Download - Effects of fluorophores environment on its spectra Lenka Beranová, Martin Hof, Radek Macháň
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)