See

discussions, stats, and author profiles for this publication at:

http://www.researchgate.net/publication/272357667

Polarization portraits of lightharvesting antennas: from single

molecule spectroscopy to imaging
THESIS JUNE 2014
DOI: 10.13140/2.1.4852.5607

READS

47
1 AUTHOR:
Rafael Camacho
University of Leuven
12 PUBLICATIONS 108 CITATIONS
SEE PROFILE

Available from: Rafael Camacho

Retrieved on: 24 September 2015

Polarization portraits of lightharvesting antennas: from single

molecule spectroscopy to imaging
Rafael Andrs Camacho Dejay

DOCTORAL DISSERTATION
by due permission of the Faculty of Science, Lund University, Sweden.
To be defended at lecture hall C, Kemicentrum, Getingevgen 60, 222 41 Lund,
Thursday 5th of June of 2014 at 09:30 am.
Faculty opponent
Prof. John Lupton
Faculty of Physics, University of Regensburg, Germany

Organization

Document name

Lund University
Division of chemical physics
Organization
Department of chemistry
Lund
Organization
Organization
P.O. Box
124 University
SE-221 00, Lund, Sweden
Division of chemical physics
Lund University
Lund University
Author(s)
Department of chemistry
Division Division
of chemical
of chemical
physics physics
P.O. BoxDejay
124 SE-221 00, Lund, Sweden
Rafael Camacho
Department
Department
of chemistry
of chemistry
Author(s)
Title
subtitle
P.O. and
Box
P.O.
124 Box
SE-221
12400,
SE-221
Lund,00,
Sweden
Lund, Sweden

DOCTORAL DISSERTATION

name
Date of Document
issue
DOCTORAL
DISSERTATION
Document
Document
name
name
2014-05-12
DOCTORAL
DOCTORAL
Date
ofDISSERTATION
issue DISSERTATION
Sponsoring
organization
Date of2014-05-12
issue
Date of issue

Sponsoring
2014-05-12
2014-05-12organization

Rafael
Camacho
Dejay
Polarization
portraits
of light-harvesting
antennas: from single molecule
spectroscopy
to imaging
Author(s)
Author(s)
Sponsoring
Sponsoring
organization
organization
Title
andCamacho
subtitle Dejay
Abstract
Rafael Camacho
Rafael
Dejay
Polarization
portraits
from singleand
molecule
spectroscopy
to imaging
Title and
Title
subtitle
and subtitle
Multichromophoric
systems of
arelight-harvesting
very importantantennas:
in photosynthesis
any device
that uses
solar energy for its
operation.
This
is
because
multichromophoric
light-harvesting
antennas
are
responsible
for
the
absorption
Abstract
Polarization
Polarization
portraits portraits
of light-harvesting
of light-harvesting
antennas:antennas:
from single
from
molecule
single molecule
spectroscopy
spectroscopy
to imaging
to imagingof light and
the efficient transfer of the absorbed energy toward distinct places where it is to be used or stored. Over the last 10
Abstract
Abstract sensitive single
Multichromophoric
systems
are very
important
in photosynthesis
any the
device
that uses organization
solar energy for its
years
polarization
molecule
methods
have been
extensively used and
to study
chromophore
operation.
Thistransfer
is because
multichromophoric
light-harvesting
antennas
are responsible
for the
absorption
of light and
and
excitation
energy
processes
in
light
harvesting
antennas.
In
general,
these
methods
probe
in
separate
Multichromophoric
Multichromophoric
systems are
systems
very are
important
very important
in photosynthesis
in photosynthesis
and any and
device
anythat
device
usesthat
solaruses
energy
solar
for
energy
its for its
the efficient
transfer of the
absorbedand
energy
towardpolarization
distinct places
where it of
is tothebe sample.
used or stored.
Over the last 10
experiments
the
fluorescence
excitation
emission
properties
This
approach
operation.
operation.
This is because
This ismultichromophoric
because multichromophoric
light-harvesting
light-harvesting
antennasantennas
are responsible
are responsible
for the absorption
for the absorption
of light and
of light and
years
polarization
sensitive
single
molecule
methods
have
been
extensively
used
to
study
the
chromophore
organisation
unfortunately
averages
out absorbed
meaningful
correlations
between
thewhere
polarization
properties
of the
chromophores
the efficient
the efficient
transfer
of
transfer
the
of the absorbed
energy
toward
energydistinct
toward
places
distinct
placesit where
is to be
it used
is to or
be stored.
used
orOver
stored.
theOver
last 10
the last 10
and excitation
energy
transfer
processes instate
light- the
harvesting
antennas. In general,
these
methods
probe in separate
preferentially
light
and
thesingle
polarization
emitted
Therefore,
inthe
2009
anorganization
alternative
years polarization
yearsabsorbing
polarization
sensitive
sensitive
single
molecule
molecule
methods methods
haveofbeen
have
extensively
beenfluorescence.
extensively
used to study
used
the
to study
chromophore
chromophore
organization
experiments
the fluorescence
excitation
and was
emission
polarization
properties
of the This
sample. done
This byapproach
method
called
two
dimensional
polarization
imaging
proposed
toIndetect
these
correlations.
and excitation
and excitation
energy
transfer
energy processes
transfer
processes
in light
harvesting
in light
harvesting
antennas.antennas.
general,
In these
general,
methods
these methods
probe isinprobe
separate
in separate
unfortunately
averages
out meaningful
correlations
between
the polarization
properties
of the chromophores
measuring
a two
function
that
describes
the fluorescence
intensity
andproperties
polarization
a single
as aapproach
experiments
experiments
thedimensional
fluorescence
the fluorescence
excitation
excitation
and emission
and
emission
polarization
polarization
properties
of the sample.
of ofthe
sample.
This object
approach
This
preferentially
absorbing
light, and
the linearly
polarization
state of the emitted
fluorescence.
indevelopment
2009 an alternative
function
of
the electric
of the
polarized
light.
However,
inTherefore,
spite
unfortunately
unfortunately
averagesfields
averages
out direction
meaningful
out meaningful
correlations
correlations
betweenexcitation
between
the polarization
the polarization
properties
properties
of of
thethechromophores
of the chromophores
method was
proposed
to detect
theseextract
correlations
called energy
two dimensional
polarization
imaging.
This
is done by
of
the
technique,
the
main
challenge
still
was
the
excitation
transfer
information
from
the
data.
In an
this
preferentially
preferentially
absorbingabsorbing
light andlight
the polarization
and the polarization
state of the
state
emitted
of the fluorescence.
emitted fluorescence.
Therefore,
Therefore,
in 2009 an
in 2009
alternative
alternative
measuring
a two
dimensional
function
that describes
the
fluorescence
intensity
and polarization
of a single
object as a
thesis
we
report
the
further
developed
understanding
of
the
theoretical
and
experimental
challenges
related
to
two
method method
called two
called
dimensional
two dimensional
polarization
polarization
imaging imaging
was proposed
was proposed
to detecttothese
detect
correlations.
these correlations.
This is done
This by
is done by
function
of
the
electric
fields
direction
of
the
linearly
polarized
excitation
light.
However,
in
spite
of
the
development
dimension
polarization
imaging.
This made
possible
characterization
of the
energy
measuring
measuring
a two dimensional
a two
dimensional
function
function
that describes
that the
describes
thequantitative
fluorescence
the fluorescence
intensity intensity
and polarization
andexcitation
polarization
of a single
ofobject
a transfer
single
as object
a
as a
of of
theindividual
technique,
theharvesting
main challenge
still through
was to extract
thebased
excitation
energyfunnel
transfer
information from
the data. In
efficiency
light
antennas
a model
on
a single
function function
of the
electric
of thefields
electric
direction
fields direction
of
the linearly
of thepolarized
linearly
polarized
excitation
excitation
light.
However,
light. However,
inapproximation
spite ofinthe
spite
development
ofproposed
the development
this
thesis
we
report
the
further
understanding
of
the
theoretical
and
experimental
challenges
developed
for
two
by
us. This
method
bethe
used
to assess
the extract
quality
ofexcitation
an the
artificial
lighttransfer
harvesting
antenna
before
it
a In this
of the
technique,
of the
technique,
thecan
main
challenge
main
challenge
still was
still
wasthe
extract
excitation
energy
energy
information
transfer
information
from
thetrying
from
data. the
Inin
this
data.
dimension
polarization
imaging.
Our development
made possible
the
quantitative
characterization
thecan
excitation
device.
Further,
we
showed
that
our methodology
is notofonly
forand
studying
of experimental
single
molecules,
butof
also
thesis we
thesis
reportwe
the
report
further
the
developed
further
developed
understanding
understanding
the beneficial
theoretical
of the theoretical
experimental
and
challenges
challenges
related
torelated
two to two
transfer efficiency
of individual
light-harvesting
antennas,
such
as the LH2
complex
and chromophore
conjugated polymers,
be
used energy
as
a fluorescence
imaging
microscopy
where
parameters
related
to characterization
energy
transfer
and
dimension
dimension
polarization
polarization
imaging.
imaging.
This
made
This
possible
made
the
possible
quantitative
the quantitative
characterization
of
the excitation
of thetheexcitation
energy transfer
energy transfer
through
a
model
based
on
a
single
funnel
approximation.
This
method
can
be
used
to
assess
the
quality
of an artificial
organization
asofimaging
contrast.
Two
dimensional
imaging
combination
with the
single
funnel
efficiencyefficiency
of serve
individual
individual
light harvesting
light harvesting
antennas
antennas
throughpolarization
through
a model abased
model
onbased
a in
single
on funnel
a singleapproximation
funnel
approximation
proposed
proposed
light harvesting
antennaused
before
tryingthin
it infilms
a device.
Further,
we showed
that
our methodology
not only
beneficial
approximation
was
study
solar
cell
material,
and
is being
tested antenna
on cellistrying
cultures
and
by us. This
by us.
method
Thissuccessfully
can
method
be used
can to
beto
assess
used
to
theassess
quality
the of
quality
ofa an
artificial
of an
artificial
light harvesting
light
harvesting
antenna
before
beforeit trying
in a it in a
for studying
of single
molecules,
but also can
be used
as atechnique
fluorescence
imaging
microscopy
wherelife
parameters related
histological
samples.
The
energy
transfer
of
our
imaging
exciting
applications
device. Further,
device.
Further,
we showed
wethat
showed
our methodology
thatsensitivity
our methodology
is not
only
is not
beneficial
only beneficial
for opens
studying
for
studying
of single
of
molecules,
single in
molecules,
but sciences
also can
but also can
to energy
transfer andrelevant
the chromophore
organisation
serve as imaging
contrast.
Two dimensional
polarization
imaging
for
the
study
of
biologically
systems,
such
as
the
aggregation
of
proteins
involved
in
the
causes
ofthe
various
be used be
as aused
fluorescence
as a fluorescence
imaging imaging
microscopy
microscopy
where parameters
where parameters
related torelated
energytotransfer
energy and
transfer
the and
chromophore
chromophore
in combination with the single funnel approximation was successfully used to study thin films of a solar cell material,
diseases.
organization
organization
serve as imaging
serve as contrast.
imaging contrast.
Two dimensional
Two dimensional
polarization
polarization
imaging in
imaging
combination
in combination
with the with
singlethe
funnel
single funnel
and is being tested on cell cultures and histological samples. The energy transfer sensitivity of our imaging technique
approximation
approximation
was successfully
was successfully
used to study
used thin
to study
filmsthin
of afilms
solarofcell
a solar
material,
cell material,
and is being
and tested
is being
ontested
cell cultures
on cell and
cultures and
Key
words
opens exciting applications in life sciences for the study of biologically relevant systems, such as the aggregation of
histological
histological
samples. samples.
The energy
Thetransfer
energysensitivity
transfer sensitivity
of our imaging
of our technique
imaging technique
opens exciting
opensapplications
exciting applications
in life sciences
in life sciences
proteins involved
in the causes
of variousExciton,
diseases.Fluorescence microscopy, Fluorescence polarization, LightEnergy
molecule
spectroscopy,
for the transfer,
study
for the
of Single
study
biologically
of
biologically
relevant
systems,
relevant systems,
such as the
suchaggregation
as the aggregation
of proteins
of proteins
involved involved
in the causes
in theofcauses
variousof various
harvesting,
Chromophore,
Conjugated
Polymer
diseases.Key
diseases.
words
Classification
system
and/or
index
terms (if
any)
Energy
transfer,
Single
molecule
spectroscopy,
Exciton, Fluorescence microscopy, Fluorescence polarization, LightKey words
Key words
harvesting, Chromophore, Conjugated Polymer, Photosynthesis
Supplementary
Language
Energy transfer,
Energybibliographical
transfer,
Single molecule
Singleinformation
molecule
spectroscopy,
spectroscopy,
Exciton, Exciton,
Fluorescence
Fluorescence
microscopy,
microscopy,
Fluorescence
Fluorescence
polarization,
polarization,
Light- Lightharvesting,
harvesting,
Chromophore,
Chromophore,
Polymer
Classification
systemConjugated
and/or Conjugated
index
terms Polymer
(if any)
English
ISSN and key title
ISBN
Classification
Classification
system and/or
system
index
and/or
terms
index
(if terms
any) (if any)
Supplementary
bibliographical
information
Language
978-91-7422-356-9
English
Recipients
notesbibliographical
Number of pages 202
Price
Supplementary
Supplementary
bibliographical
information
information
Language
Language
ISSN and key title
ISBN
English English
Security classification
978-91-7422-356-9
ISSN andISSN
key title
and key title
ISBN ISBN
Recipients notes
Number of pages 202
Price
978-91-7422-356-9
978-91-7422-356-9
Recipients
Recipients
notes notes
NumberSecurity
Number
of pagesclassification
of
202
pages 202
Price Price
Signature
Date
Security classification
Security classification

Signature
SignatureSignature

Date
Date

Date 2014-04-24

Polarization portraits of lightharvesting antennas: from single

molecule spectroscopy to imaging

Rafael Andrs Camacho Dejay

Copyright Rafael Camacho

Division of Chemical Physics
Department of Chemistry
Faculty of Science
ISBN 978-91-7422-356-9
Printed in Sweden by Media-Tryck, Lund University
Lund 2014

A part of FTI (the Packaging and

Newspaper Collection Service)

Abstract

Multichromophoric systems are very important in photosynthesis and any device that
uses solar energy for its operation. This is because multichromophoric light-harvesting
antennas are responsible for the absorption of light and the efficient transfer of the
absorbed energy toward distinct places where it is to be used or stored. Over the last 10
years polarization sensitive single molecule methods have been extensively used to study
the chromophore organisation and excitation energy transfer processes in lightharvesting antennas. In general, these methods probe in separate experiments the
fluorescence excitation and emission polarization properties of the sample. This
approach unfortunately averages out meaningful correlations between the polarization
properties of the chromophores preferentially absorbing light, and the polarization state
of the emitted fluorescence. Therefore, in 2009 an alternative method was proposed to
detect these correlations called two dimensional polarization imaging. This is done by
measuring a two dimensional function that describes the fluorescence intensity and
polarization of a single object as a function of the electric fields direction of the linearly
polarized excitation light. However, in spite of the development of the technique, the
main challenge still was to extract the excitation energy transfer information from the
data. In this thesis we report the further understanding of the theoretical and
experimental challenges developed for two dimension polarization imaging. Our
development made possible the quantitative characterization of the excitation energy
transfer efficiency of individual light-harvesting antennas, such as the LH2 complex
and conjugated polymers, through a model based on a single funnel approximation.
This method can be used to assess the quality of an artificial light harvesting antenna
before trying it in a device. Further, we showed that our methodology is not only
beneficial for studying of single molecules, but also can be used as a fluorescence
imaging microscopy where parameters related to energy transfer and the chromophore
organisation serve as imaging contrast. Two dimensional polarization imaging in
combination with the single funnel approximation was successfully used to study thin
films of a solar cell material, and is being tested on cell cultures and histological samples.
The energy transfer sensitivity of our imaging technique opens exciting applications in
life sciences for the study of biologically relevant systems, such as the aggregation of
proteins involved in the causes of various diseases.

Popular Summary

Plants, algae and some bacteria are able to harvest solar energy to run chemical reactions
by a complex process known as photosynthesis. Crucial molecules in this process are
the so-called light-harvesting antennas. These antennas contain large amounts of
pigments, such as chlorophyll, that are used for solar light absorption. Moreover, these
pigments are also involved in the efficient transport of the absorbed energy towards
special sites termed reaction centres. This occurs through a process referred to as
excitation energy transfer.
Light-harvesting antennas are not only present in natural photosynthetic
organisms but also in any device that uses solar energy as fuel for its operation.
Therefore the understanding of these molecules can help us to create new and more
efficient solar based devices. This thesis reports the study of natural and artificial light
harvesting antennas. We are particularly interested in the way the pigments are arranged
in these molecules and how efficient the exchange of energy between them is.
To obtain this information we used microscopy techniques that are able to study
just one light-harvesting antenna at a time. This allowed us to investigate how different
copies of an antenna behave. Many times important information in a system are hidden
behind what is called the ensemble average. For example, consider that you want to
study how human beings look like. One way to do this study would be to go to a large
sports event and take a picture of the whole crowd. Then by analysis of the average
appearance of humans in this picture you might be able to conclude that they have two
arms, two legs and a head. However, the presence of a few red-haired persons might
pass completely unnoticed.
To characterize the orientation of the pigments in a single antenna and the
excitation energy transfer between these pigments we used light polarization. Our
methods are based on the fact that single pigments absorb and emit light polarized
along a specific direction relative to its chemical structure. You might be familiar of the
term polarization from sunglasses that use this principle to selectively block sunlight
reflected from surfaces, such as still water. Nowadays, this property is also used in movie
theatres and TV screens to display 3D movies. The polarization of light has to do with
the orientation of its electric field vector. For example, in linearly polarized light the

oscillating electric field vector is confined to a plane along the propagation direction of
the light.
By this principle, we can use the way a single antenna absorbs light to obtain
information about the orientation of the pigments in its structure. If the pigments are
randomly oriented then the antenna would absorb all polarization orientations equally.
On the contrary, if the pigments in the antenna are preferentially aligned, then light
polarized along a specific direction is preferentially absorbed by the antenna. Further,
we can use the relationship between the polarization of the light absorbed by the
antenna and the polarization of the emitted fluorescence to study energy transfer
processes between differently oriented pigments. For example, consider an antenna that
is excited by light polarized at angle . As a result, pigments that are oriented along this
direction are preferentially excited. Therefore, the emission should also be preferentially
polarized at angle in the absence of energy exchange between different pigments. On
the other hand, if pigments that are differently oriented exchange energy, then the
emission of this antenna would be polarized at a different angle.
Using our polarization sensitive single molecule technique called two dimensional
polarization imaging we were able to measure the excitation energy transfer efficiency
of individual light-harvesting antennas. This can help us to evaluate the quality of an
antenna before using it for the construction of a solar based device. Furthermore, we
showed that our methodology can also be used as a new fluorescence imaging
microscopy that uses the energy transfer sensitivity as imaging contrast. This opens new
exciting applications for our technique in the study of systems relevant for biology, such
as the aggregation of proteins involved in the causes of various diseases.

Acknowledgements

Joy multiplies when it is shared among friends, but grief

diminishes with every division. That is life.
R. A. Salvatore, Exile.
This is probably the part of this thesis that Im most afraid of making a mistake on.
Unfortunately, I believe that I will not be able to thank every person that made this
long project possible, and for that Im sorry. Nevertheless, I will do my best, and I
would like to start by thanking Ivan Scheblykin, my supervisor. You lead by example
and honesty and for that Im very grateful. Thanks for all the support, teachings,
discussions, science, and friendship. Also I would like to give special thanks to Villy
Sundstrm, I will never forget that you were the first to give me a chance to work in
Chemical Physics, by putting me into contact with Ivan when I was a master student
looking for an interesting project. Villy I believe that your continuous work and quality
as a person are the reasons why Chemical Physics has become such a great place to
work. Many thanks also to Tnu Pullerits, my co-supervisor, for the help and the
interesting discussions we had any time I came with an idea or question to you.
I also would like to thank our collaborators: Alfred R. Holzwarth, Bo Holmqvist,
Cecilia Lundberg, Christian Hansen, Eduard Fron, Frank Wrthner, Harry L.
Anderson, Jia-Yi Li, Johan Hofkens, Olle Ingans and Peter Ekstrm; without your
great samples and support I would not have been able to do the science I did.
Many thanks to Thomas and Maria, you have done my life so much easier,
bureaucracy has never been so easy. Also many thanks to Paula for all her help with the
printing of the thesis, it is always great to come across someone that does her job as
diligently as you do.
I will be ever grateful to all the people that shared this time with me at Chemical
Physics. Arkady, Donatas, Ebbe you have never run away from a scientific discussion

and neither from a good football match, thank you very much. To all the members of
Ivans single molecule group, current and former, Oleg, Hongzhen, Daniel,
Dheerendra, Sumera, Marina, Yuxi, Matthias, Aboma and Dibakar, I could not have
done this without you guys. You have been coworkers and friends. To each one of you
that fought the Berek compensator with me, Thanks! Without you I would have
thrown it out of a window. To those that will fight with it in the future, just hold on,
there is always hope and it will work out at the end, I promise.
Regarding the quote that opens my acknowledgements, I believe it to be
completely true, and I can now tell that my life as PhD student has been great. It has
been great because of you guys, my friends! Starting from the old gang of
Mllevngsvgen: Rosa, Tiago(s), Mariano, Irem, Negar, Bruno(s), Nina thanks for
the great welcome, the party, the pasta and the beer. Passing by The House (also known
as German embassy): Janina, Miguel, Mike, Natalia, Basti, Charlotte, Sara, Sonja,
Tobias, Uta, Stefano, Serena That place felt like a great home, I will not forget the
parties, the food, and the great (and not so great) music. To all the guys that played
football with me at Victoriastadion: Mohamed, Martin, Diego, Federico, Gabriel,
Miguel, Robert without the game life would be quite empty, thank you for making
every match a great one. To the amazing people in Martas caf: Cheche, Marta, Simon,
and all of the small Venezuelan gang: Marianna, Oscar, Ale, Gabriel, Genessis, Rebeca,
Henrik; ha sido un placer! Muchas gracias por todo el apoyo, la comida y el ron, se les
quiere mucho. To my friends of old in Lund: Regina, Aldo, Stef, guys I miss you and
I hope to see you again. Last but not least, to all of the guys at Lunds Aikido: Jonas,
Lina, Jakob, Peter, Giuliano, Kazumi, Jennifer, Henrik, Johan, Piotr, Gisela, Josefine,
Lisbet, Sven, Mika, Moa, Otto, Natalie, Tommaso, Reine, Robin, Sara, Simon,
Yoshi thanks for the practice, for the friendship and for giving me a place where I
could forget about work and all my problems. TO ALL OF YOU, you have been
responsible for a great deal of joy in my life these last few years, and that has no price.
Finally I would like to thank my family, the ones here, Marianne and Steve, and
the ones back at home. I love you guys. Steve I expect that you at least try to read this
thing. Marianne thank you for all the love and support, without you I would have
probably starved in the last few days of this thesis. To my family at home: Muchas
gracias por estar all a pesar de la distancia. Se les extraa todos los das, espero nos
veamos pronto

List of publications

Papers
I

Organization of bacteriochlorophylls in individual chlorosomes from

Chlorobaculum tepidum studied by 2-dimensional polarization fluorescence
microscopy.
Tian, Y., Camacho, R., Thomsson, D., Reus, M., Holzwarth, A. R., and Scheblykin,
I. G. Journal of the American Chemical Society, 133(43), 171929 (2011).
doi:10.1021/ja2019959

II

Quantitative characterization of light-harvesting efficiency in single molecules

and nanoparticles by 2D polarization microscopy: Experimental and theoretical
challenges.
Camacho, R., Thomsson, D., Yadav, D., and Scheblykin, I. G. Chemical Physics,
406, 3040 (2012).
doi:10.1016/j.chemphys.2012.03.001

III

Cyclodextrin insulation prevents static quenching of conjugated polymer

fluorescence at the single molecule level.
Thomsson, D., Camacho, R., Tian, Y., Yadav, D., Sforazzini, G., Anderson, H. L.,
and Scheblykin, I. G. Small, 9(15), 261927 (2013).
doi:10.1002/smll.201203272

IV

Inhomogeneous Quenching as a Limit of the Correlation Between Fluorescence

Polarization and Conformation of Single Molecules.
Camacho, R., Thomsson, D., Sforazzini, G., Anderson, H. L., and Scheblykin, I. G.
The Journal of Physical Chemistry Letters, 4(6), 10531058 (2013).
doi:10.1021/jz400142x

Evidence of excited state localization and static disorder in LH2 investigated by

2D-polarization single-molecule imaging at room temperature.
Tubasum, S., Camacho, R., Meyer, M., Yadav, D., Cogdell, R. J., Pullerits, T., and
Scheblykin, I. G. Physical chemistry chemical physics, 15(45), 198629 (2013).
doi:10.1039/c3cp52127c

VI

Light Harvesting Efficiency of Artificial vs. Natural Antennas a single molecule

study.
Camacho, R., Tubasum, S., Pullerits, T., and Scheblykin, I. G. Manuscript,
(2014).

VII Excitation energy transfer and emissive charge transfer states in anisotropic
polymer/fullerene blends.
Camacho, R., Meyer, M., Vandewal, K., Tang, Z., Ingans, O., and Scheblykin, I.
G. Manuscript, (2014).

Contribution to the papers:

I

Together with co-authors, I adapted the fluorescence microscope for the

measurements. My main task was to avoid polarization artefacts or correct for
them during the data analysis. I also helped in the collection and analysis of the
experimental data. I took part in the writing of the article and did computer
simulations to test our results against theoretical models of cylindrical aggregates.

II

The article is based on a new model that I developed to extract information about
the excitation energy transfer within a single multichromophoric system. This
model is used to analyse data obtained by 2D polarization imaging. I wrote the
article based in this model and included our experience and approaches to
overcome polarization artefacts in 2D polarization imaging.

III

I helped designing some of the experiments and did part of the experimental
work. Particularly, I did measurements aimed to connect the single molecule data
with data obtained at higher concentrations. I was responsible for analysing this
part of the data and I took part in the discussion and interpretation of the results.
I participated in the writing of the article.

IV

I took raw experimental data measured by D. Thomsson and analysed it with

some of the programs that I developed for paper II. I did the interpretation of the
results and tested our hypothesis via computer simulations. Based on these
interpretations and simulations, I prepared the figures and wrote the article.

I was responsible for adapting the fluorescence microscope to overcome the very
strong polarization artefacts present when near IR excitation was used for 2D
polarization imaging measurements of the LH2 complexes. I was also responsible
for maintaining and adapting the software used for the analysis of the

experiments. I did some of the data analysis, which includes those results reported
in the final article. I took part in the interpretation of the results and in the writing
of the article.
VI I gathered different sets of raw single molecule data obtained by our group over
years. I was responsible for the tuning and maintaining of the setups used to
collect most of the experimental data. I wrote the software used for the data
analysis based in the model reported in paper II. I did the analysis of the data and
interpretation of the results. On the basis of this analysis I prepared the figures
and wrote the article.
VII I built the setup used for the experiments, which is a modification of our setup
for 2D polarization imaging of single molecules. Together with M. Meyer we
wrote software to control the experiment and analyse the data. The analysis
software is based in the model reported in paper II. I performed the fluorescence
imaging experiments and data analysis. I did the interpretation of the results
and took part on the design and realization of the simulations (M. Meyer)
used to test the interpretations. I wrote most of the manuscript.

Other related publications by the author:

Fluorescence quenching in Zn2+-bis-terpyridine coordination polymers: a single
molecule study.
Siebert, R., Tian, Y., Camacho, R., Winter, A., Wild, A., Krieg, A., Dietzek, B.
Journal of Materials Chemistry, 22(31), 16041 (2012).
doi:10.1039/c2jm31237a
Collective fluorescence blinking in linear J-aggregates assisted by long-distance exciton
migration.
Lin, H., Camacho, R., Tian, Y., Kaiser, T. E., Wrthner, F., and Scheblykin, I. G. Nano
letters, 10(2), 6206 (2010).
doi:10.1021/nl9036559

Abbreviations and Symbols

APD Avalanche PhotoDiode

CP Conjugated Polymer
CT Charge Transfer
EET Excitation Energy Transfer
EM-CCD Electron Multiplying Charge Coupled Device
ETC Energy Transfer Component
FDLD Fluorescence Detected Linear Dichroism
FRET Frster Resonance Energy Transfer
GFT- Generalised Frster Theory
HOMO Highest Occupied Molecular Orbital
IR Infra Red
LD Linear Dichroism
LH2 Light Harvesting Complex II
LUMO Lowest Unoccupied Molecular Orbital
MC Monte Carlo
MD Molecular Dynamics
MEH-PPV - Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]
Fluorescence emission modulation depth
Fluorescence excitation modulation depth
NA Numerical Aperture
NoETC No Energy Transfer Component
PCBM Phenyl-C61-butyric acid methyl ester (fullerene derivative)
PFBV nonpolar butanoylated polyfluorene bis-vinylphenylene
PFBV-Rtx nonpolar butanoylated polyrotaxane polyfluorene bis-vinylphenylene
PMMA Poly(methyl methacrylate)
PVA Polyvinyl alcohol
Fluorescence emission anisotropy
Fundamental fluorescence emission anisotropy
RET Resonance Energy Transfer
SFA Single Funnel Approximation
SMS Single Molecule Spectroscopy

 Transfer matrix
TIR Total Internal Reflection
TQ1 Poly[thiophene-bis(3-octyloxyphenyl)-quinoxaline]
2D-POLIM 2 Dimensional Polarization Imaging
Light harvesting efficiency parameter

Content

Chapter 1 Introduction

Chapter 2 Theoretical background

2.1 Single Molecule Spectroscopy ................................................................... 3

2.2 Chromophores and multichromophoric systems ....................................... 8
2.2.1 Interaction between chromophores
10
2.3 Fluorescence Polarization........................................................................ 14
2.3.1 Historical background
14
2.3.2 Principles of Fluorescence Polarization
15
2.3.3 Fluorescence polarization for an isotropic set of dipoles
16
2.3.4 Fluorescence polarization for an anisotropic set of dipoles
20
2.3.5 Relation between fluorescence excitation modulation depth, ,
and the fundamental emission anisotropy,
25
2.4 Luminescence Brightness ........................................................................ 26
2.4.1 Static and dynamic quenching
26
Chapter 3 Two dimensional polarization imaging (2D-POLIM)

27

3.1 Polarization portraits .............................................................................. 28

3.2 Modulation depths and fluorescence anisotropy ..................................... 29
3.2.2 Modulation correlation plots vs.
31
3.3 Energy transfer determination in 2D-POLIM ........................................ 32
3.3.1 Light harvesting efficiency
35
3.3.3 Single funnel approximation
36
3.4 2D-POLIM as a new contrast for fluorescence imaging .......................... 39
Chapter 4 Experimental Methods

27

4.1 Fluorescence brightness .......................................................................... 41

4.2 Fluorescence decay kinetics ..................................................................... 42
4.3 Fluorescence spectrum ............................................................................ 43

4.3 Two dimensional polarization imaging ................................................... 43

Chapter 5 Polarization artefacts

46

5.1 Polarization dependent transmittance-reflectance ................................... 46

5.2 Birefringence .......................................................................................... 50
5.3 Effects of the polarization artefacts.......................................................... 51
5.3.1 Effects of the polarization dependent transmittance-reflectance 51
5.3.2 Effects of the birefringence polarization artefacts
52
5.4 Characterization and correction of polarization artefacts in 2D-POLIM . 53
5.4.1 Polarization artefacts in the excitation path
54
5.4.2 Polarization artefacts in the emission path
55
Chapter 6 Results and discussions

58

6.1 2D-POLIM and the single funnel approximation Paper II .................. 58

6.2 Natural light harvesting systems.............................................................. 59
6.2.1 Chlorosomes (Paper I)
59
6.2.2 Light harvesting complex 2 (Paper V)
61
6.3 Conjugated Polymers ............................................................................. 64
6.3.1 Quenching of conjugated polymer fluorescence at the single
molecule level Paper III
65
6.3.2 The influence quenching to the correlation between fluorescence
polarization and conformation of a single molecule Paper IV
66
6.4 Light harvesting efficiency of artificial vs. natural antennas Paper VI ... 68
6.5 Conjugated Polymers : Fullerene Blends Paper VII .............................. 70
Chapter 7 Conclusions and future work

73

7.1 Summary of the novelties in the thesis .................................................... 73

7.2 Future work............................................................................................ 74
References

75

Chapter 1 Introduction

Vision is one of humans basic senses allowing us to perceive and interact with the
world. The idiom seeing is believing shows us the importance of imaging as source of
concrete evidence. Therefore, it is not surprising that imaging techniques are present
in the core of science and engineering. The human eye has a limited resolution, which
is better explained in terms of an example: if you place a chess board with squares of
1x1cm on a wall, then at a distance of 57 metres you will not be able to distinguish
the black and white pattern, and the board will look grey in colour. However, in science
many interesting phenomena occur at scales that require a higher resolution than that
of the naked eye in order to observe them. This generates the need for devices that allow
us to observe objects that are very far away, telescopes, or that are smaller than a tenth
of millimetre, microscopes.
In natural sciences molecular interactions and chemical reactions are generally
explained on a single-molecule basis. However, our knowledge about these processes
has come mainly from experiments on large ensembles of molecules.1 These
experiments obtain an average response of the system under study, while the individual
behaviour of each molecule stays hidden. In 1959, Richard Feynman suggested ideas
about manipulating and controlling matter on the atomic and molecular scale.2 Since
then much scientific research has been done to probe single molecules and atoms.
Since their invention, microscopes have become one of the most common tools
in scientific laboratories. Hitherto, evidence suggests that the first optical microscope
was created at the end of the sixteenth century in the Netherlands.3 In 1873, Ernst
Abbe showed that the resolution of an optical microscope is limited by light
diffraction.4 This diffraction limit depends on the wavelength of the observed light, and
for the visible is 200 nm. This imposes an important limitation for the optical
detection of single molecules, which are generally smaller than the diffraction limit.
In 1989 Moerner and Kador5 reported the first optical detection of a single
molecule based on absorption. Later, in 1990 Orrit and Bernard reported the detection
of single molecules using fluorescence emission,6 which yielded much better signal-tonoise ratios than the absorption approach did. These initial methods detected the
presence of dopant molecules in a molecular crystal at liquid-helium temperature.
However, thanks to the technical advances in optics, nowadays it is becoming common
1

practice in many laboratories to detect the fluorescence of individual molecules and

nanoparticles at room temperatures.
Fluorescence microscopes can detect single molecules on the premise that other
fluorescence molecules are at distances larger than the diffraction limit.7 Under this
condition each molecule can be seen as an individual diffraction limited spot, as one
can see individual stars on the sky. On the other hand, two molecules that are at a
distance smaller than the diffraction limit but are spectrally different can also be
independently detected.8 This is because the absorption spectral differences between
the two molecules can be used to preferentially excite one of them.
Once a single molecule is detected we can use different techniques to characterize
its properties, such as fluorescence lifetime, absorption and emission spectrum, etc.
These properties depend on the interaction between the single molecule and its local
environment. Therefore, an isolated molecule can be considered as a nanometre-scale
probe of its environment sensitive to local fields and interactions in its immediate
vicinity.2,3
Note that, in general, we think of a single molecule as a system that contains a
single chromophore the part of a molecule that can absorb and emit light. However,
many molecules contain in their structure more than one chromophore. These
multichromophoric molecules are especially important in photosynthesis and any device
that uses solar energy as fuel for its operation. This is because the larger the amount of
chromophores, the larger the amount of energy that they can acquire through their
interaction with the solar radiation. These multichromophoric systems, or light
harvesting antennas, are also involved in the efficient transfer of the absorbed solar
energy towards reaction centres for its utilisation.9
The research presented in this thesis concentrates on the study of single
multichromophoric systems. We are interested in the internal organisation of the
chromophores and the excitation energy transfer between them. For this purpose we
will use primarily a fluorescence polarization technique called two-dimensional
polarization imaging (2D-POLIM). This technique is based on two general principles:
(i) the transition dipole moments for absorption and emission lie along specific
directions within the fluorescent molecules structure, and (ii) the absorption and
emission of a single chromophore is completely polarized.10 Using these principles we
can relate the polarization properties of a single multichromophoric system to the
relative orientation of the chromophores. Further, we can study the excitation energy
transfer between differently oriented chromophores because this energy exchange
induces changes between the excitation and emission polarization properties of the
system.

Chapter 2 Theoretical background

2.1 Single Molecule Spectroscopy

Spectroscopy is the study of the interaction between matter and electromagnetic
radiation with the purpose of obtaining information about the samples photophysical
properties. Classical spectroscopy studies atoms, molecules, etc. by measuring a large
number of copies of the system of interest at the same time. The average response of
the ensemble is recorded and assigned as the behaviour of the system of interest. This
logic is completely valid if the measured ensemble consists of entirely equivalent units.
However, what if the chemical or biological system under study is inhomogeneous? For
example, the spectral properties of molecules are not defined only by their chemical
structure, but also by their interactions with the surrounding environment.2 This local
environment can be different from one molecule to another, and may fluctuate over
time inducing different changes to different groups of molecules. Furthermore,
different individual copies of a large molecule may exist in different stable
conformations.2 The existence of all these events are often unnoticed in ensemble
measurements because they are hidden by the so-called inhomogeneous broadening or
ensemble averaging.11

The most direct way to completely remove the ensemble averaging is by

measuring one molecule at a time. This allows us to obtain the distribution of the
3

molecules behaviour (histogram of the samples response). Detailed analysis of this

distribution leads to critical information about the inhomogeneities in the
environment, the diversity of the samples properties, and the interaction between
environment and sample. Therefore, through single molecule spectroscopy (SMS), an
isolated molecule can be viewed, and used, as a nanometre-scale probe of its
environment sensitive to: functional groups, atoms, ions, electrostatic charges, or other
sources of local fields in its immediate vicinity.2,3

Single atoms or molecules on a surface have been successfully studied by scanningprobe microscopes, such as scanning tunnelling microscopy12 and atomic force
microscopy13, with a spatial resolution down to the sub-nanometre-scale. As a
consequence, scanning-probe microscopy has become the tool of choice for high
resolution imaging of molecules bound to surfaces. In these microscopes a physical tip
is brought into intimate contact with the sample, which consists of a relatively flat
surface with molecules of interest attached to it. As the tip is scanned across the surface,
the presence of a molecule generates a signal, for example tunnel current or deflection
of the cantilever. However, optical methods are in general more useful to study
molecules embedded in a medium because they offer the advantage of observation from
a distance, which can result in smaller perturbations of the object under study together
with a loss in spatial resolution.2,11

The first optical detection of single molecules was published in 1989 by Moerner
and Kador,5 who detected the absorption of a single molecule. Soon after, in 1990 Orrit
and Bernard reported the optical study of single molecules by means of fluorescence,6
which yielded much better signal-to-noise ratios than the absorption method did. Far
from these initial detections of a dopant molecule in a molecular crystal at liquidhelium temperature, we are now able to study single molecules in room-temperature
solutions and surfaces. This includes, for example, biological systems of interest, which
opened a new world of possibilities.11 Remarkably, the use of fluorescence for the
detection of trace amounts of biologically important molecules under physiological
conditions can be traced back to 1976, when Hirschfeld reported the detection of a
single molecule tagged with 80 to 100 chromophores.14 Because of the low background
and high signal-to-noise ratios compared to other optical methods, fluorescence has
become the most widely used SMS technique.11
The amount of fluorescence photons is usually very small in relation to the
excitation source. However, fluorescence emission is red-shifted with respect to the
excitation light. Therefore, fluorescence photons can be efficiently separated from the
overwhelming number of photons in the excitation beam by proper filtering (Figure
1).

Figure 1: Detection of single molecules by

fluorescence. The relatively few fluorescence
photons can be detected thanks to the use of
emission filters that preferentially block the
excitation light.

The red shift between absorption and emission, called the Stokes shift, is a
consequence of the relaxations steps (energy losses) occurring during the mechanism
leading to fluorescence emission (see Figure 2). The signal of a single emitter,
fluorescence brightness, is defined by its absorption cross section () and the
fluorescence quantum yield (). Despite the potentially large signal-to-noise ratios of
single molecule emission, the biggest obstacles for experimentalists in the SMS field are
the background signal and photostability of the sample.

Figure 2: Franck-Condon energy

level diagram. S0 and S1 stand for
the singlet ground state and the
first singlet excited state,
respectively. T1 is the triplet state.
ISC: intersystem crossing from the
excited singlet state to the triplet
state. Emission is red shifted in
comparison to absorption because
of the non-emissive relaxations
processes occurring before
fluorescence.

Technological improvements over the last few decades have helped in reducing
many experimental limitations, such as dark counts of the detectors, residual
fluorescence of optical elements, or unwanted emission of excitation sources in the
spectral range of the fluorescence. However, the background photons emitted by the
sample itself are much more difficult to eliminate. This emission has two main origins:
residual fluorescence from impurities, and Raman scattering from the solvent or host
matrix.
Ultra-pure solvents and matrixes have helped to gradually overcome fluorescent
impurities. However, even in extremely clean conditions, fluorescent impurities are
present and may have spectral properties similar to that of the molecules of interest.
Furthermore, to discern whether the observed diffraction limited fluorescent spot arises
from a single molecule or from a molecular aggregate is a very challenging task. While
6

many different criteria have been used to this purpose,11 we have mainly used: (i) that
the number of detected molecules should depend linearly on concentration, while the
intensity of the diffraction limited spots (molecules) should remain the same; (ii) that
the number of fluorescence photons observed for the molecules should not exceed that
expected by their and .
On the other hand, Raman scattering, which cannot be avoided, is proportional
to the volume of the illuminated sample. Therefore, the excitation volume must be
reduced to a level that allows detection of the single fluorescent object over the Raman
scattering of the surrounding environment. A strongly absorbing molecule has an
absorption cross section on the order of , while Raman scattering of a single solvent
molecule is on the order of 10 . Therefore, Raman scattering from the solvent
limits the detectability of single chromophores to volumes smaller than 100 fL.10
Several optical configurations have been proposed to limit the excitation volume.
For example, in confocal microscopy the excitation volume is reduced by focusing the
excitation beam into its diffraction-limited size. To avoid the detection of out-of-focus
emission a small pinhole is placed at the image plane. The fluorescence signal that passes
through the pinhole is detected by a photon counting system, such as an avalanche
photodiode (APD). Focusing microscopes reduce the excitation volume to 0.5 1.0
fL, and spatially resolve 180 nm in the focal plane and about 500 800 nm along the
optical axis.8,11
The excitation volume can also be reduced by decreasing the samples thickness
during the sample preparation procedure. For example, spin casting of polymer films
doped with fluorescent molecules yields films 100 200 nm thick. In this case the
most straightforward way to detect and study single molecules is by using wide-field
microscopy with epifluorescence illumination. The detection is achieved using an
electron multiplying charge coupled device (EM-CCD), which permits to image areas
of about 100x100 m with 10 ms time resolution. Wide-field imaging is a very
flexible technique15 that allows particle-tracking3,16 and, more importantly, permits to
measure more than one molecule at the time. This reduces the amount of time needed
to accumulate a statistically significant number of events to produce a reliable frequency
histogram.3 However, the signal-to-noise ratio tends to be better in a confocal
configuration, and the time resolution of current CCD technology does not match that
of APD detectors.
One of the beautiful aspects of SMS are the different forms of fluctuations, or
stochastic behaviours, displayed by a single molecule. For example, at the SM level the
intersystem crossing becomes a digital on-off process as the system jumps between
dark triplet and emissive singlet state (Figure 3-A). The observation of this process has
been termed blinking because the transition to the dark state is manifested as an
7

interruption of the fluorescent signal.17 Moreover, the nature of the dark state depends
on the molecule and is not limited to triplet states. Other types of fluctuations include
the fluorescence excitation18 and emission frequencies19,20, which can change as the
result of, for example, changes in the local environment of the molecule (Figure 3-B).

Figure 3: Fluctuations of a single molecule over time. A) Due to intersystem crossing (ISC) a single
molecule can present discrete fluorescence intensity jumps from emissive (on) to dark (off) states. S0 is
the ground state, S1 the first singlet excited state and T the triplet non-emissive state. B) The
fluorescence spectrum of a single molecule can shift over time. This type of fluctuation is known as
spectral diffusion.

2.2 Chromophores and multichromophoric systems

A molecule is formed by the binding of two or more atoms so that the total energy is
lower than the sum of the energies of the constituents. The energy level structure of
molecules is significantly more complicated than those of atoms. This is because the
total molecular wave function contains information not only about different electronic
arrangements, but also about the relative motions of the nuclei, such as vibrational,
rotational and translational motions.21 Therefore, to obtain solutions for the
Schrdinger equation of molecules we need to make some approximations. Many
different approximations with different levels of complexity have been developed, such
as the linear combination of atomic orbitals. Although there are many states in a
molecule, a good approximation for the molecular behaviour can be accomplished by
studying the frontier molecular orbitals: highest occupied molecular orbital (HOMO),
and lowest unoccupied molecular orbital (LUMO).
8

The relationship between the structure of molecules and their colours has been
studied since the end of the 19th century. In 1845, Sir J. F. W. Herschel reported the
first observation of fluorescence from a quinine solution under sunlight illumination.22
It was later noted that quinones and aromatic azo and nitro compounds are often
coloured, and that the colours are diminished or absent when the compounds are
hydrogenated.23 This allowed to relate the occurrence of colour in organic molecules to
the presence of -conjugated systems. A -conjugated system consists of alternating
single (saturated) and double (unsaturated) bounds in carbon atoms (Figure 4).
A chromophore is defined as the part of a molecule, consisting of an atom or
group of atoms, in which the electronic transition responsible for a given spectral band
with wavelength > 200 nm is approximately localised.24 In organic molecules
chromophores are usually located in the -conjugated systems. The simple quantum
mechanical model of a particle in a box can be used to understand the electronic energy
levels of electrons in conjugated compounds. The idea behind this model is that the
electrons from the p-orbitals are particles and the length of the -conjugated system is
the size of the box. Therefore, the HOMO-LUMO gap energy can be estimated by
considering the length of the box (length of the conjugation). From this
approximation it is understandable that the larger the conjugation length the lower the
energy of the transition.

Figure 4: Conjugation length and

energy of the transition. Naphthalene
(black) has a shorter -conjugated
system than anthracene (grey). As a
result the absorption and emission of
naphthalene is blue shifted in respect
to anthracene. The spectral data was
taken from the PhotochemCAD
program.

Note that in general we think of a single molecule as a system containing a single

chromophore in its structure. However, this is not always the case. A single molecule
may contain more than one chromophore. For example, in molecules containing very
long -conjugated systems chemical defects and energetic disorder can limit the
delocalisation of the electronic wave function (break the conjugation), leading to the
formation of localised chromophores or so-called spectroscopic units. 25,26 Similarly,
individual light harvesting complexes and molecular aggregates can contain a great
number of chromophores (Figure 5). These structures are referred to as
multichromophoric systems.

Figure 5: Multichromophoric systems. The

light harvesting complex II is a good
example of a multichromophoric system,
where 27 bacteriochlorophyll a pigments are
organised in two coaxial rings.27 One of the
rings contains 9 pigments while the second
contains 18.

2.2.1 Interaction between chromophores

Interactions can arise between chromophores that are separated by distances smaller
than a few tens of nanometres. This is the case for many multichromophoric systems,
which leads to, for example, excitonic delocalisation of the wave function and/or
transfer of the electronic excitation between the chromophores. The type of interaction
depends on the electronic coupling and the relative positions of the chromophores.
The study of the interaction between chromophores can be tracked to the middle
of the 20th century when researchers noted that the fluorescence quantum yield of a
concentrated dye solution is smaller than that of a dilute solution.28,29 To explain this
phenomena, it was proposed that the excitation energy can be transferred from an
excited molecule to a nearby ground state molecule. This excitation energy transfer
(EET) enables a single non-fluorescent species to gain access to a larger amount of
excited states, coming from different molecules, increasing the quenching efficiency. It
was established that this EET does not involve the simple radiative mechanism by
which the fluorescence emission of a donor is reabsorbed by an acceptor, so-called inner
filter effects.10 It was also found that collision between the donor and acceptor
molecules is not required to transfer the excitation energy.
10

In 1929, different groups described the EET process based on dipole-dipole

interactions and resonance concepts.30,31 Kallmann and London proposed a
dependence of the resonant energy transfer (RET) rate as a function of the donoracceptor distance, , while Perrin reported an
dependence. Therefore, these
theories explained energy transfer over sufficiently long distances to not require the
collision of molecules.
A full account of the theory of resonance energy transfer is beyond the scope of
this thesis. I will describe only some of the general equations. Moreover, those
interested in the experimental and theoretical antecedents are referred to the paper by
Masters,32 while for the current understanding and new directions on RET theory see
the review by Olaya-Castro and Scholes.33 The electronic coupling that transfers
excitation from the donor D to the acceptor A can be described by:
=
|
= 2(

) 2(

)+

Following the notation of Ref. 34, is the HOMO and is the LUMO of the
donor (D), and similarly and for the acceptor (A). The bar means that the orbital
contains an electron of spin , while in its absence the orbital contains an electron of
represent the overlap integrals.
spin .
,
The term 1 of equation 1 is the quantum mechanical inductive resonance,
which is a long-range electrodynamic coupling between the transition densities.35
the de-excitation of the donor resonates with the excitation of the
Through
acceptor. The terms 2 and 3 of equation 1 depend on the overlap of the donor and
acceptor molecular orbitals. These terms are typically negligible for inter-chromophore
separations greater than 5 due to their exponential dependence with distance. This is
the reason why they are neglected in Frster theory. Equation 1 can be simplified by
combining terms 2 and 3 to:
=

where
is the overall short-range interaction that depends on the degree of
overlap between the D and A molecular orbitals.
When the two molecules D and A are separated by a distance large in comparison
can be approximated as a dipole-dipole interaction between
to their size, the
transition dipole moments of the donor ( ) and acceptor ( ):
= 2(

| )

1
4

11

where is the orientation factor and

A.

is the centre-to-centre distance between D and

= 3

Frster united equation 4 with careful experimental observations29 that showed

that the EET efficiency depends on the overlap between the emission spectrum of the
donor and the absorption spectrum of the acceptor. To do so, Frster used the Fermi
golden rule to estimate the energy transfer rate in the limit of very weak electronic
coupling:28
=

( )d

where is the refractive index of the medium, which is used to describe the screening
of the electrodynamic interaction. Following the approach of the generalised Frster
theory (GFT), ( ) is the overlap between the area-normalized spectra.
The genius of Frster was his ability to relate the theory, equation 5, to purely
spectroscopic observables, which could be measured by experimentalists. This allowed
the Frster resonance energy transfer (FRET) theory to move from the field of physics
to that of chemistry, biology and applied sciences.
Within FRET the energy transfer rate can be written as:
( )=

9000(ln 10)
128

( ) ( )

where is the fluorescence quantum yield of the D in the absence of the A, is

Avogadros number, is the distance between D and A, and
is the lifetime of the
D in absence of the A. ( ) is the area normalized fluorescence spectrum of the D.
( ) is the extinction coefficient of the A, in units of M-1cm-1. Term 1 is the Frster
spectral overlap.
The Frster radius, , is defined as the inter-chromophore distance at which the
energy transfer efficiency is 50%. In other words, half of the times the energy will be
transferred from D to A, and the other half of the time the D will go back to the ground
state by its usual radiative or non-radiative decay. has proven to be particularly useful
as it allows experimentalists to think about distances rather than transfer rates:
1

=
1+
where
12

is the energy transfer efficiency.

Around the same time Frster was working on his theory, Dexter was interested
in EET processes involving dipole- or spin-forbidden transitions, such as triplet-triplet
energy transfer.36 Dexter proposed that higher multipole transition moments could
support EET. Furthermore, Dexter showed that when the donor and acceptor are very
close to each other, their molecular orbitals can overlap and this can significantly affect
the electronic coupling (term 2 in equation 2). The spectral overlap has an exponential
dependence on the D-A distance and its effect is important for separations of less than
about 5 . Although many studies present the Frster and Dexter theories as
independent, these mechanisms coexist as shown by equation 2.
In the weak electronic coupling limit, Frster theory and its modifications hold.
In this limit the EET process involves the hopping of the excitation energy between
chromophores. On the other hand, in the limit of very strong coupling exciton effects
are observed. This generally occurs in molecular aggregates and crystals. The excited
states are delocalised over several chromophores, forming a Frenkel exciton.37,38 As a
result, the absorption bands of the component molecules undergo strong spectral shifts
or splitting.
The study of these spectral shifts can be traced back to Scheibe and Jelley who
studied pseudoisocyanine (PIC) molecular aggregates.39,40 They found that the
absorption band of concentrated PIC solutions, depending on the polarity of the
solvent, could become very narrow and red-shifted with respect to that of dilute
solutions. These red-shifted aggregates receive the name of J-aggregates. In 1937, after
experimenting with different dyes, solvents, concentrations, and temperatures, Scheibe
concluded that the change in the absorption spectrum was due to the aggregation of
the dye monomers.41 On the other hand, other dyes show a shift towards the blue and
are referred to as H-aggregates (hypsochromic). Contrary to J-aggregates, H-aggregates
have broader absorption bands and are in general non-emissive.
In comparison to other organic systems J-aggregates are known to possess
extremely efficient energy migration properties. In 1939, Scheibe showed that the
fluorescence quenching of J-aggregates at room temperature was extremely efficient.42
This led to the conclusion that the excitons could move over 106 monomer dyes.
In ordered systems, like crystals, Frenkel excitons can undergo coherent transport.
This means that the exciton moves like a wave, with some scattering on defects and
phonons.43 On the other hand, in the case of very strong exciton-phonon coupling,
small resonance intermolecular interaction, or large disorder, coherence can be
completely lost.44 In this case the initially extended exciton becomes localised and
transport becomes incoherent (hopping mechanism). Thus, in most systems at room
temperature the exciton transport has been treated as incoherent.
On the other hand, hybrid systems can be found where different groups of
strongly coupled chromophores coexist. The excitation energy is delocalised within one
13

of these groups, but it can hop incoherently through space from one group (donor) to
another (acceptor).33,4549

2.3 Fluorescence Polarization

Fluorescence polarization has proven to be a powerful tool to study the conformation
and EET processes of many fluorescent systems. In this section we first present a
historical account to show how the term polarized light came to be, and the
importance of this property on the study of light. This is based on the more detailed
accounts of references 50 and 51.

2.3.1 Historical background

In the 17th century, studies about light became separated from questions about vision.
Due to the way light travels, studies concluded that light must be something that
moves, such as liquids, particles, vibrations of a fluid, or waves. The idea of liquids was
introduced as an explanation to the diffraction phenomena reported by Grimaldi in the
middle of the 1600s. Apart from its flaws, the idea of fluids introduced the notion of
the wave properties of light.
After the publications of Newtons Principia in 1687, the theory of light as a
particle became widely accepted. Newton suggested that light was made out of particles
susceptible to attractive and repulsive forces. It was at this point in time that
polarization effects were reported and included in the theory of light. In 1669
Bartholinus reported, for the first time, that light passing through Icelandic spar
(calcite) splits into two rays. This process received the name of double refraction, or
birefringence. In 1672, Huygens started to experiment with double refraction, and he
later explained this effect through a vibration theory of light. According to this theory
light was produced by vibrations of an ethereal fluid. Moreover, his findings were used
by others to support a particle theory. They used the fact that longitudinal waves cannot
show birefringence as a counter argument against Huygens theory.
In 1808 Malus reports his finding on partially reflected light. The following is an
extract from Malus memoir:
We shall begin by the simplest example and by the easiest experiment. If we let a beam
of light fall upon a surface of still water at an angle of 52o 54 to the vertical, the reflected
light has all the characteristics of one of the beams produced by the double refraction of
a crystal.

14

From these observations Malus then expanded Newtons ideas about the
asymmetry of light particles to explain both double refraction and partial reflection.
Malus suggested that the light particles had poles, in analogy to magnetic bodies.
According with this theory, when initially randomly oriented light particles pass
through calcite, they align and become ordered. Similarly, Malus explained, reflecting
surfaces could have the ability to orient the bipoles. Malus called this output polarized
light, a term that is still used today. Moreover, the name of Malus might be better
remembered for the Malus law, which describes the relationship between the intensity
of light that passes through a polarizer, and the orientation of the polarizer relative to
the incoming beam of light.
During the middle of the 1700s the vibration theory of light was supported by
scientists like Euler and Young, gaining renown thanks to the double-slit experiment
of Young. During the 19th century Fresnel supported a wave theory of light. One of the
differences between the vibrational and wave theories was that, in the former light was
a longitudinal vibration, while in the later light moved in a transverse manner. In this
question, polarization experiments supported the wave theory of light because
transverse waves could explain the fact that no light passed through crossed polarizers.
In 1873 Maxwell proposed the theory of light as an electromagnetic wave. Some
years later, Hertz carried out experiments that confirmed many of the predictions done
by the electromagnetic theory of light. The next step was a change in the paradigm of
light. In the beginning of the 20th century Einstein, Plank and others showed that the
notion of light as a continuous electromagnetic wave was not enough to explain many
phenomena related to light. They proposed that light was made from packets (quanta)
of discrete amount of energy called photons. This proposition explained how light
could have both wave-like and particle-like properties. This wave-particle duality is the
basis of the modern quantum mechanical description of light. In 1986, Grangier et al.
reported a well-designed experiment that supports the photon duality.52

2.3.2 Principles of Fluorescence Polarization

Let us first consider the interaction between a chromophore and linearly polarized
excitation light. The excitation probability ( ) of a chromophore depends on the on
the angle, (Figure 6), between the electric field vector of the incident light ( ) and
the absorption transition dipole moment of the chromophore ( ):
~(

) ~ cos

Furthermore, the transition dipole moments for absorption and emission of most
chromophores lie along specific directions within the structure.10,53 As consequence,
15

when the system is illuminated by linearly polarized light, those chromophores with
transition dipoles oriented along the electric vector of the incident light are
preferentially excited. This is known as excitation photoselection.
Now consider the fluorescence emission of a single chromophore. Based on the
specific direction of the emission dipole, the fluorescence of a single chromophore fixed
in space is linearly polarized. As a consequence, the fluorescence intensity of a
chromophore after passing through a polarizer is given, according to Malus law, by:
=

cos

where,
is the angle between the emission transition dipole moment of the
chromophore ( ) and the transmission axis of the polarizer (Figure 6).

Figure 6: A single transition dipole excited with linearly polarized light, and observed through a linear
polarizer. The single dipole is oriented with angle relative to the electric field of the excitation light
relative to the transmission axis of the polarizer.
and angle

2.3.3 Fluorescence polarization for an isotropic set of dipoles

There are two main parameters used to characterize the fluorescence polarization
resulting from the excitation of an isotropic ensemble by linearly polarized light:
emission anisotropy ( ) and polarization ratio ( ). is defined as:24
=

+2

10

where and are the intensities measured with the transmission axis of the emission
polarizer parallel and perpendicular, respectively, to the electric field of the excitation
light (Figure 7). The denominator in equation 10 is proportional to the total
16

fluorescence intensity of the sample due to the cylindrical symmetry of the

chromophore distribution around the excitation axis.

Figure 7: Emission anisotropy

measurements. Excitation travels along
the axis with electric field oriented
along . Observation is carried out from
the axis (90 degrees to excitation
propagation).

On the other hand,

is defined as:24
=

11

Generally, emission anisotropy is preferable over the polarization ratio because

is additive.10,24 Assume a sample containing several emitting species with contribution
and fractional intensities , the average anisotropy (r) is given by:
=

12

To describe the effects of photoselection on an isotropic sample we follow the

approach of Galanin.54 Let us assume a set of randomly oriented chromophores fixed
in space. The excitation is carried out by linearly polarized light with electric field
oriented along the axis, and observation is carried out from the axis. The orientation
of the transition dipole moment is determined by the polar angle and the azimuthal
angle (Figure 8). interacts with the component of the electric field directed along
the direction of the chromophore according to equation 8. To calculate the radiation
intensity of the dipole along each Cartesian axis it is convenient to split the dipole
moment into:

17

cos

sin cos

sin sin

13

Figure 8: Emission intensities for a single

chromophore in a Cartesian coordinate
system. The electric field is oriented
along the axis. The orientation of the
dipole is determined by the polar angle
and the azimuthal angle .

The radiation intensity along each axis is proportional to:

~

cos

= cos

cos

cos

= cos

sin

cos

cos

= cos

sin

sin

14

To calculate the contribution from all the dipoles in the system we average the
dipole distribution over the polar and azimuthal angles. After excitation the population
of excited chromophores is symmetrically distributed around the axis. Hence, the
average value of cos
is given by:
cos

cos

1
2

15

On the other hand, the number of dipoles between the polar angles and +
is proportional to sin
(surface angle of the sphere). Thus, the average value of
and cos sin are given by:
cos

18

cos

cos

sin

sin

1
5
16

cos

sin

cos

sin

sin

sin

2
15

Using equations 14-16 we are able to calculate the emission anisotropy and
polarization ratio for isotropically oriented chromophores fixed in space:
=
=

+2

= 2 5 = 0.4
17

= 1 2 = 0.5

= 0.4 is obtained for isotropic ensembles of dipoles where no depolarizing

events occur after the initial formation of the excited state, and receives the name of
fundamental emission anisotropy.
Note that the fluorescence emission of isotropic samples can be polarized after
excitation by natural light, if the observation is carried out at an angle to the excitation
light. The highest degree of polarization is obtained when the observation is
perpendicular to the direction of the excitation.
Let us assume a parallel beam of natural light traveling along the direction of the
axis, while the observation is carried out from the axis. In order to simplify the
equations, the excitation light can be represented as two equal incoherent components
with electric fields directed along the and axes (Figure 9). Consequently, the total
intensity of the system along the and axis is:
=
=

+
+

18

Figure 9: Dipole representation for natural illumination. Natural light, C, can be represented as the
sum of A + B.

19

The fluorescence intensity generated by the component along the and axis
is equivalent to our previous calculation (equation 14), and will be labelled
=
and
= (Figure 9-A). On the other hand, the fluorescence intensity generated
component corresponds to our previous calculation but observing from the
by the
axis. Therefore,
and
are equal to each other and will be labeled (Figure
9-B):

= +

=2

19

Combining equation 19 with equations 14-16, the emission anisotropy and

polarization ratio for isotropically oriented chromophores under normal illumination
(Figure 10) is given by:
= 14
=1 3

20

Figure 10: Fluorescence anisotropy and

polarization ratio for normal illumination.
Excitation is done by natural light traveling
along the axis. Observation is carried out
from the axis (90 degrees to excitation
propagation).

2.3.4 Fluorescence polarization for an anisotropic set of dipoles

For isotropic samples all directions in space are equivalent. On the other hand, for
anisotropic samples different results will be obtained for different polarization
orientations of the linearly polarized excitation light. Linear dichroism (LD) is one of
the most used polarization sensitive techniques to study anisotropic samples. LD
measures the difference in absorption for two perpendicular linearly polarized beams:24
LD =

20

21

and
are the absorption spectra measured with the excitation polarized
where
parallel and perpendicular to the samples orientation axis, respectively. LD yields
information about molecular orientations and structural properties related to molecular
symmetry.
LD measurements employ different alignment methods in order to generate
anisotropic samples of known orientation axis. For example, laminar flow of solvents
can be used to orient molecules depending on their physical shape, the stretching of a
film causes molecules to orient in the direction of the stretch, and electric fields can
orient molecules that have permanent dipole moments.
Isolated single multichromophoric systems tend to have unknown anisotropic
organisation. Therefore, measurements of the fluorescence detected linear dichroism
(FDLD) could be used to obtain information about their structure. Unfortunately the
spin casting procedure, generally used for preparing SMS samples, generates a random
orientation of the isolated multichromophoric systems in relation to the laboratory axis.
This makes the FDLD approach insufficient as the orientation axis of the anisotropic
single molecule is unknown. To solve this problem, the fluorescence intensity of the
sample, , as a function of the excitation polarization angle,
, must be
unambiguously determined. This is accomplished by measuring the fluorescence
) is obtained at more than two excitation
excitation modulation depths where (
angles (Figure 11).

Figure 11: Fluorescence intensity of an

isotropic collection of chromophores as a
function of the excitation polarization
angle used.

Let us assume a small collection of randomly oriented chromophores fixed in

space. The excitation is carried out by linearly polarized light traveling along the axis
with electric field that can be rotated in the - plane (Figure 11). The orientation of
the linearly polarized light is defined by its angle respective to the axis,
. The
21

observation of the fluorescence intensity is carried out from the axis. Therefore, the
component of the chromophores does not contribute to the measured intensity.
According to equation 8, (
) is proportional to:
(

)~

sin ( )

cos (

22

where the orientation of each dipole, , is determined by its polar, , and azimuthal,
, angle. Term 1 is the component of the transition dipole moment able to interact
with the electric field. Term 2 is the angular dependence (
) of the excitation
probability.
Any linear combination of cosines having the same frequency, , but different
phase shifts, , is also a cosine with frequency , but a potentially different phase shift
( ):
cos (

)=

cos ( ) +

23

where A and B are constants.

We are interested in rewriting the previous equation in terms of the maximum
and minimum intensities:
=
cos ( ) +

=(

+ ;

Equation 25, after using the identity cos

rearranging, takes the form:
cos ( ) +

+
2

1+

24

) cos ( ) +

25

= (1 + cos(2 ))/2 and some

cos(2

26

where term 1 is the average value of the cosine function and term 2 is the modulation
depth.
Using equation 26, equation 22 is rewritten as:
(

)= 1+

cos(2

27

where is the average fluorescence intensity of the system over

,
is the
fluorescence excitation modulation depth, and
is the fluorescence excitation phase.
values range from 0 1 for large ensembles of isotropically oriented chromophores
22

and perfectly oriented (collinear) systems, respectively. On the other hand,

represents the main orientation axis of the system.
is equivalent to the FDLD obtained when: (i) the observation is carried out
from the same direction as the incident light, and (ii) the parallel orientation is defined
as
. Note that a small ensemble of randomly oriented chromophores is not able to
equally probe all angles in the solid sphere. Therefore, this type of ensembles are in
general anisotropic and have
> 0.
To the best of our knowledge the first fluorescence excitation polarization study
on single molecules was reported in 1993 by Gttler and co-workers.55 They studied
the transition dipole moment orientation of pentacene molecules with respect to the
crystallographic structure of the host p-terphenyl crystal. Since then different groups
have tried to use the fluorescence excitation polarization to obtain structural
information of multichromophoric systems. In particular, much research has been done
on the chain morphology of conjugated polymers.
In 2000, the research group led by Paul Barbara used Monte Carlo (MC)
simulations, employing a bead-on-a-chain model, to show that the
histograms
obtained for isolated MEH-PPV molecules was consistent with stiff polymers adopting
a highly ordered, collapsed conformation.56 Later molecular dynamics (MD) methods
were also used to simulate the conformation of single conjugated polymers.57 MC and
MD simulations have been used extensively to try to connect the conjugated polymer
chain morphology to
histograms5662 and further rationalise the conformational
impact of different chain lengths63 and defects6466. Similarly,
histograms have been
used to distinguish between aggregated and non-aggregated forms of conjugated
polymers.67,68
As for the case of FDLD, it is difficult to define , or , for anisotropic samples
of unknown organisation and unknown main axis of orientation. To overcome this
difficulty we can define the fluorescence emission modulation depth. Let us assume
again a small collection of randomly oriented chromophores fixed in space. In this case,
the excitation is carried out by natural light traveling along the axis (Figure 12). The
observation of the fluorescence intensity is carried out from the axis with a polarizer
placed before the detector. The transmission axis of the polarizer is rotated in the plane, and its orientation is defined by its angle respective to the axis,
. According
) is proportional to:
to Malus law, (
(

)~

sin( )

cos (

28

where term 1 is proportional to the excitation probability of each dipole.

By using equation 26, equation 28 can be written as:
23

)= 1+

cos(2

29

where is the average intensity of the system over

,
is the fluorescence
emission modulation depth, and
is the fluorescence emission phase.
values
range from 0 1 and
represents the main emission axis of the system. Note that
is equivalent to the polarization ratio obtained for natural illumination when: (i)
the observation is carried out from the same direction as the incident light, and (ii) the
(Figure 12).
parallel orientation is defined as

Figure 12: Relation between

and the polarization ratio under natural illumination for an
anisotropic object. Observation is carried out from the same axis as the excitation light is propagating.

(
) and (
) have the same functional form and
=
if each
chromophore in the system has equal fluorescence quantum yield and collinear
transition dipole moments for absorption and emission. However, interactions between
chromophores in multichromophoric systems can result in EET. As a consequence, the
effective transition dipole moment responsible for the fluorescence emission can be
different than the initially excited one. This can induce a difference in the orientation
of
relative to . Therefore, for single multichromophoric systems fixed in space,

indicates the presence of EET between differently oriented

chromophores.
Already 60 years ago, S. I. Vavilov introduced fundamental ideas for the
polarization studies of anisotropic samples.69,70 Vavilov discussed the possible
dependency of the emission polarization on the orientation of the linearly polarized
excitation light in organic crystals. These experiments were later performed for several
molecular crystals and concluded that the EET in these systems is very efficient 71.

24

2.3.5 Relation between fluorescence excitation modulation depth,

and the fundamental emission anisotropy,

and
depend on the angular distribution of the dipoles in space, as described by
equations 14 and 22, respectively. We are interested in characterizing the relationship
between
and for an ensemble of dipoles anisotropically oriented in the absence
of EET. To do so the fluorescence signal arising from a large collection of identical
dipoles was calculated numerically. The angular distribution of the dipoles can be
arbitrarily changed from isotropically oriented to uniaxial towards the axis. Moreover,
the angular distribution of the dipoles is always cylindrically symmetric around the
axis. For calculation of both
and
the linearly polarized excitation propagates
along the axis and observation is carried from the axis.
To obtain we determine and and calculate the emission anisotropy according
to equation 10. The sample is excited with the electric field oriented along the main
orientation axis, i.e., the axis. is the radiation intensity of all dipoles along the
axis, while
is the radiation intensity of all dipoles along the axis. On the other
hand, for the
calculation we have to determine
and
(see equation 26).
Due to the cylindrical symmetry of the dipole distribution,
and
are obtained
when the excitation light is polarized with electric field along the and
axis,
respectively. In both
and
we calculate the sum of radiation intensity of all
dipoles along the and axis. The results of this numerical calculation are presented
in Figure 13.

Figure 13: Fluorescence excitation

modulation depth vs. fundamental emission
anisotropy for an ensemble of dipoles
cylindrically symmetric around the
alignment direction.

25

2.4 Luminescence Brightness

The ability of a fluorescent object to emit light due to photoexcitation is proportional
to the product between the absorption cross section ( ) and the fluorescence quantum
yield (), also known as the fluorescence excitation cross section:
= . The
later can be experimentally characterized via the luminescence brightness ( ):68,72,73
=

30

where is the fluorescence intensity detected in units of

, and
is
the excitation power density given in units of
. depends on the detection
efficiency of the setup and thus is setup-specific. The way to compare the measured
in different laboratories is to measure a standard dye and use it as a reference.
measurements are used to estimate the number of effective chromophores in a
multichromophoric system. An effective chromophore: (i) absorbs excitation light, and
(ii) the energy absorbed by it is, at least partly, emitted later as fluorescence. On the
other hand, if the excitation energy initially absorbed at a chromophore is quenched
(no matter where in the system) then its = 0 and we refer to this chromophore as
dark.

2.4.1 Static and dynamic quenching

Fluorescence quenching can be the result of different processes. These can be divided
into two: static and dynamic quenching. Dynamic quenching involves processes that
provide a non-radiative decay for an excited state. As consequence, the fluorescence
intensity of the quenched chromophore is reduced and the lifetime becomes shorter.
On the other hand, static quenching involves the interaction of a fluorescent molecule
with a quencher in the ground state. Due to this interaction, the properties of the
chromophore change and it becomes non-fluorescent at the excitation wavelength.
In order to illustrate one important difference between dynamic and static
quenching, let us consider a group of identical chromophores where some are
quenched. In the case of dynamic quenching the lifetime of the overall system becomes
shorter. On the contrary, in the case of static quenching the lifetime of the systems does
not change as the quenched molecules do not go to the excited state.

26

Chapter 3 Two dimensional

polarization imaging (2D-POLIM)

The use of solar radiation for the

production of energy is thought to become one
of the main sources of renewable energy for
human kind. To accomplish this challenge
efficient devices that use light as fuel must be
produced. Natural photosynthetic organisms
are great examples of efficient machineries that
harvest solar energy and use it to run a range of
chemical reactions.9 Any device, natural or Figure 14: Light harvesting antennas.
artificial, that uses light as fuel for its operation
contains a light-harvesting antenna. This antenna is responsible for the absorption of
light and the efficient transport of this energy to special sites where it is to be used or
stored (Figure 14). In photosynthetic devices, light-harvesting antennas contain
multichromophoric ensembles having large absorption cross section.
Throughout this thesis we address the study of light harvesting
multichromophoric systems. Using SMS we obtain information about the internal
configuration of the chromophores and the excitation energy transfer (EET) efficiency
between them. We concentrate on the EET between energetically similar
chromophores, so-called homo-EET.7476 Therefore, we cannot use spectral differences
between donors and acceptors to access EET.
Fluorescence anisotropy is one of the few parameters that is sensitive to homoEET.54,77 It is widely used in ensemble spectroscopy to obtain information about the
relative orientation of absorbing and emitting dipole transitions, and some information
about the EET. However, as mentioned in chapter 2 there are principle differences
between using fluorescence anisotropy at the ensemble and single molecule level.
Basically, for isolated multichromophoric systems a contribution of unknown spatial
orientation, unknown geometrical organisation and unknown internal EET makes this
approach very unpractical.
27

There are single molecule techniques probing in independent experiments

polarization properties in fluorescence excitation,56,58,62,7880 emission8187 or even linear
dichroism for large individual natural light-harvesting antennas.88 Comparison of
statistical information (histograms) on the fluorescence excitation (
) and emission
(
) modulation depths can provide some information about the efficiency of
internal EET.60,89,90 However, the quantitative sensitivity of this approach is very low
because correlations of the emission polarization properties as function of the excitation
polarization, which are the most sensitive to EET, are missing. To overcome this issue,
in 2009 a new method called two-dimensional (2D) polarization single-molecule
imaging was reported.91
2D polarization imaging (2D-POLIM) is a powerful polarization technique that
can be seen as an improvement of the fluorescence anisotropy and fluorescence detected
linear dichroism experiments. 2D-POLIM characterizes the correlation between the
emission polarization of the sample and the electric fields direction of the linearly
polarized excitation light. The fluorescence intensity of the sample is measured as a
function of the excitation and emission polarization analyser angles,
and
,
,
combinations a 2D polarization portrait
respectively. Using several different
is built.91,92 The polarization portraits data depends on the internal organisation of the
multichromophoric-object, in terms of the orientation and number of chromophores,
and the EET between them. In spite of the existence of the technique, the main
challenge is to extract the energy transfer efficiency from the data. In paper II we
reported our efforts to solve this problem, which we summarize in this chapter.
It is worth mentioning that, to our knowledge, the first simultaneous
characterization of fluorescence excitation and emission polarization properties of a
single molecule was done by Hofkens et al., in 1998.93 In this approach the emission
polarization was characterized only at two fixed emission polarization orientations,
contrary to 2D-POLIM. Later this technique was used to obtain information about the
EET properties of a multichromophoric system, which consisted of eight
chromophores.94

3.1 Polarization portraits

When an isolated multichromophoric system is measured through 2D-POLIM, the
single object is excited by linearly polarized light with polarization angle at the sample
plane
. The fluorescence intensity is measured through a polarizer with transmission
axis at angle
. Both
and
are measured relative to the laboratory axis.
28

The fluorescence intensity measured is a 2D

) that receives the name of
function (
,
polarization portrait (Figure 15). The numerical
aperture of the objective lens must be kept low to
avoid: (i) excitation of the out of plane component
of the transition dipole moments, and (ii) collection
of light polarized perpendicular to the sample plane.
The task of a 2D-POLIM measurement is to
unambiguously determine the polarization portrait.
Let us consider the degrees of freedom of a Figure 15: Polarization portrait
polarization portrait. Any cross section of the portrait
at a fixed
, or
, contains the contribution of a cos dependency for each dipole
in the system, according to equations 8 and 9. Therefore, in the most general case a
cross section can be seen as the summation over many cos dependencies with the same
frequency. According to equation 23 the cross sections then have the functional form:
(

cos (

)+

cos (

)+

31

where , and
are constants, and define the 3 degrees of freedom of each cross
section. Theoretically, measuring the fluorescence intensity at three different angles is
enough to calculate unambiguously the parameters in equation 31. Consequently, a
polarization portrait can be determined by measuring the fluorescence intensity at a
). Experimentally,
minimum of 3 3 = 9 different combinations of angles (
,
the quality of a polarization portrait depends on the goodness of fit of each cross section,
which can be obtained by measuring a few points with very good accuracy, or many
points with lower signal to noise ratio.

3.2 Modulation depths and fluorescence anisotropy

As discussed in chapter 2, the modulation depths for fluorescence excitation (
) and
emission (
) are the equivalents of the fluorescence detected linear dichroism, and
polarization degree under natural illumination for single anisotropic objects. The
modulation depths describe the dependence of the fluorescence intensity over one of
the polarization angles,
or
. These can be obtained by integration of the
polarization portrait (Figure 16).
29

Figure 16: Visualization of the

polarization parameters extracted
from a polarization portrait. The
modulation depths and phases are
calculated through integration.
On the other hand, the
fluorescence anisotropy is
calculated from two points in the
portrait, once the main axis of
orientation is known ( ).

For example, integration over

(

) =

yields

= 1+

cos(2

):
32

This equation is equivalent to equation 29, where is the average intensity of the
,
is the fluorescence emission modulation depth, and
is the
system over
fluorescence emission phase.
On the other hand, integration of the polarization portrait over the emission angle
):
(Figure 16) yields (

) =

= 1+

)
cos(2

33

This equation is equivalent to equation 27, where is the average intensity of the
,
is the fluorescence excitation modulation depth, and
is the
system over
fluorescence emission phase. The difference between the fluorescence excitation and
30


. In
emission phases receives the name of luminescence phase shift,
=
the absence of the systems rotation during the excited lifetime,
0 indicates the
presence of EET between differently oriented chromophores.
The main axis of orientation of the transition dipoles that participate in the
fluorescence excitation is given by
. Therefore, we are able to calculate the
fluorescence anisotropy under the assumption that the fluorescence emission of the
system is cylindrically symmetric around the axis of the sample plane (Figure 16).

(
(

,
,

) (
)+2 (

,
,

+ )
+ )

34

Note that this is generally not the case for single multichromophoric systems. The
excitation anisotropy of a nanoparticle can be approximated using the so-called
absorption ellipsoid. In the simplest case, this ellipsoid is rotationally symmetric around
long axis of the object. However, this axis does not need to lie parallel to the sample
plane. Optical microscopy methods have been used to measure the 3D orientation of
single molecule absorption transition moments.95,96 Besides the far-field excitation used
to obtain the projection of the dipole moments into the sample plane, the molecules
can also be excited with optical near-fields by total internal reflection (TIR). The
component of the electric field for TIR is then used to also calculate the component
of the transition dipoles.

3.2.2 Modulation correlation plots

vs.

As mentioned in section 2.3.4,

occurs in systems where the emission

dipoles are differently oriented than the initially excited ones. For multichromophoric
systems fixed in space, this effect is generally the result of EET between differently
oriented chromophores. Typical representations of this information in literature consist
of modulation depth histograms.62,90,97 Moreover, when
and
are obtained
simultaneously for each molecule we can generate the
,
correlation plot
(Figure 17). The modulation correlation plot shows the relationship between the
excitation and emission polarization properties of each individual molecule. This
correlation, which is lost in the histograms, can be used to obtain extra information
about the systems EET properties.89,91

31

,
correlation plots. The scatter plot is the most common representation.
Figure 17:
However, for large data the probability density is more illustrative. The data presented in this figure
belongs to isolated light harvesting complex II and was used for paper V and VI.

3.3 Energy transfer determination in 2D-POLIM

The following is a general description of the fluorescence intensity function
(
) of multichromophoric systems measured by 2D-POLIM. We are
,
particularly interested in nanoparticles immobilized in a solid matrix. Consequently,
we assume that the transition dipole moments for fluorescence excitation and emission
do not change their orientation in the time frame of the measurements.
To simplify our equations, we assume that in
each chromophore the transition dipole moment for
fluorescence excitation and emission are collinear.
Further, 2D-POLIM measurements are done using
low NA objectives lenses and thus we consider the
projections of the dipole moments onto the sample
plane (Figure 18). Let us assume a single system that Figure 18: Projection of the
consists of
chromophores without EET between transition dipole moments onto
them. Following our previous notation, the function the sample plane.
(
) can be described by:
,

(
32

)=

cos (

) cos (

35

is proportional to the fluorescence excitation cross section of the dipole

where
(

sin ( )
).
Although
can be different for each dipole in the system, every single
can
always be represented by an arbitrary number ( ) of identical chromophores ( )
oriented at
such as:

36

where the notation is given in magnitude, angle , and is an integer. This principle
can be used to find a standard in plane dipole ( ) that satisfies the following
condition for all dipoles (Figure 19):

37

is an integer.
can be seem as the lowest common denominator of the
where
initial system of
potentially different dipoles. Using this logic any
multichromophoric system can be represented by a new set of = standard
dipoles.
Figure 19: Example of a 3-dipole
system where the projections onto the
sample plane can be represented by a
new system of = 6 identical dipoles.
For simplicity the length of the vectors
is related to their fluorescence excitation
cross section.

Using this standard dipole system equation 35 can be rewritten as:

(

)=

cos (

) cos (

38

33

However, if there are EET processes between the chromophores, then the
excitation energy of chromophore has some probability of being emitted from a
different chromophore . Therefore, to fully describe the fluorescence emission two
) takes the form:
summations are needed, and (
,

)=

cos (

cos (

39

where,
is a matrix describing the EET between the different chromophores, and
receives the name of transfer matrix. Each element of
has values between 0 1 and
designates the probability of the excited state created at chromophore to be emitted
from chromophore . In this simple description non-radiative decays can be omitted
because all chromophores have the same fluorescence excitation yields. Therefore,
energy conservation leads to:

40

Note that so far the mechanism behind the EET processes occurring within the
single multichromophoric system has not been specified. The transfer matrix describes
the EET process regardless of its nature. However, if there is enough knowledge about
the internal organisation of the chromophores in the system, the elements of the
transfer matrix can be defined according to specific transfer mechanisms, such as Frster
resonance energy transfer.
Two different models have been used to extract EET information from a
polarization portrait. The first, introduced in 2009, characterizes the EET through the
| /2.91 This angle describes how
so-called average rotation angle, 0 |
large the average net change of the emission vs. excitation polarization orientations is
due to the EET process. The second, introduced in paper II, characterizes the EET
through the light harvesting efficiency, 0 1.92 In this model quantitatively
describes the amount of energy that each dipole in the system transfers to a single pool
of dipoles, responsible for emission after the EET processes (EET-emitter). In the
following section we present a full description of this model.

34

3.3.1 Light harvesting efficiency

The principle behind this approach is to split the
) into two
fluorescence intensity function (
,
components having clear physical meaning: energy
transfer component (ETC), and no energy transfer
component (NoETC). Let us consider the elements of
the transfer matrix,
, introduced in equation 39.
The diagonal elements of
describe the amount of
energy that each dipole does not transfer, while the offFigure 20: Transfer matrix
diagonal elements describe the ET behaviour (Figure
20).
Therefore, by splitting the diagonal and off-diagonal components of the transfer
matrix we obtain:
(

)=

41

42

43

Note that in equations 42 and 43

1 for simplicity, which can be seen as a
fluorescence intensity normalization. We can define the energy transfer efficiency (0
1) of chromophore as:
=

44

After some rearrangements and using equation 23-26, equations 42 and 43 can
be rewritten as:
=

(1 )

1
1+
2

cos 2

45

46

35

where is the energy transfer efficiency of chromophore to a pool of chromophores

responsible for the fluorescence emission after EET, referred as EET-emitter. The EETemitter has modulation depth
and orientation .
The general equations 45 and 46 help us to understand the complexity of the EET
processes in multichromophoric systems. For each dipole in a multichromophoric
system there are 4 degrees of freedom: (i) orientation, (ii) amount of energy transfer,
(iii) modulation of the EET-emitter, and (iv) orientation of the EET-emitter. In
practice, the situation is even more difficult because the number and orientation of each
chromophore is unknown in most nanoparticles.
Equation 44 unambiguously describes the energy transfer efficiency of each
chromophore through . However, to define the energy transfer efficiency of a
multichromophoric system as a whole is not a trivial task. One reasonable approach is
to average over all chromophores ( ). While specifically describes the average EET
efficiency of the chromophores, it does not describe transfer efficiency of a
multichromophoric system as it is generally understood. For example, consider a large
multichromophoric system where there is very efficient local EET between
neighbouring chromophores (small domains), but no EET among different local
domains. In this case,
would be very large, while the EET over the whole system is
absent. Therefore, it is very important to think about the characteristics of the EET
processes that we would like to have in single multichromophoric antennas.
A property of paramount importance, when considering light-harvesting devices,
is the ability of the system to transport the excitation energy towards a special site for
its utilisation or storage. This property is generally thought to arise from: (i) preferential
EET towards a localised acceptor made of one or several chromophores, so called EET
funnelling, or from (ii) efficient energy migration over all the chromophores the system
is made of. Both situations can be explained by EET towards a single energy acceptor,
where the EET-emitter is either a specific pool of chromophores or the whole system
itself. Therefore, to characterize the EET efficiency of multichromophoric
nanoparticles we compare the experimental data with the behaviour expected from a
light-harvesting antenna with a single EET-emitter. This description receives the name
of single funnel approximation.

3.3.3 Single funnel approximation

In a multichromophoric system with a single funnel, the EET-emitter of all
chromophores have same phase, , and polarization modulation,
. Therefore
equation 46 takes the form:
36

1+

cos 2

47

where the degrees of freedom have been considerably reduced. However, we are not
able to solve equations 45 and 47 for the most general case, where the number of
chromophores and their orientation is unknown, because the degrees of freedom of the
equations overwhelm those of the polarization portrait.
To overcome this problem we propose a simplified model where all chromophores
transfer the same amount of energy ( ) towards a single EET-emitter. This model
receives the name of single funnel approximation (SFA). Within the SFA the
and
are described by:

= (1 )

1+

cos 2

48

49

where characterizes the light harvesting efficiency of the whole nanoparticle towards
the single EET-emitter. One of the main advantages of this approach is that we can
unambiguously relate the equations to experimental observables, such as
and
,
as we will see below.
To use the SFA to characterize the light harvesting properties of nanoparticles,
there is one last limitation to overcome: the unknown organisation of the
chromophores ( and ). The chromophore organisation is responsible for the
fluorescence excitation polarization properties of the system, and also it describes the
. Therefore, a geometrical model is designed to fit these properties.
We use a geometrical model based on a symmetric three-dipole-model (Figure
21), which is based in our previous research.91 The orientation of the main dipole ()
defines the orientation axis of the system. Both side dipoles have the same fluorescence
excitation cross section and angle respective to the main dipole (). Thus, the
geometrical model has 3 degrees of freedom: , , and the ratio between the
fluorescence excitation cross section of the main and side dipoles ().

37

Figure 21: Three-dipole-model. For simplicity the

length of the vectors is related to the fluorescence
excitation cross section of the dipoles. The oval is
proportional to the fluorescence intensity that would
be measured as a function of the emission analyser
orientation.

defines the possible values for and :

The experimentally determined

(,

)=

( + 2)
1
arccos
2
2

50

)
)

2(1 +
(1

51

On the other hand, is related to the main orientation axis of the

multichromophoric system, and thus =
. Note that equations 50 and 51 are
defined only for 0
< 1. For
= 1 a special case is defined and modelled by
a geometrical configuration with a single dipole oriented at =
.
The second summation in equation 49 can be written in terms of
and
(using equation 26), while equation 48 is rewritten in terms of the three-dipole-model
to obtain:

(1 )
2+
(
+
(
+

1
1+
4

)
+

cos(2

)
(

) 1+

cos 2

52

53

where the +3 indicates the use of the three-dipole-model and (2 + ) is a

normalization term that ensures:
1
1

38

54

3.4 2D-POLIM as a new contrast for fluorescence

imaging
EET applications have grown substantially over the years and found use in
biology, biochemistry and polymer science.98 While we have discussed the role of EET
within the field of conjugated polymers and light harvesting systems, we would like to
briefly address the use of EET in life sciences. Particularly, fluorescence imaging
techniques sensitive to EET have been used in medical research, to visualize and track
physiological and pathological processes.75 For example, FRET imaging has been used
for tracking protein-protein interactions, which are involved in many cellular activities
as well as in the underlying causes of various diseases, such as Alzheimers, Parkinson
and mad cow/Creutzfeldt-Jakob.
Most of the available EET microscopies rely on the interaction between two
components, the EET pair, generally labelled with different dye molecules.99101 After
excitation of the donor, EET induces emission from the acceptor molecule, which
occurs only when both molecules are in close proximity. Due to the distance
dependence of EET, this is limited to distances smaller than 10 nm.
Fluorescence anisotropy microscopy has been used to study samples of randomly
oriented chromophores.102 As mentioned in chapter 2, EET results in a decrease of the
measured . In these techniques molecular rotations are detrimental. However, this can
be usually ignored since EET and fluorescence emission take place more rapidly than
the molecular motions of the large fluorescence proteins used for labelling.75 Probably
the greatest advantage of using polarization sensitive microscopies is that they are the
only method capable of measuring homo-EET. This property has been used to measure
monomer-dimer transition of GFP-tagged proteins,103 and to quantify protein cluster
sizes.76,104 On the other hand, fluorescence anisotropy measurements are difficult to use
for quantitative discrimination of EET due to the instrumental artefacts, and the
intrinsic anisotropy of some samples.75
It is precisely for these reasons that our experience and methodologies developed
for single molecule measurements can be used to improve the current status of the
fluorescence polarization microscopies. A 2D-POLIM measurement involves the
recording of a sequence of fluorescence images. This data can be used not only to
analyse the signal from diffraction limited spots, but also to generate the polarization
portrait for each pixel in the image. After analysis of these portraits, the 2D-POLIM
parameters can be used as new imaging contrasts. Thus, in addition to imaging the
samples intensity, we can generate images for:
1. Fluorescence excitation modulation depth (
) and phase ( ).
2. Fluorescence emission modulation depth (
) and phase (
).
39

3.
4.
5.
6.

Luminescence phase shift ( ).

Fluorescence anisotropy ( ).
Light harvesting efficiency ( ).
Properties of the EET-emitter (

and

).

This promising application for 2D-POLIM is the latest development of our work.
We have so far used 2D-POLIM for the successful analysis of the EET properties of
polymer films (paper VII). On the other hand, its use for the study of biologically
relevant systems, such as aggregation of proteins, although promising, is in the stage of
proof of principle and thus is not included in this thesis.

40

Chapter 4 Experimental Methods

In this chapter we do a small overview of the fluorescence microscopy setups used for
the studies reported in this thesis. All setups are home-built modifications of the
commercially available Olympus IX71 inverted microscope. Although the setups are
adapted for the specific spectral requirements of the samples, the general scheme of each
experiment can be divided into two parts: excitation control, and emission collection.
Both of these units are specially built depending on the purpose of the setup.

4.1 Fluorescence brightness

The purpose of this setup is to carefully measure the fluorescence intensity of the sample
as a function of the excitation power. Therefore, the excitation control is the most
critical element of the experiment. A Berek polarization compensator was used to
obtain circularly polarized excitation at the sample plane and avoid photoselection of
the chromophores (Figure 22). In order to obtain large statistics we imaged the sample
in wide-field configuration, and thus, the excitation light is defocused at the sample
plane. The excitation power profile of the laser was carefully characterized at the sample
plane to account for inhomogeneities. The sample was measured under a nitrogen
atmosphere to avoid photo-oxidation. The fluorescence detection was done by an
electron multiplying charge coupled device (EM-CCD). In order to diminish the
photo-damage of the samples the excitation power was kept low and the exposure times
large, 10 s.

41

Figure 22: Setup for fluorescence brightness measurements.

4.2 Fluorescence decay kinetics

The fluorescence excitation kinetics of the sample were measured using a pulsed laser
as excitation source together with a time correlated single-photon counting (TCSPC)
unit and an avalanche photodiode (Figure 23). In order to keep the flexibility of the
setup the microscope was coupled to an EM-CCD camera and the excitation light is
defocused to accomplish wide-field illumination. The EM-CCD camera is used to
image the sample plane and locate the nano-object of interest for the lifetime
measurement. Then the light of this object was selected by a pinhole and sent towards
the avalanche photodiode using a flipping mirror.

Figure 23: Setup for fluorescence decay kinetics.

42

4.3 Fluorescence spectrum

For spectral measurements the microscope was used in wide-filed configuration.
The fluorescence was collected, imaged onto a slit, passed through a transmission
holographic diffraction grating, and imaged onto an EM-CCD camera (Figure 24). In
principle the slit is not needed to obtain the spectrum of a single molecule as its image
is diffraction limited. However, the slit was used to obtain the spectrum of larger
luminescent objects and to ensure that the spectrum of different single nano-objects
did not overlap with each other. The spectral sensitivity was calculated using a standard
tungsten incandescent lamp. The instrumental response function had a spectral full
width at half-maximum (FWHM) 5 nm.

Figure 24: Setup for

fluorescence spectrum. The
holographic diffraction
grating is placed in a
flipping mount. The zero
order of diffraction
produces an image while
the first order gives the
spectrum

4.3 Two dimensional polarization imaging

The purpose of a 2D-POLIM setup is to determine the polarization portrait of
). This portrait is then used for the calculation of the
the sample, (
,
polarization parameters as explained in chapter 3. In the 2D-POLIM setup the
excitation unit was used to control the orientation of the linearly polarized excitation
light,
, while the emission unit was used to probe the polarization state of the
emission that reaches the detector (Figure 25).

43

Figure 25: Setup for 2D polarization imaging. The physical orientation of the excitation controller
( ) and emission analyser (
) is controlled by stepper motors. Both stepper motors and the EMCCD camera are controlled by a home-written LabVIEW software.

Within the excitation unit, the device that prepares the linearly polarized light at angle
receives the name of excitation controller. The excitation controller can be
constructed in various ways. Hitherto, we have experimented with two different
constructions (Figure 26). In the first, randomly or circularly polarized light goes
through a polarizer. The orientation of the polarizers transmission axis ( ) defines
the orientation of the linearly polarized output light,
=
. In the second
approach, linearly polarized light goes through a half-wave (/2) plate. The /2 plate
orientation (
according to:

44

) defines the orientation of the linearly polarized output light

=2

Figure 26: The excitation controller. We have used two constructions for the polarization controller: a
polarizer and a /2 plate. When a polarizer is used the input must be circularly or randomly polarized
is the physical orientation of the
light, while for the /2 plate the input is linearly polarized light.
optical element in the excitation controller.

When using laser light for excitation, the /2 plate approach has proven to be the
most practical because the output of lasers tends to be highly polarized. However, for
unpolarized light sources we recommend to use the polarizer approach.
In the emission collection unit, the emission analyser consists of a polarizer that
is used to probe the polarization state of the collected fluorescence. The orientation of
the polarizers transmission axis (
) defines the emission polarization angles probed
by the detector,
=
.
Although the principles behind polarization measurements are simple, in practice
it is quite difficult to accomplish reliable experimental results due to the presence of
detrimental polarization artefacts. The characterization and correction of these
experimental artefacts are of paramount importance for the success of a 2D-POLIM
experiments and are discussed in detail in chapter 5.

45

Chapter 5 Polarization artefacts

Polarization artefacts are the most difficult experimental challenge that polarization
sensitive fluorescence microscopes have to overcome. Polarization artefacts can affect
the polarization state of both the excitation light and the collected emission. These
artefacts can be divided into two according to their origin: (i) polarization dependent
transmittance-reflectance, and (ii) birefringence. In this chapter we describe the origin
of these artefacts, and explain our approaches to characterize and correct for these
problems in the excitation and emission paths.

5.1 Polarization dependent transmittance-reflectance

Let us consider reflection and transmission in terms of geometrical optics. The mirrorlike reflection of light from a surface (specular reflection) is described by the law of
reflection,105 which states that: (i) the incident ray, reflected ray, and the normal to the
surface are in the same plane known as plane of incidence (Figure 27); (ii) The incident
and reflected rays have equal angle against the normal to the surface; (iii) The reflected
and incident rays are on opposite sides of the normal to the surface.
Figure 27: Reflection and transmission in terms of
geometrical optics. The figure depicts the plane of
incidence containing the incident, i, reflected, r,
and refracted, t, rays. According to the law of
reflection the incident ( ) and reflected ( ) rays
have the same angle against the normal. The
velocity of the light in both media is different
because of the different indices of refraction.
Therefore, the angle of incidence ( ) is different
than the angle of refraction ( ). This is described
by the The Snell-Descartes law, see text.

Regarding transmission, the relationship between the angles of incidence ( ) and

refraction ( ) is described by the Snell-Descartes law:
46

sin( )
=
sin( )

55

where, and are the indices of refraction in the two media (Figure 27). According
to this law, < for > , and vice versa.
On the other hand, Fresnels equations describe the amount of light that is
reflected and transmitted when it encounters uniform planar interfaces.105 These
depend on the polarization state of the incident light and the angle of incidence ( ).
Within Fresnels description light of different polarization states is described as a linear
combination of s- and p-polarized light. Light polarized with its electric field parallel
to the plane of incidence is said to be p-polarized. On the contrary, light polarized
perpendicular to the plane of incidence is said to be s-polarized. For s-polarized light
the reflected ( ) and refracted ( ) electric field amplitudes are related to the incident
electric field amplitude ( ) by:
=

56

=
where:
=

cos
cos

cos
cos

2
cos

cos
+ cos

57

On the other hand, for p-polarized light the relationships are:

=

58

=
where:
=

cos
cos

cos
cos

2
cos

cos
+ cos

59

Figure 28 shows the Fresnel amplitude equations for < . Note that and
can have negative values due to the phase shift of the electric field after reflection on
the interface. Furthermore, there is an angle of incidence for which the p-polarized light
is not reflected, = 0. This angle receives the name of Brewsters angle ( ).
47

Figure 28: Fresnel amplitude equations for < . Left: Electric field amplitude reflected for s- and
p-polarized light as a function of the angle of incidence. Right: Electric field amplitude transmitted
for s- and p-polarized light as a function of the angle of incidence. For this calculation = 1 and
= 2.

Using the Fresnels equations and Snell-Descartes law we find that the Brewsters
angle is determined by:
= tan

60

To understand how this effect acts as a polarization artefact it is more intuitive to

work with the Fresnels power equations. We are interested in calculating the
reflectance ( ), which is the ratio between the reflected and the incident power, and
the transmittance ( ), which is the ratio between the transmitted and incident power:

61

62

where:
63
| |
2
The reflected and incident rays propagate through the same medium and have the
same angle with the normal. As a consequence, both beams have the same area ( =
) and the reflectance for the s- and p-polarized components can be written as:

48

=| |

64

On the other hand, the refracted and incident rays travel in different media (
) and consequently have a different angle to the normal. Therefore, :
=

cos
cos

65

Thus we have:
=

cos
cos

cos
cos

| |
66

According to the Snell-Descartes law, when light encounters a medium of lower

index of refraction > (Figure 29, > ). As a result, there is a critical angle of
incidence, , for which
is equal to 90 degrees, and
=
= 1. For incident
angles larger than total internal reflection occurs and the light is trapped inside the
medium. We can calculate using the Snell-Descartes law:
= arcsin

67

Figure 29: Fresnel power equations for > . Left: Reflectance for s- and p-polarized light as a
function of the angle of incidence. Right: Transmittance for s- and p-polarized light as a function of
the angle of incidence. For this calculation = 1.5 and = 2.

49

In fluorescence microscopy the excitation and emission light goes through

different optical elements. Each of these elements has an index of refraction different
than air. As a consequence the polarization state of the light transmitted by the optical
elements can be different to that of the incident light, according to the Fresnels
equations. Equations 66 shows that this effect occurs for optical elements which optical
axis does not coincide with the direction of the incident light (Figure 29). For example,
flat filters and mirrors which planes are not perpendicular to the light propagations can
introduce polarization artefacts. Therefore, such optical elements should be avoided in
polarization sensitive setups as much as possible.

5.2 Birefringence
Light travels through a transparent material by interacting with the atoms within the
medium. In a classical description, the electric field of the light interacts with the
electrons in the material and they radiate.105 This secondary radiation interferes with
the initial wave resulting is the refracted wave that moves on. The speed of the light in
the material is determined by the difference between the frequency of the electric field
and the natural frequency of the atoms. This is characterized experimentally by the
refractive index of the material ( ). Thus, the phase velocity of the light inside the
material is given by: = / .
The simplest class of materials are those with cubic symmetry. In this material the
index of refraction does not depend on the polarization and propagation direction of
light. Thus, for any set of reference axes
=
= . As a consequence, light travels
with the same phase velocity in all directions and its polarization properties remain
unchanged after travelling through the optically isotropic material.
On the other hand, some materials have anisotropic optical properties and are
referred to as birefringent. The simplest birefringent material is known as uniaxial,
meaning that only one axis has different refractive index,
=
. Generally,
this unique axis is called the extraordinary axis ( ), while the other two are referred to
as ordinary ( ). When light goes through a birefringent material, the polarization
component of the light that propagates in the plane defined by the extraordinary axis
has a different phase velocity. Thus, this polarization component of the light acquires
a phase retardation relative to the light components polarized along the ordinary
directions. As a result the polarization properties of the light change as it travels through
the material.

50

5.3 Effects of the polarization artefacts

The polarization dependent transmittance-reflectance and the birefringence artefacts
have different effects on the light that goes through the microscope. Using these
differences, experiments can be designed to characterize and accurately correct the
depolarization artefacts of a particular experimental setup.

5.3.1 Effects of the polarization dependent transmittance-reflectance

When linearly polarized light encounters an interface with polarization dependent
transmittance the transmitted light is also linearly polarized (Figure 30). Let us consider
the incident linearly polarized light as a linear combination of s- and p-polarized
components. These components are transmitted to a different extent, according to
Fresnels equations (see Figure 29). As a consequence we observe two effects: (i) the
polarization orientation of the transmitted linearly polarized light ( ) can be different
than that of the incident light ( ), and (ii) the intensity of the transmitted light is
dependent on the polarization orientation of the incident light ( , Figure 30).

Figure 30: Effects of polarization dependent transmittance on linearly polarized light. The incident (I,
black) linearly polarized light arrives at three different angles. The corresponding transmitted light is
can be
portrayed in grey colour (T). Due to the lower transmittance of the s-polarized component
different than . However, the transmitted light remains linearly polarized. Further, the intensity of the
transmitted light (T) changes with the polarization orientation of the incident light ( ), in the figure
>
> .

On the other hand, when elliptically or randomly polarized light encounter a

polarization dependent transmittance interface, the transmitted light has a different
polarization degree than the incoming light. The effect on randomly polarized light is

51

of particular interest as it can be used to characterize the extent of the transmittance

artefacts in the setup.
Let us consider randomly polarized light that reaches an interface with
polarization dependent transmittance. The transmitted light is polarized with a
modulation depth that depends on the difference between the transmittance of the sand p-polarized components (Figure 31). Further, the polarization phase of the
transmitted light ( in Figure 31) defines the orientation dependence of the
transmittance depolarization effect.

Figure 31: Effects of transmittance

depolarization on an incident beam of
randomly polarized light. Due to the different
transmittance of the s- and p-polarized
components the transmitted light is elliptically
polarized. The modulation depth of the
transmitted light ( ) defines how large the
depolarization effect is. The polarization phase
( ) defines the orientation dependence of the
transmittance.

5.3.2 Effects of the birefringence polarization artefacts

Wave plates are one of the most common applications of birefringent materials in
spectroscopy. For example, a half-wave plate (/2) changes the orientation of linearly
polarized light by an angle of 2 , where is the angle between the input polarization
orientation and the wave plates ordinary axis (fast axis). A properly oriented quarterwave (/4) plate converts linearly polarized light into circularly polarized light.
However, some of the optical elements used in polarization sensitive setups are
unexpectedly birefringent. The sum of all these unwanted birefringent effects acts as a
/ plate, where is unknown. As a consequence, when polarized light goes through
the setup the polarization state of the light changes. These effects are particularly
difficult to characterize and correct because they are wavelength dependent.
52

5.4 Characterization and correction of polarization

artefacts in 2D-POLIM
Based on our experience we suggest three general rules when constructing a polarization
sensitive fluorescence microscope. First, avoid optical elements that are not
perpendicular against the propagation of light because these elements introduce
transmittance polarization artefacts (see Fresnels equations). Second, mechanical stress
can make optical elements birefringent (photoelasticity). This birefringence can be
quite large and should not be underestimated. Third, experiments must be designed to
test the quality of the setup.
Beam splitters are the heart of fluorescence microscopes that work with
epifluorescence illumination. The most common beam splitter consists of a dichroic
mirror placed 45 degrees to the optical path (Figure 32). The dichroic mirror is
designed to reflect the excitation light towards the sample, while only transmitting the
collected emission towards the detector. Unfortunately dichroic mirrors can introduce
strong polarization artefacts to both excitation and emission due to their complicated
design (many thin layers of dielectric material). An alternative approach will be
discussed below.

Figure 32: Filter cube and dichroic mirror. A) Physical appearance of a filter cube. B) Structure of a
filter cube: The dichroic mirror rests at 45 degrees to the excitation beam, it reflects the excitation
light and transmits the fluorescence emission. Extra excitation and emission filters are mounted in the
filter cube. Note that the emission filter is not perpendicular to the collected fluorescence in most
filter cubes to avoid back reflections. This can result in polarization artefacts.

In a 2D-POLIM experiment the excitation and emission light have important

differences. While excitation is linearly polarized and spectrally narrow, emission can
have any degree of polarization and is spectrally broad. Due to these differences we
characterize and correct the polarization artefacts in the excitation and emission paths
separately. Let us first consider the polarization artefacts in the excitation path.
53

5.4.1 Polarization artefacts in the excitation path

In a 2D-POLIM experiment we must excite the sample at specific excitation
polarization angles (
) with linearly polarized light and constant excitation intensity.
This excitation polarization control is especially sensitive to birefringence artefacts and
also, to a lower degree, to polarization dependent transmittance.
The first critical step is to ensure the proper functioning of the excitation
controller (see section 4.3). For example, if a polarizer is used, then partially polarized
light before the analyser would result in a dependence of the excitation intensity with
the orientation of the polarizer ( ). On the other hand, if a /2 plate is used, then
an imperfect wave plate or partially polarized light as input to the plate would result in
an elliptically polarized output.
After the excitation controller there are several optical elements placed before the
sample plane that can affect the polarization state of the excitation light. To test for
these polarization artefacts a polarizer is used to determine the polarization degree of
the excitation light at the sample plane as a function of the orientation of the excitation
controller, . We usually observe that the linearly polarized output of the excitation
controller becomes depolarized on its way to the sample plane. Such depolarization of
linearly polarized light is a consequence of birefringence artefacts. This birefringence is
corrected using a Berek polarization compensator placed after the excitation controller
(Figure 33). The Berek is tuned to introduce a phase retardance opposite to that of the
optics. This procedure makes the light at the sample plane linearly polarized regardless
of .

Figure 33: Berek polarization compensator for

birefringence correction. The optics between the
excitation controller and the sample plane work
as a / plate. The Berek compensator is used
to introduce a retardance opposite to that of the
optics. This way the polarization at the sample
plane remains linear.

54

On the other hand, transmission artefacts may still be present. As a result: (I) the
excitation polarization angle,
, obtained at the sample plane may be different than
that expected by , and (II) the excitation intensity may depend on (Figure 30).
To correct for artefact (I) we experimentally find what orientation of the excitation
controller produces the best polarized excitation at each desired
. During this
procedure it is important to realize that imperfections in the /2 plates make the
/

polarization state of the excitation light different for

and
+ /2. To correct
. This
for artefact (II) we measure the excitation intensity ( ) as a function of
function is used during the analysis procedure to correct the undesired dependence of
the fluorescence intensity with
due to the excitation polarization artefacts:
(

)=

(
max

)
(

68

5.4.2 Polarization artefacts in the emission path

After correct excitation of the sample, the fluorescence must reach the emission analyser
without changes in its polarization properties. In addition, the sensitivity of the detector
on the polarization properties of the incident light must also be considered. The control
of the polarization artefacts in the emission path presents an extra complication in
comparison to the excitation. Namely, the fluorescence emission is spectrally broad and
thus: (i) the polarization artefacts can affect differently different parts of the emission
spectrum, and (ii) a Berek compensator cannot be used to correct for birefringence
artefacts.
To characterize the birefringence polarization artefacts present in our setup we
developed the so-called artificial molecule (AM). The AM consists of a polarizer placed
on the sample plane with a solution of the sample of interest on top of it (Figure 34,
paper II). The solution is used to produce fluorescence emission with the same spectral
properties as the sample to be measured. The name artificial molecule is given to this
device because it absorbs and emits light as a single dipole. Therefore, the polarization
portrait of an AM must have
=
= 1 and
=
=
, where
is
the orientation of the AM on the sample plane.

55

Figure 34: Artificial molecule (AM). When the

excitation light reaches the AM only the
component polarized parallel to the transmission of
the polarizer goes through and interacts with the
sample. Thus the AM absorbs light as a single
chromophore. On the other hand, the isotropic
emission of the solution becomes completely
polarized after passing through the polarizer.
Therefore, the AM emits as a single chromophore.

The AM procedure consists of measuring the polarization portrait of the AM at

different
. Emission birefringence results in
< 1, and

. The way
to correct for these artefacts is to carefully choose and reduce the number of the optical
elements used in the setup. Experience demonstrates that dichroic mirrors can
introduce large emission birefringence. For example, for excitation in the near IR region
the dichroic mirror behaved almost as a /4 plate (paper II). This birefringence tends
to be larger at wavelengths close to the transmission edge of the dichroic. Therefore,
using a long pass filter to remove this spectral region from the detection has proven to
be very useful.
In some cases a dichroic mirror cannot be used due to its extremely large
depolarization artefact. To solve this problem, we have successfully used a so-called
polarization preserving universal beam splitter, which replaces the dichroic mirror in
the standard filter cube (Figure 35, paper II). The universal beam splitter consists of a
glass plate with anti-reflection coatings on both sides and very small metal mirror in
the middle (diameter 1 mm). The excitation light is focused on the plane of the small
mirror by a carefully chosen wide-field lens (WFL), to a diameter small enough to be
fully reflected by the small mirror. The focal distance of the WFL is selected in such a
way that the excitation light is focussed by the microscope objective lens above its focal
distance, and generates wide-field illumination of the sample. The fluorescence
collected by the microscope objective results in a beam of much larger diameter than
the small mirror. Therefore, the light obstruction of the mirror is negligible. The
birefringence of the universal beam splitter has proven to be much smaller than that of
dichroic mirrors for the near IR region of the spectrum (paper II). Further, this beam
splitter can be used for a very broad range of excitation wavelengths, which increases
the flexibility of the microscope.
56

Figure 35: Polarization preserving

universal beam splitter. For this
beam splitter it is very important to
have high quality emission filters as
the excitation light can be reflected
back to the detector. For single
molecule detection usually more
than one filter is needed.

On the other hand, to characterize the transmittance artefacts in emission we

measure the polarization portrait of an isotropic emitter (section 5.3.1). The isotropic
emitter consists of a solution of the sample of interest. A correction function (
) is
calculated from the
and
obtained for the isotropic emitter:
(

)=1+

cos 2

69

Note that this function also accounts for the sensitivity of the detector.
Finally equations 68 and 69 are used during the analysis procedure to obtain the
fluorescence intensity corrected for the excitation and emission transmittance artefacts,
:
(

)=

(
(

,
)

70

) is the polarization portrait obtain from the experiments.

where (
,
Using these procedures in the visible region using dichroic mirrors we were able
0.01,
to obtain, for example, for excitation at 488 nm, the following accuracies:
0.03 and phase uncertainty of about 1-2 degrees. For AM experiments it
corresponds to 0.96
1.00 and 0.95
1.00. On the other hand, for
the near IR using the universal beam splitter we obtained accuracies of:
0.03,
0.05 and a phase uncertainty of about 2-5 degrees. For AM experiments it
corresponds to 0.94
0.98 and 0.95
0.99. For these values the
excitation controller consisted of an achromatic waveplate (Thorlabs), while the
emission analyser consisted of a wire-grid polarizer (Edmund optics).

57

Chapter 6 Results and discussions

In this chapter we describe the study of light harvesting multichromophoric systems.

The results presented are based on the papers attached at the end of this thesis. We
concentrate on the internal configuration of the chromophores and the excitation
energy transfer (EET) processes among them. For this purpose we use the methods
described in chapter 5. Although our focus has been mainly in the field of single
molecule spectroscopy, our latest contribution uses 2D-POLIM as a fluorescence
imaging tool to study preferentially oriented bulk polymer/fullerene films.

6.1 2D-POLIM and the single funnel approximation

Paper II
In paper II we describe the general problem of extracting intramolecular excitation
energy transfer (EET) from fluorescence polarization experiments at the single molecule
level. We derive a general model to obtain the EET efficiency of an individual
multichromophoric system from the polarization data obtained by two dimensional
polarization imaging (2D-POLIM). This model does not require any previous
knowledge about the samples structure or energy transfer mechanism, and receives the
name single funnel approximation (SFA, fully described in chapter 3). The SFA
quantitatively describes the EET efficiency of individual antennas through a parameter
called light-harvesting efficiency, . We also present important technical details about
2D-POLIM and practical methods to avoid polarization artefacts in fluorescence
microscopy. These artefacts are the most difficult experimental challenge of polarization
sensitive techniques (see chapter 5).

58

6.2 Natural light harvesting systems

Nature uses complex machineries to harvest solar energy and run chemical reactions
that are necessary for sustaining life. This process is known as photosynthesis and occurs
in plants, algae and some bacterial organisms. All of these organisms contain so-called
light-harvesting antennas. These antennas are involved in two key processes of
photosynthesis: the absorption of solar energy and the efficient transfer of the absorbed
energy towards reaction centres for its utilisation.9
Due to the increasing demand of energy, much research is now focused onto using
solar light as a source of renewable energy. In this regard there are numerous efforts to
mimic the natural process of photosynthesis. 106114 One of the crucial elements of these
studies is the understanding of photosynthetic organisms. Particularly, any device that
intends to use light for its operation must contain a light-harvesting antenna. Therefore,
the study of natural light-harvesting antennas can help us to ultimately create new and
more efficient solar based devices.

6.2.1 Chlorosomes
Chlorosomes are the natural light harvesting systems of green sulfur bacteria.115 The
ability to efficiently harvest light energy is of paramount importance for these bacteria
because they live in extremely low light conditions. Chlorosomes are ellipsoidal bodies,
of approximately 200x100x30 nm, containing hundreds of thousands of
bacteriochlorophyll (BChl) molecules enveloped in a lipid membrane, which makes
them the largest antenna systems in nature.116,117
The light harvesting structures of chlorosomes consists of BChl c, d, or e
molecules, which self-organise in supramolecular structures without the inclusion of
proteins. The energy absorbed by these pigments is efficiently transferred to the base
plate, which is located in the membrane and consists of BChl a and proteins. This
energy is then further transferred towards the reactions centre.
The closely packed BChl of the light harvesting structure form excitonically
coupled systems, similar to J-aggregates, giving rise to a strongly red-shifted absorption
spectrum and large exciton diffusion length.118120 However, there has been a long
debate on the supramolecular structure of the self-assembled BChl in chlorosomes. To
date, several structural models have been proposed: rod-shaped, lamellar and rolled-up
models.117,121,122 One of the main problems to study these structures is that chlorosomes
come in many different sizes and thus crystallization approaches are not possible.
Therefore, optical spectroscopies have been used to reveal structural information.
59

Organisation of the light harvesting supramolecular assemblies of chlorosomes

Paper I
Despite many efforts to elucidate the structure of the light harvesting supramolecular
assemblies of chlorosomes, the issue of large structural disorder is still discussed. In
paper I we report the fluorescence spectrum and 2D-POLIM study of individual
chlorosomes. The chlorosomes were grown and isolated under very mild and carefully
controlled conditions from w.t. C. tepidium bacteria. For single chlorosome studies the
stock solution was diluted (~50 times) and spin-cast onto a glass substrate.
We did not observe evidence of large structural disorder between individual
chlorosomes. The fluorescence spectra of individual chlorosomes were nearly identical
to that of chlorosomes in solution, and were independent of the excitation wavelength
used. The fluorescence emission polarization properties of all individual chlorosomes
were essentially identical (
~0.77, Figure 36) and independent on the excitation
wavelength. On the other hand, the fluorescence excitation modulation depth were
always narrowly distributed, and the mean value of this distribution depended on the



excitation wavelength (
~0.69,
~0.38 and
~0.78, Figure
36).

Figure 36: Modulation correlation plot for chlorosomes excited at 633 (A), 458 (B) and 750 nm (C).
Figure adapted from paper I.123

After the spin-casting procedure, chlorosomes lay on the surface with their long
axis parallel to it because of their elongated shape. These individual chlorosomes are
also expected to orient more or less randomly around their long axis due to the
hydrophobicity of the lipid membrane. Considering these spatial constrains and the
uniformity of the polarization properties, we concluded that the total excitonic
transition dipole of chlorosomes possesses a cylindrical symmetry (Figure 37). This is
60

in agreement with previously suggested supramolecular structures of the self-assembled

BChl.

Figure 37: Chlorosomes on the

sample plane with their long axis
parallel to the surface and transition
dipole with cylindrical symmetry.

The optical properties of chlorosomes are defined by the strong excitonic coupling
between the BChls arranged in a J-type aggregate. The dependence of the
values
with the excitation wavelength is in good agreement with previously reported linear
dichroism data.124 Further, we could demonstrate that the
values are also in
agreement with theoretical calculations based on a concentric tubular organisation of
the BChls.

6.2.2 Light harvesting complex 2

The light-harvesting complex 2 (LH2) is the peripheral light-harvesting system present
in most species of purple bacteria. The structure of the LH2 complex has been
determined through X-ray crystallography27,125 with a resolution of ~2 . The LH2
consists of a hollow cylinder with C9 symmetry which contains two coaxial rings of
BChl a (Figure 38). The BChl a are the pigments involved in the light capture of the
complex. These two rings are known as B800 (9 BChl) and B850 (18 BChl) due to
their Qy absorption maximum. The BChl molecules in the B800 ring are separated by
a distance of ~20 , and thus each pigment can be seen as an individual chromophore.
On the other hand, the B850 BChls are ~9 away from each other and are
excitonically coupled. This conformation leads to efficient (1 2 ) EET from the
B800 chromophores to the B850 excitonic states.126128

61

Figure 38: The light harvesting complex II (LH2). Left: Organisation of the pigment in the LH2.
Right: Absorption spectrum of LH2 in solution (dash line) and emission spectrum of an isolated LH2
complex on the surface.

Excited state localisation and static disorder in LH2 at room temperature Paper V
In paper V we report the polarization properties of individual LH2 complexes studied
by 2D-POLIM. Two separate experiments were performed to study the symmetry of
the B800 and B850 states. In each experiment the B800 or B850 ring was preferentially
excited using a spectrally narrow laser line that matched its Qy transition (Figure 38).
Moreover, the fluorescence detected in both experiments is emitted by the B850
excitonic states due to the previously mentioned EET from B800 to B850, regardless
of the excitation wavelength (Figure 38). Therefore, each experiment selectively probed
the excitation polarization of the B800 or B850, while probing the emission
polarization of the indirectly or directly populated excitonic states of the B850 ring,
respectively.
For both experiments a modulation correlation plot was obtained. Both plots
display a clear departure above the diagonal: emission occurs from states significantly
more polarized than excitation (
>
, Figure 39 A-B). On the other hand, the
B850 ring was a more isotropic absorber than the B800 (
<
, Figure 39
C).

62

Figure 39: Polarization properties of isolated LH2 complexes. A) Modulation correlation plot for
LH2 using 800 nm for excitation. B) Modulation correlation plot for LH2 using 850 nm for
excitation. C) Histogram for the excitation modulation depth of LH2 excited at 800 and 850 nm.

The three lower exciton states of the B850 ring form a very isotropic absorber
according to the spectrally disordered exciton model.129,130 Due to the broadening of
these exciton states at room temperature, excitation around the maximum of absorption
has very little selectivity over these states. On the other hand, the spectral selectivity of
the individual chromophores in the B800 ring can be significantly larger, even at room
temperature, due to their small electron-phonon interaction.131,132 Therefore, we
suggest that the excitonic nature of the B850 excited states is the reason behind its more
isotropic absorption.
The more polarized emission of the B850 ring compared to the fluorescence
excitation of both B800 and B850 (
>
) is the consequence of selective EET
towards more anisotropic states. We attribute this behaviour to preferential localisation
of the emitting state in a part of the B850 ring. In emission the energetically lowest
exciton state is preferentially populated, which can lead to a larger anisotropy of the
emissive state in comparison to excitation. Another localisation mechanism is exciton
self-trapping, where the originally delocalised exciton state localises in a picosecond
timescale due to excitation-induced lattice deformation. However, in a disorder-free
system this trapping can happen at any location. Therefore, static disorder and/or
deformation of the complex is needed to increase
. Regardless of the localisation
mechanism, the particular disorder of a single LH2 must be stable for minutes to
explain our observations. On the contrary, several studies have reported fluctuations of
the photophysical properties of LH2 occurring on much shorter timescales.130,133,134
Thus, we suggest that these fast fluctuations do not blur away the localisation of the
emissive state.

63

6.3 Conjugated Polymers

Before the mid-1970s, conjugated polymers (CP) attracted little attention, despite the
fact that a few papers were published describing unusually high conductivity for some
of these polymers.135 In 1977 Heeger, MacDiarmid and Shirakawa reported that
poly(acetylene) can present large conductivities when doped by bromide or iodine
vapours.136,137 Since then much research has been devoted to the chemistry and physics
of CPs both in their neutral (undoped) and charged (doped) states.135,138 As a result, in
2000, Heeger, MacDiarmid and Shirakawa were awarded a Nobel Prize in
chemistry.139141 Currently, CPs are emerging as very attractive elements for different
applications in organic-based devices. One of the main reasons for this is that polymerbased photovoltaic elements introduce the potential of obtaining cheap and easy
methods to harvest solar energy.142
In the framework of this thesis we are interested in the optical properties of
undoped CPs. Let us consider the case of poly(acetylene), which is one of the simplest
conjugated systems.143 Each carbon atom is sp2-hybridized and has four valence
electrons. Three of those valence electrons are involved in the strong -bonds, while
the fourth valence electron occupies the non-hybridized p-orbital ( -electron). The
overlapping of the -electron wave functions forms a -band.
Since the delocalised molecular -orbitals are half-filled, one might expect
metallic behaviour according to the finite density of states at the Fermi level. However,
the incompletely filled band distorts due to the Peierls instability and a band gap
appears between the filled bonding ( ) states and the empty antibonding ( )
states.135,143 As a result, the semiconducting state for the polymer is energetically
favourable.
CPs are coloured due to the absorption of the - optical transition.135 The
HOMO-LUMO energy gap of CP varies from 0.8 to 4 eV covering the visible and
infrared region of the spectrum. This energy depends on the chemical constitution of
the conjugated backbone, the nature of the substituents (electron donating or
withdrawing character) and the extent of delocalisation of the -electrons. Therefore,
structure plays a major role on the photophysical properties of CP, which can vary to a
very large extent144.
The optical properties of CP have shown in many instances to be strongly
dependent on interchain interactions.145 For example, transport properties of CP and
luminescence of light-emitting devices are highly sensitive to the local ordering of the
polymer chains.145,146 Further, intermolecular interactions are known to reduce the
fluorescence quantum yield of CP in films in comparison to that of solutions.145
64

Even at the single chain level CPs are intrinsically disordered materials.147 For
example, the size of a CP is broadly distributed (polydispersity) and as a consequence
several different conformations of an individual chain exist in the ensemble.56,148
Further, a single CP can be considered as a multichromophoric systems because
disorder disrupts and localises the -electrons at random intervals, leading to the
formation of spectroscopic units (or chromophores).26 The different local environment
that each chromophore unit is subject to (inhomogeneous broadening) leads to the very
broad spectral features observed for CPs (see also chapter 2). Therefore, the field of
plastic electronics can benefit from the ability of SMS to overcome the inhomogeneous
broadening present in CPs. This approach allows in a very fundamental level to relate
structure and conformation to the photophysical properties of CPs.

6.3.1 Quenching of conjugated polymer fluorescence at the single

molecule level Paper III
In paper III we present a study of the photophysical processes that decrease the
fluorescence brightness of many CPs in single molecule condition (single CP embedded
in a solid host) compared to solutions and pristine films. For this purpose we measured
the fluorescence properties of a nonpolar butanoylated polyrotaxane polyfluorene bisvinylphenylene (PFBV-Rtx) and its non-insulated counterpart (PFVB) in different
environments: liquid solutions, pristine films and single chains (Figure 40).

Figure 40: Chemical structures and spectra of PFBV and PFBV-Rtx. Absorption is depicted in dash
lines, while fluorescence emission is in full lines. The spectra for PFBV and PFBV-Rtx are in black
and grey colour, respectively.

The insulated PFBV-Rtx and unprotected PFBV have the same length, very
similar absorption cross section per monomer unit and fluorescence quantum yield ()
65

in solution. On the contrary, single PFBV chains have about 4 times lower fluorescence
brightness ( , see section 2.4) than PFBV-Rtx, whose fluorescence intensity is close to
that expected for a chain with = 100%. This suggests the presence of an important
amount of fluorescence quenching in single PFBV. However, PFBV and PFBV-Rtx
dispersed in a solid host showed very similar fluorescence decays, which were also very
similar to the fluorescence decay obtained in liquid solutions. Therefore, we concluded
that the quenching of single PFBV chains is ultrafast or static in nature (see section
2.4.1).
Although the actual quenching mechanism of PFBV in not completely
understood, it can be avoided by the cyclodextrin insulation. We proposed that the
cyclodextrin insulation of the PFBV-Rtx absorbs the forces originated from the
polymer host. This allows the conjugated backbone of PFBV-Rtx to be in a relaxed
state similar to that in solution, contrary to the more constrained PFBV. It has been
suggested previously that the CPs in a solid matrix can end up frozen in an
inconvenient conformation, which reduces its freedom to relax.68,149 This constraint
makes it very difficult for the chain to acquire the usually more planar configuration150
needed to reach the highly emissive state geometry. For example, MEH-PPV chains in
viscous polystyrene solutions decrease their fluorescence brightness upon an increase of
the viscosity.151
Many single molecule studies try to relate the photophysical properties of CPs,
measured via fluorescence, to the chain length, measured by solution chromatography.
However, fluorescence techniques are sensitive only to those active chromophores that
contribute to the florescence excitation cross section of the chain. Therefore, if the
presence of large quenched polymer segments (dark chromophores) is not considered,
then the properties of small fluorescent regions can be erroneously assigned to the whole
micrometre-long chain.

6.3.2 The influence quenching to the correlation between fluorescence

polarization and conformation of a single molecule Paper IV
The fluorescence excitation modulation depth (
) of a multichromophoric system
depends on the number and orientation of the active chromophores in the system. This
correlation has been used in many single molecule studies to obtain information about
the conformation of single CPs. As discussed in the previous section, there has been
different reports dealing with the important decrease of the fluorescence brightness ( )
of some CPs isolated in a solid matrix. This reduction is related to the interactions
between the isolated CP and the polymer host, and thus, it is highly dependent on the
sample preparation conditions. However, the influence of the dark chromophores in
66

the polarization properties of the chains has not been considered so far. As we will show
below, if the fraction of dark chromophores is large, then this can break the correlation
between the physical shape of the polymer chain and its fluorescence polarization
properties.
Our previous study showed that the insulated PFBV-Rtx does not present any
decrease in when embedded in PMMA matrix, while its unprotected counterpart
PFBV possess 4 times lower (paper III). This gives us a unique possibility to study
the nature of the fluorescence quenching of CPs at the single molecule level, and how
this quenching affects the polarization properties of single CPs. In paper IV we report
the study of isolated PFBV and PFBV-Rtx in a solid host by 2D-POLIM.
Both PFBV and PFBV-Rtx have the same 10-monomer-unit long ( 18 )
backbone with polydispersity 2.21 and 1.76, respectively. The main difference
between both structures is the presence of the cyclodextrin rings in PFBV-Rtx.
Molecular mechanic calculations have shown that polyrotaxanes very similar to PFBVRtx have rod-shaped structures with straighter geometry than that of the PFBV
backbone.152 Therefore, the conformation of the PFBV-Rtx chains embedded in the
polymer matrix should be straighter (or very similar) than PFBV chains.153
On the other hand, the
histograms reveal that single PFBV molecules were
more polarized than their protected counterpart PFBV-Rtx (Figure 41). However, if
the number of effective chromophores in PFBV-Rtx and PFBV were similar, as
expected by the chain length, then their
values should also be very similar or larger
for the straighter PFBV-Rtx chains. Therefore, we proposed that the more polarized
fluorescence excitation of the PFBV is not the result of the organisation of the backbone
but the result of the inhomogeneous quenching of the PFBV chromophores. Through
simulations, we estimated that the extent of inhomogeneous quenching in PFBV was
70-80% (Figure 41 - Simulations). This is in agreement with the ultrafast/static
quenching of PFBV reported in paper III. Our experimental observations demonstrate
that recognizing the absence or presence of quenched regions in isolated nanoparticles
is of paramount importance to accurately correlate the polarization properties to the
objects conformation.

67

Chapter 7 Conclusions and future

work

7.1 Summary of the novelties in the thesis

We consider for the first time the influence of the fluorescence brightness decrease
of single conjugated polymers embedded in a solid matrix on the polarization
properties of the chains. We demonstrate that this fluorescence quenching can be:
static or ultrafast in nature, and inhomogeneous along the polymer chain. This
breaks the correlation between the fluorescence excitation polarization and the
conjugated polymer chain conformation.
2D-POLIM proved to be a powerful tool to assess the internal organisation of
natural light harvesting antennas. Using 2D-POLIM we found that:
1. The total excitonic transition dipole moment of single chlorosomes possesses
a cylindrical symmetry relative to their physical long axis.
2. We were able to obtain for the first time very reliable measurements of the
fluorescence polarization properties of isolated light harvesting complex II.
3. The polarization properties of the light harvesting complex II are consistent
with static energy disorder and/or deformation of the complex stable over
minutes at room temperature.
We developed a model based on a single funnel approximation to quantitatively
determine the light harvesting efficiency (0 1) of a single molecule
measured by 2D-POLIM. This model was successfully used to characterize the
light harvesting efficiency of natural and artificial antennas. To use this model no
previous knowledge about the conformation and excitation energy transfer
mechanisms of the nanoparticle is needed.
2D-POLIM and the single funnel approximation are not only beneficial for single
molecules studies, but also can be used as a fluorescence imaging tool for bulk
samples, such as thin polymer films, cell cultures, histological samples, etc. 2DPOLIM was used to study the charge transfer processes occurring in thin films of
conjugated polymer/fullerene blends, a solar cell material, and we found that:
73

1. The emissive charge transfer states borrow more dipole moment from the
donor material (conjugated polymer) than previously thought.
2. The emissive charge transfer (CT) states are spatially and/or energetically
isolated from the other states in the system, such as, non-emissive CT states
and other emissive CT states.

7.2 Future work

One exciting direction for 2D-POLIM at the single molecule level is to measure a time
resolved polarization portrait. This could allow us to observe the evolution of the intraEET processes during the life time of the multichromophoric system, and how the
energy migrates from one polarized state to another differently polarized. On the other
hand the polarization portrait of a single molecule could be measured during a very
long time (minutes to hours). Analysing this data, we could obtain different
polarization portraits for different stable states of a single molecule. These states could
be related to different intensity levels, spectrum or polarization properties. The analysis
of the correlation between the polarization portrait and the state properties could lead
to new insights to the photophysical properties of the molecule.
Further theoretical calculations on the excitation energy transfer processes at the
single molecule level could help us to better understand the limitations of the single
funnel approximation, and to develop improvements to the model. Moreover, the
single funnel approximation was designed to obtain information about
multichromophoric systems with very little prior knowledge. However, if we measure
a system with very well defined properties, such as number of chromophores and
orientation in space, we could develop more specific models of the general equations
introduced in chapter 3. This could allow us to experimentally obtain more detailed
information about the energy transfer processes.
The latest development in our research is the use of 2D-POLIM as a fluorescence
imaging tool. In this regard, one of the most exciting possibilities is the use of 2DPOLIM in life sciences as an improvement of the currently available excitation energy
transfer (EET) microscopies, such as FRET microscopy. Current techniques based on
fluorescence anisotropy are not able to quantitatively discriminate EET due to the
presence of instrumental artefacts, and the intrinsic anisotropy of some samples. We
are currently trying to use 2D-POLIM for the study of biological relevant systems, such
as the aggregation of proteins involved in the causes of various diseases.

74

References

(1)

Xie, X. S.; Trautman, J. K. Optical Studies of Single Molecules at Room Temperature.

Annu. Rev. Phys. Chem. 1998, 49, 441480.

(2)

Moerner, W. E.; Orrit, M. Lluminating Single Molecules Matter in Condensed. Science

(80-. ). 1999, 283, 16701676.

(3)

Michalet, X.; Kapanidis, A. N.; Laurence, T.; Pinaud, F.; Doose, S.; Pflughoefft, M.;
Weiss, S. The Power and Prospects of Fluorescence Microscopies and Spectroscopies.
Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 161182.

(4)

Abbe, E. Beitrge Zur Theorie Des Mikroskops Und Der Mikroskopischen

Wahrnehmung. Arch. mikrosk. Anat. Entwichlungsmech. 1873, 9, 413468.

(5)

Moerner, W.; Kador, L. Optical Detection and Spectroscopy of Single Molecules in a

Solid. Phys. Rev. Lett. 1989, 62, 25352538.

(6)

Orrit, M.; Bernard, J. Single Pentacene Molecules Detected by Fluorescence Excitation

in a P-Terphenyl Crystal. Phys. Rev. Lett. 1990, 65, 27162719.

(7)

Single-Molecule Optical Detection, Imaging and Spectroscopy; Basch, T.; Moerner, W.

E.; Orrit, M.; Wild, U. P., Eds.; VCH Verlagsgesellschaft mbH: Weinheim, Germany,
1996.

(8)

Hell, S. W. Toward Fluorescence Nanoscopy. Nat. Biotechnol. 2003, 21, 13471355.

(9)

Van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstrom, V. Energy Transfer and
Trapping in Photosynthesis. Biochim. Biophys. Acta - Bioenerg. 1994, 1187, 165.

(10)

Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 3rd ed.; Springer Science:

Singapore, 2006; p. 954.

(11)

Nie, S.; Zare, R. N. Optical Detection of Single Molecules. Annu. Rev. Biophys. Biomol.
Struct. 1997, 26, 567596.

(12)

Binnig, G.; Rohrer, H. Scanning Tunneling Microscopyfrom Birth to Adolescence.

Rev. Mod. Phys. 1987, 59, 615625.

(13)

Binnig, G.; Quate, C. F. Atomic Force Microscope. Phys. Rev. Lett. 1986, 56, 930
933.

75

(14)

Hirschfeld, T. Optical Microscopic Observation of Single Small Molecules. Appl. Opt.

1976, 15, 29652966.

(15)

Weiss, S. Fluorescence Spectroscopy of Single Biomolecules. Science (80-. ). 1999, 283,

16761683.

(16)

Kulzer, F.; Orrit, M. Single-Molecule Optics. Annu. Rev. Phys. Chem. 2004, 55, 585
611.

(17)

Basch, T.; Kummer, S.; Bruchle, C. Direct Spectroscopic Observation of Quantum

Jumps of a Single Molecule. Nature 1995, 373, 132134.

(18)

Ambrose, W. P.; Moerner, W. E. Fluorescence Spectroscopy and Spectral Diffusion of

Single Impurity Molecules in a Crystal. Nature 1991, 349, 225227.

(19)

Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Near-Field Spectroscopy of Single
Molecules at Room Temperature. Nature 1994, 369, 4042.

(20)

Lu, H. P.; Xie, X. S. Single-Molecule Spectral Fluctuations at Room Temperature.

Nature 1997, 385, 143146.

(21)

Svanberg, S. Atomic and Molecular Spectroscopy. In Atomic and Molecular Spectroscopy:

Basic Aspects and Practical Applications; Springer-Verlag: New York, 2004; pp. 3140.

(22)

Herschel, J. F. W. Formula No. I. On a Case of Superficial Colour Presented by a

Homogeneous Liquid Internally Colourless. Philos. Trans. R. Soc. London 1845, 135,
143145.

(23)

Encyclopdia
Britannica
Online.
chromophore
http://www.britannica.com/EBchecked/topic/116032/chromophore (accessed Jan 28,
2014).

(24)

Braslavsky, S. E. Glossary of Terms Used in Photochemistry, 3rd Edition (IUPAC

Recommendations 2006). Pure Appl. Chem. 2007, 79, 293465.

(25)

Wong, K. F.; Skaf, M. S.; Yang, C.-Y.; Rossky, P. J.; Bagchi, B.; Hu, D.; Yu, J.; Barbara,
P. F. Structural and Electronic Characterization of Chemical and Conformational
Defects in Conjugated Polymers. J. Phys. Chem. B 2001, 105, 61036107.

(26)

Beenken, W. J. D.; Pullerits, T. Spectroscopic Units in Conjugated Polymers: A

Quantum Chemically Founded Concept? J. Phys. Chem. B 2004, 108, 61646169.

(27)

McDermott, G.; Prince, S. M.; Freer, A. A.; Hawthornthwaite-Lawless, A. M.; Papiz,

M. Z.; Cogdell, R. J.; Isaacs, N. W. Crystal Structure of an Integral Membrane LightHarvesting Complex from Photosynthetic Bacteria. Nature 1995, 374, 517521.

(28)

Frster, T. Zwischenmolekulare Energiewanderung Und Fluoreszenz. Ann. Phys. 1948,

437, 5575.

76

(29)

Galanin, M. D. The Problem of the Effect of Concentration on the Luminescence of

Solutions. Sov. Phys. JETP-USSR 1955, 1, 317325.

(30)

Kallmann, H.; London, F. ber Quantenmechnische Energiebertragung Zwischen

Atomaren Systemen. Ein Beitrag Zum Problem Der Anomalgrossen
Wirkungsquerschnitte. Z. Phys. Chem. 1929, B2, 207.

(31)

Perrin, F. La Fluorescence Des Solutions. Induction Molculaire-Polarisation et Dure

Dmission ; Photochimie. Ann. Phys. 1929, 12, 169275.

(32)

Masters, B. R. Paths to Frsters Resonance Energy Transfer (FRET) Theory. Eur. Phys.
J. H 2013, 39, 87139.

(33)

Olaya-Castro, A.; Scholes, G. D. Energy Transfer from FrsterDexter Theory to

Quantum Coherent Light-Harvesting. Int. Rev. Phys. Chem. 2011, 30, 4977.

(34)

Harcourt, R. D.; Scholes, G. D.; Ghiggino, K. P. Rate Expressions for Excitation

Transfer. II. Electronic Considerations of Direct and Throughconfiguration Exciton
Resonance Interactions. J. Chem. Phys. 1994, 101, 1052110525.

(35)

Andrews, D. L.; Curutchet, C.; Scholes, G. D. Resonance Energy Transfer: Beyond the
Limits. Laser Photon. Rev. 2011, 5, 114123.

(36)

Dexter, D. L. A Theory of Sensitized Luminescence in Solids. J. Chem. Phys. 1953, 21,

836850.

(37)

Frenkel, J. On the Transformation of Light into Heat in Solids. I. Phys. Rev. 1931, 37,
1744.

(38)

Kasha, M.; Rawls, H.; El-Bayoumi, M. The Exciton Model in Molecular Spectroscopy.
Pure Appl. Chem 1965, 371392.

(39)

Jelley, E. E. Spectral Absorption and Fluorescence of Dyes in the Molecular State.

Nature 1936, 138, 10091010.

(40)

Scheibe, G. ber Die Vernderlichkeit Des Absorptionsspektrums Einiger

Sensibilisierungsfarbstoffe Und Deren Ursache. Angew. Chemie 1936, 49, 563.

(41)

Scheibe, G. ber Die Vernderlichkeit Der Absorptionsspektren in Lsungen Und Die

Nebenvalenzen Als Ihre Ursache. Angew. Chemie 1937, 50, 212219.

(42)

Scheibe, G.; Schontag, A.; Katheder, F. Fluorescence and Energy Transmission in

Reversible Polymerised Pigments. Naturwissenschaffen 1939, 27, 499501.

(43)

Pope, M.; Swengerg, C. Electronic Processes in Organic Solids. Ann. Rev. Phys. Chem.
1984, 35, 613655.

(44)

Scheblykin, I. G. Temperature Dependence of Exciton Transport in J-Aggregates. In JAggregates Volume 2; Kobayashi, T., Ed.; World Scientific: Singapore, 2012; pp. 247
271.

77

(45)

Sumi, H. Theory on Rates of Excitation-Energy Transfer between Molecular

Aggregates through Distributed Transition Dipoles with Application to the Antenna
System in Bacterial Photosynthesis. J. Phys. Chem. B 1999, 103, 252260.

(46)

Sumi, H. Structural Strategies in the Antenna System of Photosynthesis on the Basis of

Quantum-Mechanical Coherence among Pigments. J. Lumin. 2000, 87-89, 7176.

(47)

Scholes, G. D. Long-Range Resonance Energy Transfer in Molecular Systems. Annu.

Rev. Phys. Chem. 2003, 54, 5787.

(48)

Jang, S.; Newton, M.; Silbey, R. Multichromophoric Frster Resonance Energy

Transfer. Phys. Rev. Lett. 2004, 92, 218301.

(49)

Jang, S.; Newton, M. D.; Silbey, R. J. Multichromophoric Frster Resonance Energy

Transfer from b800 to b850 in the Light Harvesting Complex 2: Evidence for Subtle
Energetic Optimization by Purple Bacteria. J. Phys. Chem. B 2007, 111, 68076814.

(50)

Ronchi, V. The Nature Of Light; Heinemann Educational Books Ltd: London, 1970.

(51)

Kliger, D. S.; Lewis, J. W.; Randall, C. E. Polarized Light in Optics and Spectroscopy;
Academic Press, Inc.: Santa Cruz, 1990.

(52)

Grangier, P.; Roger, G.; Aspect, A. Experimental Evidence for a Photon Anticorrelation
Effect on a Beam Splitter: A New Light on Single-Photon Interferences. Europhys. Lett.
1986, 1, 173179.

(53)

Valeur, B. Molecular Fluorescence: Principles and Applications; 1st ed.; WILEY-VCH:

Weinheim, 2002.

(54)

Galanin, M. D. Luminescence of Molecules and Crystals; Cambridge International

Science Publishing, 1996.

(55)

Gttler, F.; Sepiol, J.; Plakhotnik, T.; Mitterdorfer, A.; Renn, A.; Wild, U. P. Single
Molecule Spectroscopy: Fluorescence Excitation Spectra with Polarized Light. J. Lumin.
1993, 56, 2938.

(56)

Hu, D.; Yu, J.; Wong, K.; Bagchi, B.; Rossky, P.; Barbara, P. Collapse of Stiff
Conjugated Polymers with Chemical Defects into Ordered, Cylindrical
Conformations. Nature 2000, 405, 19982001.

(57)

Ebihara, Y.; Vacha, M. Relating Conformation and Photophysics in Single MEH-PPV

Chains. J. Phys. Chem. B 2008, 112, 1257512578.

(58)

Sun, W.-Y.; Yang, S.-C.; White, J. D.; Hsu, J.-H.; Peng, K. Y.; Chen, S. A.; Fann, W.
Conformation and Energy Transfer in a Single Luminescent Conjugated Polymer.
Macromolecules 2005, 38, 29662973.

78

(59)

Adachi, T.; Brazard, J.; Chokshi, P.; Bolinger, J. C.; Ganesan, V.; Barbara, P. F. Highly
Ordered Single Conjugated Polymer Chain Rod Morphologies . J. Phys. Chem. C
2010, 114, 2089620902.

(60)

Bolinger, J. C.; Traub, M. C.; Brazard, J.; Adachi, T.; Barbara, P. F.; Vanden Bout, D.
A. Conformation and Energy Transfer in Single Conjugated Polymers. Acc. Chem. Res.
2012, 45, 19922001.

(61)

Traub, M. C.; DuBay, K. H.; Ingle, S. E.; Zhu, X.; Plunkett, K. N.; Reichman, D. R.;
Vanden Bout, D. a. Chromophore-Controlled Self-Assembly of Highly Ordered
Polymer Nanostructures. J. Phys. Chem. Lett. 2013, 4, 25202524.

(62)

Stangl, T.; Bange, S.; Schmitz, D.; Wrsch, D.; Hger, S.; Vogelsang, J.; Lupton, J. M.
Temporal Switching of Homo-FRET Pathways in Single-Chromophore Dimer Models
of -Conjugated Polymers. J. Am. Chem. Soc. 2013, 135, 7881.

(63)

Lammi, R. K.; Barbara, P. F. Influence of Chain Length on Exciton Migration to LowEnergy Sites in Single Fluorene Copolymers. Photochem. Photobiol. Sci. 2005, 4, 95
99.

(64)

Hu, D.; Yu, J.; Padmanaban, G.; Ramakrishnan, S.; Barbara, P. F. Spatial Confinement
of Exciton Transfer and the Role of Conformational Order in Organic Nanoparticles.
Nano Lett. 2002, 2, 11211124.

(65)

Bounos, G.; Ghosh, S.; Lee, A. K.; Plunkett, K. N.; DuBay, K. H.; Bolinger, J. C.;
Zhang, R.; Friesner, R. a; Nuckolls, C.; Reichman, D. R.; et al. Controlling Chain
Conformation in Conjugated Polymers Using Defect Inclusion Strategies. J. Am. Chem.
Soc. 2011, 133, 1015510160.

(66)

Adachi, T.; Brazard, J.; Ono, R. J.; Hanson, B.; Traub, M. C.; Wu, Z.-Q.; Li, Z.;
Bolinger, J. C.; Ganesan, V.; Bielawski, C. W.; et al. Regioregularity and Single
Polythiophene Chain Conformation. J. Phys. Chem. Lett. 2011, 2, 14001404.

(67)

Hofkens, J.; Verheijen, W.; Shukla, R.; Dehaen, W.; De Schryver, F. C. Detection of
a Single Dendrimer Macromolecule with a Fluorescent Dihydropyrrolopyrroledione
(DPP) Core Embedded in a Thin Polystyrene Polymer Film. Macromolecules 1998, 31,
44934497.

(68)

Lin, H.; Hania, R. P.; Bloem, R.; Mirzov, O.; Thomsson, D.; Scheblykin, I. G. Single
Chain versus Single Aggregate Spectroscopy of Conjugated Polymers. Where Is the
Border? Phys. Chem. Chem. Phys. 2010, 12, 1177011777.

(69)

Vavilov, S. I. The Microstructure of Light; USSR Academy of Sciences: Moscow, 1950.

(70)

Vavilov, S. I.; Schultz, G. Die Mikrostruktur Des Lichtes; Akademie-Verlag: Berlin,

1954.

79

(71)

Zhevandrov, N. D. Optical Anisotropy and Energy Migration in Molecular Crystals;

Nauka: Moscow, 1987.

(72)

Lin, H.; Camacho, R.; Tian, Y.; Kaiser, T. E.; Wrthner, F.; Scheblykin, I. G.
Collective Fluorescence Blinking in Linear J-Aggregates Assisted by Long-Distance
Exciton Migration. Nano Lett. 2010, 10, 620626.

(73)

Thomsson, D.; Camacho, R.; Tian, Y.; Yadav, D.; Sforazzini, G.; Anderson, H. L.;
Scheblykin, I. G. Cyclodextrin Insulation Prevents Static Quenching of Conjugated
Polymer Fluorescence at the Single Molecule Level. Small 2013, 9, 26192627.

(74)

Runnels, L. W.; Scarlata, S. F. Theory and Application of Fluorescence Homotransfer

to Melittin Oligomerization. Biophys. J. 1995, 69, 15691583.

(75)

Sun, Y.; Wallrabe, H.; Seo, S.-A.; Periasamy, A. FRET Microscopy in 2010: The Legacy
of Theodor Frster on the 100th Anniversary of His Birth. Chemphyschem 2010, 462
474.

(76)

Bader, A. N.; Hoetzl, S.; Hofman, E. G.; Voortman, J.; van Bergen en Henegouwen,
P. M. P.; van Meer, G.; Gerritsen, H. C. Homo-FRET Imaging as a Tool to Quantify
Protein and Lipid Clustering. Chemphyschem 2011, 12, 475483.

(77)

Agranovich, V. M.; Galanin, M. D. Electronic Excitation Energy Transfer in Condensed

Matter; North-Holland Publishing Company, 1982.

(78)

Muller, J. G.; Lupton, J. M.; Feldmann, J.; Lemmer, U.; Scherf, U. Ultrafast
Intramolecular Energy Transfer in Single Conjugated Polymer Chains Probed by
Polarized Single Chromophore Spectroscopy. Appl. Phys. Lett. 2004, 84, 1183.

(79)

Richter, M. F.; Baier, J.; Cogdell, R. J.; Khler, J.; Oellerich, S. Single-Molecule
Spectroscopic Characterization of Light-Harvesting 2 Complexes Reconstituted into
Model Membranes. Biophys. J. 2007, 93, 183191.

(80)

Feist, F. a; Zickler, M. F.; Basch, T. Origin of the Red Sites and Energy Transfer Rates
in Single MEH-PPV Chains at Low Temperature. ChemPhysChem 2011, 12, 1499
1508.

(81)

Hu, D.; Yu, J.; Barbara, P. F. Single-Molecule Spectroscopy of the Conjugated Polymer
MEH-PPV. J. Am. Chem. Soc 1999, 121, 69366937.

(82)

Huser, T.; Yan, M.; Rothberg, L. J. Single Chain Spectroscopy of Conformational

Dependence of Conjugated Polymer Photophysics. Proc. Natl. Acad. Sci. U. S. A. 2000,
97, 1118711191.

(83)

Sartori, S. S.; De Feyter, S.; Hofkens, J.; Van Der Auweraer, M.; De Schryver, F.;
Brunner, K.; Hofstraat, J. W. Host Matrix Dependence on the Photophysical
Properties of Individual Conjugated Polymer Chains. Macromolecules 2003, 36, 500
507.

80

(84)

Lammi, R. K.; Fritz, K. P.; Scholes, G. D.; Barbara, P. F. Ordering of Single

Conjugated Polymers in a Nematic Liquid Crystal Host. J. Phys. Chem. B 2004, 108,
45934596.

(85)

Yu, Z.; Barbara, P. F. Low-Temperature Single-Molecule Spectroscopy of MEH-PPV

Conjugated Polymer Molecules. J. Phys. Chem. B 2004, 108, 1132111326.

(86)

Dedecker, P.; Muls, B.; Deres, A.; Uji-i, H.; Hotta, J.; Sliwa, M.; Soumillion, J.-P.;
Mllen, K.; Enderlein, J.; Hofkens, J. Defocused Wide-Field Imaging Unravels
Structural and Temporal Heterogeneity in Complex Systems. Adv. Mater. 2009, 21,
10791090.

(87)

Forster, M.; Thomsson, D.; Hania, P. R.; Scheblykin, I. G. Redistribution of Emitting

State Population in Conjugated Polymers Probed by Single-Molecule Fluorescence
Polarization Spectroscopy. Phys. Chem. Chem. Phys. 2007, 9, 761766.

(88)

Furumaki, S.; Vacha, F.; Habuchi, S.; Tsukatani, Y.; Bryant, D. a; Vacha, M.
Absorption Linear Dichroism Measured Directly on a Single Light-Harvesting System:
The Role of Disorder in Chlorosomes of Green Photosynthetic Bacteria. J. Am. Chem.
Soc. 2011, 133, 67036710.

(89)

Lin, H.; Tabaei, S. R.; Thomsson, D.; Mirzov, O.; Larsson, P.-O.; Scheblykin, I. G.
Fluorescence Blinking, Exciton Dynamics, and Energy Transfer Domains in Single
Conjugated Polymer Chains. J. Am. Chem. Soc. 2008, 130, 70427051.

(90)

Traub, M. C.; Lakhwani, G.; Bolinger, J. C.; Vanden Bout, D.; Barbara, P. F.
Electronic Energy Transfer in Highly Aligned MEH-PPV Single Chains. J. Phys. Chem.
B 2011, 115, 99419947.

(91)

Mirzov, O.; Bloem, R.; Hania, P. R.; Thomsson, D.; Lin, H.; Scheblykin, I. G.
Polarization Portraits of Single Multichromophoric Systems: Visualizing Conformation
and Energy Transfer. Small 2009, 5, 18771888.

(92)

Camacho, R.; Thomsson, D.; Yadav, D.; Scheblykin, I. G. Quantitative

Characterization of Light-Harvesting Efficiency in Single Molecules and Nanoparticles
by 2D Polarization Microscopy: Experimental and Theoretical Challenges. Chem. Phys.
2012, 406, 3040.

(93)

Hofkens, J.; Verheijen, W.; Shukla, R.; Dehaen, W.; Schryver, F. C. De. Detection of
a Single Dendrimer Macromolecule with a Fluorescent Dihydropyrrolopyrroledione (
DPP ) Core Embedded in a Thin Polystyrene Polymer Film. 1998, 81, 44934497.

(94)

Gensch, T.; Hofkens, J.; Khn, F.; Vosch, T.; Herrmann, A.; Mllen, K.; De Schryver,
F. Polarisation Sensitive Single Molecule Fluorescence Detection with Linear Polarised
Excitation Light and Modulated Polarisation Direction Applied to Multichromophoric
Entities. Single Mol. 2001, 2, 3544.

81

(95)

Vacha, M.; Kotani, M. Three-Dimensional Orientation of Single Molecules Observed

by Far- and near-Field Fluorescence Microscopy. J. Chem. Phys. 2003, 118, 5279.

(96)

Ebihara, Y.; Vacha, M. A Method for Determining the Absorption Ellipsoid of Single
Conjugated Polymer Molecules and Single Luminescent Nanoparticles. J. Chem. Phys.
2005, 123, 244710.

(97)

Becker, K.; Lupton, J. M. Efficient Light Harvesting in Dye-Endcapped Conjugated

Polymers Probed by Single Molecule Spectroscopy. J. Am. Chem. Soc. 2006, 128,
64686479.

(98)

Ha, T.; Enderle, T.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Probing
the Interaction between Two Single Molecules: Fluorescence Resonance Energy
Transfer between a Single Donor and a Single Acceptor. Proc. Natl. Acad. Sci. U. S. A.
1996, 93, 62646268.

(99)

Mattheyses, A. L.; Hoppe, A. D.; Axelrod, D. Polarized Fluorescence Resonance Energy

Transfer Microscopy. Biophys. J. 2004, 87, 27872797.

(100)

Piston, D. W.; Kremers, G.-J. Fluorescent Protein FRET: The Good, the Bad and the
Ugly. Trends Biochem. Sci. 2007, 32, 407414.

(101)

Piston, D. W.; Rizzo, M. a. FRET by Fluorescence Polarization Microscopy. Methods

Cell Biol. 2008, 85, 415430.

(102)

Varma, R.; Mayor, S. GPI-Anchored Proteins Are Organized in Submicron Domains

at the Cell Surface. Nature 1998, 394, 798801.

(103)

Gautier, I.; Tramier, M.; Durieux, C.; Coppey, J.; Pansu, R. B.; Nicolas, J. C.; Kemnitz,
K.; Coppey-Moisan, M. Homo-FRET Microscopy in Living Cells to Measure
Monomer-Dimer Transition of GFP-Tagged Proteins. Biophys. J. 2001, 80, 3000
3008.

(104)

Bader, A. N.; Hofman, E. G.; Voortman, J.; en Henegouwen, P. M. P. van B.;

Gerritsen, H. C. Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes
with Subcellular Resolution. Biophys. J. 2009, 97, 26132622.

(105)

Hecht, E. Optics; 4th ed.; Addison Wesley: San Francisco, 2002.

(106)

Balaban, T. S. Tailoring Porphyrins and Chlorins for Self-Assembly in Biomimetic

Artificial Antenna Systems. Acc. Chem. Res. 2005, 38, 612623.

(107)

Rger, C.; Mller, M. G.; Lysetska, M.; Miloslavina, Y.; Holzwarth, A. R.; Wrthner,
F. Efficient Energy Transfer from Peripheral Chromophores to the Self-Assembled Zinc
Chlorin Rod Antenna: A Bioinspired Light-Harvesting System to Bridge the Green
Gap. J. Am. Chem. Soc. 2006, 128, 65426543.

82

(108)

Rger, C.; Miloslavina, Y.; Brunner, D.; Holzwarth, A. R.; Wrthner, F. SelfAssembled Zinc Chlorin Rod Antennae Powered by Peripheral Light-Harvesting
Chromophores. J. Am. Chem. Soc. 2008, 130, 59295939.

(109)

Ganapathy, S.; Sengupta, S.; Wawrzyniak, P. K.; Huber, V.; Buda, F.; Baumeister, U.;
Wrthner, F.; de Groot, H. J. M. Zinc Chlorins for Artificial Light-Harvesting SelfAssemble into Antiparallel Stacks Forming a Microcrystalline Solid-State Material.
Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 1147211477.

(110)

Miyatake, T.; Tamiaki, H. Self-Aggregates of Natural Chlorophylls and Their Synthetic

Analogues in Aqueous Media for Making Light-Harvesting Systems. Coord. Chem. Rev.
2010, 254, 25932602.

(111)

Chappaz-Gillot, C.; Marek, P. L.; Blaive, B. J.; Canard, G.; Brck, J.; Garab, G.; Hahn,
H.; Jvorfi, T.; Kelemen, L.; Krupke, R.; et al. Anisotropic Organization and
Microscopic Manipulation of Self-Assembling Synthetic Porphyrin Microrods That
Mimic Chlorosomes: Bacterial Light-Harvesting Systems. J. Am. Chem. Soc. 2012, 134,
944954.

(112)

Sengupta, S.; Ebeling, D.; Patwardhan, S.; Zhang, X.; von Berlepsch, H.; Bttcher, C.;
Stepanenko, V.; Uemura, S.; Hentschel, C.; Fuchs, H.; et al. Biosupramolecular
Nanowires from Chlorophyll Dyes with Exceptional Charge-Transport Properties.
Angew. Chem. Int. Ed. Engl. 2012, 51, 63786382.

(113)

Eisele, D. M.; Cone, C. W.; Bloemsma, E. A.; Vlaming, S. M.; van der Kwaak, C. G.
F.; Silbey, R. J.; Bawendi, M. G.; Knoester, J.; Rabe, J. P.; Vanden Bout, D. A. Utilizing
Redox-Chemistry to Elucidate the Nature of Exciton Transitions in Supramolecular
Dye Nanotubes. Nat. Chem. 2012, 4, 655662.

(114)

Furumaki, S.; Vacha, F.; Hirata, S.; Vacha, M. Bacteriochlorophyll Aggregates SelfAssembled on Functionalized Gold Nanorod Cores as Mimics of Photosynthetic
Chlorosomal Antennae: A Single Molecule Study. ACS Nano 2014, 8, 21762182.

(115)

Blankenship, R.; Olson, J.; Miller, M. Antenna Complexes from Green Photosynthetic
Bacteria. In Anoxygenic photosynthetic bacteria; Springer, 1995; pp. 399435.

(116)

Oostergetel, G. T.; Reus, M.; Gomez Maqueo Chew, A.; Bryant, D. A.; Boekema, E.
J.; Holzwarth, A. R. Long-Range Organization of Bacteriochlorophyll in Chlorosomes
of Chlorobium Tepidum Investigated by Cryo-Electron Microscopy. FEBS Lett. 2007,
581, 54355439.

(117)

Saga, Y.; Shibata, Y.; Tamiaki, H. Spectral Properties of Single Light-Harvesting

Complexes in Bacterial Photosynthesis. J. Photochem. Photobiol. C Photochem. Rev.
2010, 11, 1524.

83

(118)

Prokhorenko, V.; Steensgaard, D.; Holzwarth, A. Exciton Dynamics in the

Chlorosomal Antennae of the Green Bacteria Chloroflexus Aurantiacus and
Chlorobium Tepidum. Biophys. J. 2000, 79, 21052120.

(119)

Prokhorenko, V.; Steensgaard, D.; Holzwarth, A. Exciton Theory for Supramolecular

Chlorosomal Aggregates: 1. Aggregate Size Dependence of the Linear Spectra. Biophys.
J. 2003, 85, 31733186.

(120)

Linnanto, J. M.; Korppi-Tommola, J. E. I. Investigation on Chlorosomal Antenna

Geometries: Tube, Lamella and Spiral-Type Self-Aggregates. Photosynth. Res. 2008, 96,
227245.

(121)

Ganapathy, S.; Oostergetel, G. T.; Wawrzyniak, P. K.; Reus, M.; Gomez Maqueo
Chew, A.; Buda, F.; Boekema, E. J.; Bryant, D. a; Holzwarth, A. R.; de Groot, H. J.
M. Alternating Syn-Anti Bacteriochlorophylls Form Concentric Helical Nanotubes in
Chlorosomes. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 85258530.

(122)

Psenck, J.; Collins, A. M.; Liljeroos, L.; Torkkeli, M.; Laurinmki, P.; Ansink, H. M.;
Ikonen, T. P.; Serimaa, R. E.; Blankenship, R. E.; Tuma, R.; et al. Structure of
Chlorosomes from the Green Filamentous Bacterium Chloroflexus Aurantiacus. J.
Bacteriol. 2009, 191, 67016708.

(123)

Tian, Y.; Camacho, R.; Thomsson, D.; Reus, M.; Holzwarth, A. R.; Scheblykin, I. G.
Organization of Bacteriochlorophylls in Individual Chlorosomes from Chlorobaculum
Tepidum Studied by 2-Dimensional Polarization Fluorescence Microscopy. J. Am.
Chem. Soc. 2011, 133, 1719217199.

(124)

Tamiaki, H.; Tateishi, S.; Nakabayashi, S.; Shibata, Y.; Itoh, S. Linearly Polarized Light
Absorption Spectra of Chlorosomes, Light-Harvesting Antennas of Photosynthetic
Green Sulfur Bacteria. Chem. Phys. Lett. 2010, 484, 333337.

(125)

Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. The Crystal Structure of the
Light-Harvesting Complex II (B800850) from Rhodospirillum Molischianum.
Structure 1996, 4, 581597.

(126)

Freiberg, A.; Godik, V.; Pullerits, T.; Timpman, K. Picosecond Dynamics of Directed
Excitation Transfer in Spectrally Heterogeneous Light-Harvesting Antenna of Purple
Bacteria. Biochim. Biophys. Acta - Bioenerg. 1989, 973, 93104.

(127)

Shreve, A. P.; Trautman, J. K.; Frank, H. A.; Owens, T. G.; Albrecht, A. C.

Femtosecond Energy-Transfer Processes in the B800850 Light-Harvesting Complex
of Rhodobacter Sphaeroides 2.4.1. Biochim. Biophys. Acta - Bioenerg. 1991, 1058, 280
288.

(128)

Pullerits, T.; Hess, S.; Herek, J. L.; Sundstrm, V. Temperature Dependence of

Excitation Transfer in LH2 of Rhodobacter Sphaeroides. J. Phys. Chem. B 1997, 101,
1056010567.

84

(129)

Matsushita, M.; Ketelaars, M.; van Oijen, a M.; Khler, J.; Aartsma, T. J.; Schmidt, J.
Spectroscopy on the B850 Band of Individual Light-Harvesting 2 Complexes of
Rhodopseudomonas Acidophila. II. Exciton States of an Elliptically Deformed Ring
Aggregate. Biophys. J. 2001, 80, 16041614.

(130)

Novoderezhkin, V. I.; Rutkauskas, D.; van Grondelle, R. Dynamics of the Emission

Spectrum of a Single LH2 Complex: Interplay of Slow and Fast Nuclear Motions.
Biophys. J. 2006, 90, 28902902.

(131)

Hofmann, C.; Michel, H.; van Heel, M.; Khler, J. Multivariate Analysis of SingleMolecule Spectra: Surpassing Spectral Diffusion. Phys. Rev. Lett. 2005, 94, 195501.

(132)

Zigmantas, D.; Read, E. L.; Mancal, T.; Brixner, T.; Gardiner, A. T.; Cogdell, R. J.;
Fleming, G. R. Two-Dimensional Electronic Spectroscopy of the B800-B820 LightHarvesting Complex. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 1267212677.

(133)

Damjanovi, A.; Kosztin, I.; Kleinekathfer, U.; Schulten, K. Excitons in a

Photosynthetic Light-Harvesting System: A Combined Molecular Dynamics,
Quantum Chemistry, and Polaron Model Study. Phys. Rev. E 2002, 65, 031919.

(134)

Rutkauskas, D.; Novoderezkhin, V.; Cogdell, R. J.; van Grondelle, R. Fluorescence

Spectral Fluctuations of Single LH2 Complexes from Rhodopseudomonas Acidophila
Strain 10050. Biochemistry 2004, 43, 44314438.

(135)

Pron, A.; Rannou, P. Processible Conjugated Polymers: From Organic Semiconductors

to Organic Metals and Superconductors. Prog. Polym. Sci. 2002, 27, 135190.

(136)

Chiang, C.; Fincher, C.; Park, Y.; Heeger, A.; Shirakawa, H.; Louis, E.; Gau, S.;
MacDiarmid, A. Electrical Conductivity in Doped Polyacetylene. Phys. Rev. Lett. 1977,
39, 10981101.

(137)

Shirakawa, H.; Louis, E. J.; MacDiarmid, A. G.; Chiang, C. K.; Heeger, A. J. Synthesis
of Electrically Conducting Organic Polymers: Halogen Derivatives of Polyacetylene,
(CH) X. J. Chem. Soc. Chem. Commun. 1977, 578580.

(138)

Friend, R. H.; Gymer, R. W.; Holmes, A. B.; Burroughes, R. N.; Marks, R. N.; Taliani,
C.; Bradley, D. D. C.; Dos Santos, D. A.; Bredas, J. L.; Logdlund, M.; et al.
Electroluminescence in Conjugated Polymers. Nature 1999, 397, 121128.

(139)

Heeger, A. Semiconducting and Metallic Polymers: The Fourth Generation of

Polymeric Materials (Nobel Lecture). Angew. Chemie Int. Ed. 2001.

(140)

MacDiarmid, A. Synthetic Metals: A Novel Role for Organic Polymers (Nobel

Lecture). Angew. Chemie Int. Ed. 2001.

(141)

Shirakawa, H. The Discovery of Polyacetylene Film: The Dawning of an Era of

Conducting Polymers (Nobel Lecture). Angew. Chemie Int. Ed. 2001.

85

(142)

Sariciftci, N. S.; Smilowitz, L.; Heeger, A. J.; Wudl, F. Photoinduced Electron Transfer
from a Conducting Polymer to Buckminsterfullerene. Science 1992, 258, 14741476.

(143)

Jaiswal, M.; Menon, R. Polymer Electronic Materials: A Review of Charge Transport.

Polym. Int. 2006, 55, 13711384.

(144)

Rossi, G.; Chance, R. R.; Silbey, R. Conformational Disorder in Conjugated Polymers.

J. Chem. Phys. 1989, 90, 7594.

(145)

Cornil, J.; Beljonne, D.; Calbert, J.-P.; Brdas, J.-L. Interchain Interactions in Organic
-Conjugated Materials: Impact on Electronic Structure, Optical Response, and
Charge Transport. Adv. Mater. 2001, 13, 10531067.

(146)

Cornil, J.; Calbert, J. P.; Beljonne, D.; Silbey, R.; Brdas, J.-L. Charge Transport versus
Optical Properties in Semiconducting Crystalline Organic Thin Films. Adv. Mater.
2000, 12, 978983.

(147)

Lupton, J. M. Single-Molecule Spectroscopy for Plastic Electronics: Materials Analysis

from the Bottom-Up. Adv. Mater. 2010, 22, 16891721.

(148)

Collison, C. J.; Rothberg, L. J.; Treemaneekarn, V.; Li, Y. Conformational Effects on

the Photophysics of Conjugated Polymers: A Two Species Model for MEHPPV
Spectroscopy and Dynamics. Macromolecules 2001, 34, 23462352.

(149)

Lin, H.; Tian, Y.; Zapadka, K.; Persson, G.; Thomsson, D.; Mirzov, O.; Larsson, P.O.; Widengren, J.; Scheblykin, I. G. Fate of Excitations in Conjugated Polymers:
Single-Molecule Spectroscopy Reveals Nonemissive Dark Regions in MEH-PPV
Individual Chains. Nano Lett. 2009, 9, 44564461.

(150)

Beenken, W. J. D. Excitons in Conjugated Polymers: Do We Need a Paradigma

Change? Phys. Status Solidi A 2009, 206, 27502756.

(151)

Onda, S.; Kobayashi, H.; Hatano, T.; Furumaki, S.; Habuchi, S.; Vacha, M. Complete
Suppression of Blinking and Reduced Photobleaching in Single MEH-PPV Chains in
Solution. J. Phys. Chem. Lett. 2011, 2, 28272831.

(152)

Frampton, M. J.; Sforazzini, G.; Brovelli, S.; Latini, G.; Townsend, E.; Williams, C.
C.; Charas, A.; Zalewski, L.; Kaka, N. S.; Sirish, M.; et al. Synthesis and Optoelectronic
Properties of Nonpolar Polyrotaxane Insulated Molecular Wires with High Solubility
in Organic Solvents. Adv. Funct. Mater. 2008, 18, 33673376.

(153)

Becker, K.; Gaefke, G.; Rolffs, J.; Hger, S.; Lupton, J. M. Quantitative Mass
Determination of Conjugated Polymers for Single Molecule Conformation Analysis:
Enhancing Rigidity with Macrocycles. Chem. Commun. (Camb). 2010, 46, 4686
4688.

86

(154)

Ford, J. S.; Andrews, D. L. Geometrical Effects on Resonance Energy Transfer between

Orthogonally-Oriented Chromophores, Mediated by a Nearby Polarisable Molecule.
Chem. Phys. Lett. 2014, 591, 8892.

(155)

Miranda, P.; Moses, D.; Heeger, A. Ultrafast Photogeneration of Charged Polarons in

Conjugated Polymers. Phys. Rev. B 2001, 64, 081201.

(156)

Gnes, S.; Neugebauer, H.; Sariciftci, N. S. Conjugated Polymer-Based Organic Solar

Cells. Chem. Rev. 2007, 107, 13241338.

(157)

Wudl, F. The Chemical Properties of Buckminsterfullerene (C60) and the Birth and
Infancy of Fulleroids. Acc. Chem. Res. 1992, 25, 157161.

(158)

Kroto, H. W.; Heath, J. R.; OBrien, S. C.; Curl, R. F.; Smalley, R. E. C60:
Buckminsterfullerene. Nature 1985, 318, 162163.

(159)

Allemand, P. M.; Koch, A.; Wudl, F.; Rubin, Y.; Diederich, F.; Alvarez, M. M.; Anz,
S. J.; Whetten, R. L. Two Different Fullerenes Have the Same Cyclic Voltammetry. J.
Am. Chem. Soc. 1991, 113, 10501051.

(160)

Yu, G.; Gao, J.; Hummelen, J. C.; Wudl, F.; Heeger, A. J. Polymer Photovoltaic Cells:
Enhanced Efficiencies via a Network of Internal Donor-Acceptor Heterojunctions.
Science (80-. ). 1995, 270, 17891791.

(161)

Campoy-Quiles, M.; Ferenczi, T.; Agostinelli, T.; Etchegoin, P. G.; Kim, Y.;
Anthopoulos, T. D.; Stavrinou, P. N.; Bradley, D. D. C.; Nelson, J. Morphology
Evolution via Self-Organization and Lateral and Vertical Diffusion in Polymer:fullerene
Solar Cell Blends. Nat. Mater. 2008, 7, 158164.

(162)

Chen, H.; Hou, J.; Zhang, S.; Liang, Y.; Yang, G.; Yang, Y. Polymer Solar Cells with
Enhanced Open-Circuit Voltage and Efficiency. 2009, 3, 649653.

(163)

Park, S. H.; Roy, A.; Beaupre, S.; Cho, S.; Coates, N.; Moon, J. S.; Moses, D.; Leclerc,
M.; Lee, K.; Heeger, A. J. Bulk Heterojunction Solar Cells with Internal Quantum
Efficiency Approaching 100%. Nat. Photonics 2009, 3, 297303.

(164)

Liang, Y.; Xu, Z.; Xia, J.; Tsai, S.-T.; Wu, Y.; Li, G.; Ray, C.; Yu, L. For the Bright
Future-Bulk Heterojunction Polymer Solar Cells with Power Conversion Efficiency of
7.4%. Adv. Mater. 2010, 22, E1358.

(165)

Huang, Y.; Westenhoff, S.; Avilov, I.; Sreearunothai, P.; Hodgkiss, J. M.; Deleener, C.;
Friend, R. H.; Beljonne, D. Electronic Structures of Interfacial States Formed at
Polymeric Semiconductor Heterojunctions. Nat. Mater. 2008, 7, 483489.

(166)

Vandewal, K.; Tvingstedt, K.; Ingans, O. Polarization Anisotropy of Charge Transfer

Absorption and Emission of Aligned Polymer:fullerene Blend Films. Phys. Rev. B 2012,
86, 035212.

87