H 3 C

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

9 Experimental characterization of solvent relaxation The most comprehensive information is obtained form Time Resolved Emission Spectra (TRES) S 1 FC S 0 FC S 1 Rel S0S0 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 ns 0.1 ns

10 Intensity decays (TRES) D(t,λ) 10 ns 400 nm 440 nm 470 nm 500 nm Steady-state emission spectrum S 0 (λ) 5 ns 2 ns 0.1 ns Time Resolved Emission Spectra (TRES)

11 Time-zero estimation 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 A p () 5.Calculation of time-zero spectrum using f(), g(), p(δ) J. Sykora et al. Chem. Phys. Lipids (2005)

12 static (spectral shift) 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 fluorophores environment TRES and description of the relaxation

13 is directly proportional to the polarity function F example: C 1 OH: F = 0.71; = 2370 cm -1 C 5 OH: F = 0.57; = 1830 cm -1 Horng et al., J Phys Chem :17311 [cm -1 ] F = [( s -1)/ ( s +2)] - [(n 2 -1)/ (n 2 +2)] E2 Dependence of spectral shift on fluorophoes environment polarity Coumarin 153

14 Reflects local viscosity of the fluorophores surroundings TRES and description of the relaxation Kinetic (correlation function and relaxation time) )()0( )()( )( t tC

15 Kinetics of the relaxation reflect local viscosity surrounding the fluorophore R. Richert et al. Chem. Phys. Lett. (1994) 229:302 Ru(bpy) 2 (CN) 2 92 K 170 K τ F = 20 ns τ CT = 4 s P = 0.25 s dyes in tetrahydrofuran K } } } Probed by S 1S 0 fluorescence Probed by charge- transfer emission Probed by phosphorescence E3

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

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 ( 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) S 1 FC S 0 R* S 1 Rel S0S0 F solvent relaxation R

18 Red-edge excitation spectra The effect of excitation wavelength depends on ration SR / (whether the emission spectrum is closer to 0 or ) S 1 FC S 0 R* S 1 Rel S0S0 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

19 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 Defined localization of the fluorophores A local polarities and viscosities in all regions backboneheadgroup region external interface

21 : 3750 cm -1 (Prodan); 3000 cm -1 (Patman) Prodan probes larger polarity τ SR : 1.0 ns (Prodan); 1.7 ns (Patman) Prodan probes lower micro-viscosity Headgroup labels (DOPC - fluid bilayer) A1

22 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 ) τ SR(average) (ns) n.d. % SR (<50 ps) Sykora, Kapusta, Fidler, Hof (2002) Langmuir A1 Summary of SR in DOPC vesicles

23 Large unilamellar vesicles (LUV) = low curvature Small unilamellar vesicles (SUV) = high curvature d200nm d30nm Membrane curvature and headgroup hydration A2A2 Motivation: membrane fusion, vesiculation, formation of new organelles,... are intermediated via highly deformed bilayer structures.

24 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 τ SR = 1.2 ns τ SR = 0.9 ns J. Sýkora et al. Chem. Phys. Lipids (2005) A2A2 Membrane curvature and headgroup hydration d200nm d30nm

25 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)

26 B Solvent relaxation in proteins (haloalkan dehalogenases) DbjA DhaA (cm -1 ) SR (ns) % observed7090 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: a)Cyclohexane (nonpolar) b)Diethyl ether (medium polar) c)Ethyl acetate (polar)

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