Single molecule spectroscopy of macromolecules and aggregates ...

Single molecule spectroscopy of macromolecules
and aggregates
Ivan Scheblykin
Single Molecule Spectroscopy group
Department of Chemical Physics, Lund University, SWEDEN

Sample
Very low surface concentration
Laser excitation
Ivan Scheblykin
5 µ
Single molecule spectroscopy

Ivan Scheblykin

Wide-field excitation and detection,
The same as in a normal epi-fluorescence microscope.
Vacuum or
gas chamber
or
cryostat (5K-300K)
Ivan Scheblykin
CCD
Camera
Objective lens
Excitation
blocking filter
Images with 10 ms time resolution
Movie – 100 frames per second
Intensity(t), Fluorescence Spectrum (t)
Fluorescence lifetime (20 ps time resolution)
Polarization microscopy
Excitation clean-up filter
Excitation light:
Ar-ion laser
514 nm
488 nm
458 nm
Ti-Sph
750 – 850 nm
Diode lasers (CW and pulsed 50 ps)
405 nm
480 nm
640 nm
He-Ne laser
546 nm
592 nm
633 nm

Ivan Scheblykin
Dye molecule
Structure,
Energy transfer ?
Molecular aggregate
Luminescent polymer chain
(conjugated polymer)
SMS for material sciences !
Fluorescence Polarization

Traditional characteristic of polarization - Modulation Depth
Excitation
polarization
M
=
I
I
max
max
−
+
I
I
min
min
I max
I min
Emission
Modulation in
EXCITATION
Rotated linear
M ex
Circular
Rotated analyzer
(linear polarization)
Modulation in
FLUORESCENCE
M fl

Single molecule
λ/2
CCD camera Ivan Scheblykin
I T
Rotated linear
Rotated analyzer
ϕ (linear polarization)
ex ϕ fl
I T (ϕ ex , ϕ fl )
Polarization ”portraites” of molecules
2D polarization single molecule imaging
Emission
polarization
angle
2D plot = Topology of the molecule
Excitation polarization angle
Energy transfer
between differently oriented
chromophores
O. Mirzov, R. Bloem, P. R. Hania, D. Thomsson, H. Lin, and I. G. Scheblykin, Small 5 (2009) 1877

Conjugated polymers
Ivan Scheblykin
Organic electronics
Poly-phenylene-vinylene derivative
MEH-PPV
Flexible displays
Transistors ...
Spin coating from a diluted solution
chains
In polymer host matrix
Quartz

“Chromophore size”
~ 3-5 nm
Ivan Scheblykin
5 ÷ 20 nm
5 - 500 “chromophores”
Polymer chain – ensemble of chromophores

Modulation depth correlation plot
Absorption by en ensemble,
Emission from a single chromophore
(energy funnel)
1
Single
chromophore
M
=
I
I
max
max
−
+
I
I
min
min
M EM
0 1
Absorption and Emission
by a large ensemble of
chromophores
M EX
M ex – geometry of the whole object
M em – geometry of the emitting sites

Modulation depth correlation plot
Prof. Harry Anderson, Oxford, UK
H.Anderson et al, Adv. Funct. Mater. 2008,18, 3367
M EM
1.0
0.8
0.6
0.4
PFBV.Me, Modulation Depth
1.74 nm
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
M EX
N ≈ 9 monomer units
Emitting chromophores are
different from the
absorbing chromophores

When N→∞ , M EX → M EM
H.Lin et al, JACS 2008
The whole object absorbs light
Continues distribution of the dipole moment orientations is assumed
M EX
Only N chomophores emit light
N dipoles are chosen randomly from the given distribution
M EM

Number of chromophores
1,0
1,0
N=2 4
Y Axis Title
0,8
0,6
0,4
Y Axis Title
0,8
0,6
0,4
0,2
N2
0,2
N4
6
Y Axis Title
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0
X Axis Title
X Axis Title
1,0
1,0
0,8
0,8
0,6
0,6
0,4
0,4
Y Axis Title
8
0,2
0,2
M EM
1,0
0,8
0,6
0,4
PFBV.Me, Modulation Depth
Y Axis Title
N6
N8
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0
X Axis Title
X Axis Title
1,0
1,0
N10
N12
10 12
0,8
0,6
0,4
0,4
Y Axis Title
0,8
0,6
0,2
0,2
0,2
0,0
0,0 0,2 0,4 0,6 0,8 1,0
M EX
Y Axis Title
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0
1,0
X Axis Title
1,0
X Axis Title
N14
N18
0,8
0,6
0,4
Y Axis Title
0,8
14 16
0,6
0,4
0,2
0,2
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0
X Axis Title
X Axis Title
Y Axis Title
1,0
1,0
N18
N20
F9
F10
18 20
0,8
0,6
0,4
Y Axis Title
0,8
0,6
0,4
0,2
0,2
Y Axis Title
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0
1,0
X Axis Title
1,0
X Axis Title
N22
N24
F11
F12
0,8
0,6
0,4
Y Axis Title
0,8
22 24
0,6
0,4
0,2
0,2
0,0
0,0
0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0
X Axis Title
X Axis Title

0.0 0.2 0.4 0.6 0.8 1.0
1.0
0.8
250K
0.0 0.2 0.4 0.6 0.8 1.0
1.0
200K
0.8
0.6
0.6
0.4
0.2
0.0
MEH-PPV
single chains
(toluene)
MEH-PPV
aggregates
(200 nm)
0.4
0.2
0.0
1.0
300K
1.0
Huge aggregates of
chlorophyll molecules.
0.8
0.8
200 nm long
M F
0.4
0.2
MEH-PPV
from aged
solution
0.0
0.0 0.2 0.4 0.6 0.8 1.0
M E
M F
O. Mirzov, R. Bloem, P. R. Hania, D. Thomsson, H. Lin, and I. G. Scheblykin, Small 5 (2009) 1877
M em
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
M ex
Very good internal organization
In collaboration with Prof. Alfred Holzwarth,
Mülheim an der Ruhr, Germany
0.6

Can we have more information than just polarization modulation depth ?
Yes!
We just need to learn how to read 2D plots
Conjugated polymer MEH-PPV

ϕ ex
ϕ fl
Ivan Scheblykin
λ/2
I T
CCD camera
I R
I T
I R
We have the total intensity I=I T +I R
Normalized 2D plot: I T /( I T +I R )
ET
I T
I T /I
better signal/noise
enhanced polarization pattern
Small 5 (2009) 1877

Perfect energy transfer to a fixed set of emitting chromophores
Paul Barbara, 1997
Energy funneling in conjugated polymers
Yes or No ?
Funnel
Set of absorbing
chromophores
Energy transfer
Funnel
Set of emitting
chromophores is
INDEPENDENT
on excitation
Excitation
polarization
I T /I
ϕ emission
ϕ Excit
I
I
I
I
T
R
T
T
=
=
+
IT
+ I
2
2
( 1−
mex
cos [ ϕex
+ α ]) 1−
mem
cos [ ϕem
+ β ]
2
2
( 1−
mex
cos [ ϕex
+ α ]) 1−
mem
sin [ ϕem
+ β ]
2
I = 2 ( 1−
m ) 1−
m cos [ ϕ + α ]
R
R
=
1
2
( )
( )
(
ex ex
)
( 1−
m
2
cos ϕ + β )
em
( 1−m
) [ ]
em
em
em

Two independent chromophores
Several chromophores +
some ET between them
ET
ET
Ensemble of
independent chromophores
(solution)
Funnel
ϕ emission
photoselecton
Polarization of emission is
DEPENDENT on polarization of
excitation
ϕ Excit
NO photoselecton
Polarization of emission is
INDEPENDENT on
polarization of excitation

Continuously distributed dipoles
0 0 – 90 0 I T /I I T
Energy Transfer is 0, no funnel

Continuously distributed dipoles
0 0 – 90 0 I T /I I T
Energy Transfer is 0.6 to funnel at 0 0

Continuously distributed dipoles
0 0 – 90 0 I T /I I T
Energy Transfer is 0.6 to funnel at 45 0

Continuously distributed dipoles
0 0 – 90 0 I T /I I T
Energy Transfer is 0.6 to funnel at 90 0

Continuously distributed dipoles
0 0 – 90 0 I T /I I T
Energy Transfer is 0.6 to funnel at 135 0

Polymer chain

I T /I
I T
Energy Transfer = 0
Polymer chain

I T /I
I T
Energy Transfer = 0.4
Polymer chain

I T /I
I T
Energy Transfer = 0.8
Polymer chain

I T /I
I T
Energy Transfer = 0.9
Polymer chain

I T /I
I T
ET=1 Energy Transfer = 1
Polymer chain

Almost 100% energy transfer to a funnel
Experiment
Fitting

Almost 100% energy transfer to a funnel
Experiment
Fitting

2 polarized regions (2 dipoles)
Experiment
Fitting

Independend chromophores + some funneling
Experiment
Fitting

I T (ϕ ex , ϕ fl )
Polarization ”portraites” of molecules
2D polarization single molecule imaging
Emission
polarization
angle
2D plot = Topology of the molecule
Excitation polarization angle
Energy transfer
between differently oriented
chromophores
Molecular aggregate
Luminescent macromolecules
O. Mirzov, R. Bloem, P. R. Hania, D. Thomsson, H. Lin, and I. G. Scheblykin, Small 5 (2009) 1877

SMS group
Daniel Thomsson
Rafael Camacho
Dr. Yuxi Tian
Former members
Dr. Hongzhen Lin
Dr. Oleg Mirzov
Acknowledgements
Conjugated polymers
Prof. Harry Anderson, Oxford, UK
Giuseppe Sforazzini
Chemical Physics
Prof. Villy Sundström
Prof. Tönu Pullerits