Nanoscopic Visualization of Cross-Linking Density in Polymer Networks with Diarylethene Photoswitches

Article abstract of DOI:10.1002/anie.201807741

The in situ nanoscopic imaging of soft matter polymer structures is of importance to gain knowledge of the relationship between structure, properties, and functionality on the nanoscopic scale. Cross-linking of polymer chains effects the viscoelastic prop

Full text of DOI:10.1002/anie.201807741

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Accepted Article
Title: Nanoscopic visualization of cross-linking density in polymer
networks with diarylethene photoswitches
Authors: Eric Siemes, Oleksii Nevskyi, Dmytro Sysoiev, Sarah Kristin
Turnhoff, Alex Oppermann, Thomas Huhn, Walter Richtering,
and Dominik Wöll
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201807741
Angew. Chem. 10.1002/ange.201807741
Link to VoR: http://dx.doi.org/10.1002/anie.201807741
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10.1002/anie.201807741
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Nanoscopic visualization of cross-linking density in polymer
networks with diarylethene photoswitches
Eric Siemes,+[a] Oleksii Nevskyi, +[a] Dmytro Sysoiev,[b] Sarah K. Turnhoff,[a] Alex Oppermann,[a] Thomas
Huhn,[b] Walter Richtering[a] and Dominik Wöll*[a]
Abstract: The in situ nanoscopic imaging of soft matter polymer
structures is of paramount importance to gain a detailed knowledge of
the relationship between structure, properties and functionality on the
nanoscopic scale. Cross-linking of polymer chains has tremendous
effects on the viscoelastic properties of gels, and the correlation of
mechanic properties on the distribution and amount of cross-linkers is
of huge relevance for applications as well as for a detailed
understanding of polymers on the molecular scale. We introduce a
super-resolution fluorescence microscopy based methodology
enabling the visualization and quantification of cross-linker points in
polymer systems. A novel diarylethene-based photoswitch with a
highly fluorescent closed and a non-fluorescent open form is used as
a photoswitchable cross-linker in a polymer network. Its photophysical
properties, switching behaviour, and high photostability make the dye
an ideal candidate for photoactivation localization microscopy (PALM)
without further modification of the structure of interest. As an example
for its capability to nanoscopically visualize cross-linking, we
investigate pNIPAM microgels as system known with variations in
internal cross-linking density.
information even in the subnanometer range for sufficient electron
density contrast.[12-13] However, scanning probe and electron
microscopy methods have the drawback, that they are invasive or
need high-vacuum conditions, respectively. Additionally, many
polymer systems possess poor electron contrast. The advantages
of super-resolution fluorescence imaging techniques are that they
are non-invasive and allow for real-time imaging of structures.[14-
15] For addressing many relevant questions in materials science,
however, further developments concerning sample preparation,
measurement conditions and especially designing switchable
dyes with suitable photophysical and chemical properties, and
especially selective labeling capability of functionalities or
compartments, are necessary. Direct visualization of the position
of single cross-links and their distribution in polymer networks is
one such dream of polymer physicists and chemists. It is known
that in many polymer networks and hydrogels such cross-links
exhibit heterogeneities on the nanoscale[16] which are often
already introduced by the polymerization conditions.[17] The high
complexity of such cross-linked soft matter systems prevents their
quantitative structural analysis by common spectroscopic
techniques.[18]
Super-resolution fluorescence microscopy methods allow
for in situ visualization and investigation of various biological
structures at the nanoscale level.[1-7] In recent years, also an
increasing number of soft matter systems and materials science
questions can be addressed, since the commonly employed
protocols for biological systems could be adapted for materials
science.[8-9] Super-resolution fluorescence microscopy has the
potential to give new insights that complement those obtained by
well-known and recognized techniques in materials science, such
as scanning probe microscopy techniques[10] and modern electron
microscopy methods.[11] The former gives access to the
nanometer range and determines softness and surface properties,
such as topology, and the latter techniques can yield structural
Polymer networks extensively swollen with water are the basis of
hydrogels,[19] both of macroscopic sizes and in their particle form
as microgels in a size range from nanometers to several
micrometers.[20] In the swollen state, the volume of such a
microgel is in many cases filled by less than 10% polymer.[21]
Choosing appropriate monomers such as N-isopropyl acrylamide
(NIPAM), these microgels can be made stimuli-responsive and
possess a huge potential for a wide variety of applications, for
example as smart surface coatings and membranes,[22-23]
controlled release of pharmaceuticals or guest-molecules,[24-25]
model systems for colloidal chemistry,[26] (bio)catalysis,[27-28]
optical applications[29-30] and emulsion stabilization.[31-32] The
physical properties of microgels are mainly governed by the
cross-linker content and distribution.[33] Scattering experiments
indicate a complex morphology, often described by a so-called
“fuzzy-sphere” model with dangling chains,[21] for the internal
structure of such a microgel, which is governed by polymerization
kinetics.[34] External stimuli such as change in temperature, ionic
strength, pH, pressure, external magnetic and electric fields or
light provide the opportunity to reversibly change the
physicochemical properties. However, also this responsive
behavior is to a large extent governed by the network structure.
Thus, a direct visualization and quantification of the cross-linker
positions is a key point to understand the microgel complex
architectures and characterize their properties.[35] It has been
previously shown that super-resolution fluorescence microscopy
is highly suitable for investigations of microgels with covalently
[a]
[b]
E. Siemes, O. Nevskyi, S. K. Turnhoff, A. Oppermann, Prof. Dr.
Walter Richtering, Prof. Dr. D. Wöll
Institute for Physical Chemistry
RWTH Aachen University
Landoltweg 2, 52074 Aachen
E-mail: woell@pc.rwth-aachen.de
Dr. D. Sysoiev, Dr. T. Huhn
Department of Chemistry
University of Konstanz
Universitätsstr. 10, 78464 Konstanz
[+] These authors contributed equally
Supporting information for this article is given via a link at the end of
the document.
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bound labels[36-38] or freely diffusing dye molecules penetrating the
microgel network.[39] However, in all of these cases, switching
buffers were essential to obtain the fluorescence blinking
conditions required for localization-based superresolution
methods. Such buffers can affect the swelling especially in the
case of microgels that contain ionic and pH-sensitive moieties.
2c are presented in Figure 1. The bathochromic shift of the
absorption band of the closed form 2c in comparison to 1c is due
to an increase of the π-system length. Photoswitch 2oc has two
styrene units on both sides which allows for its incorporation as
cross-linker in radical polymerizations. The diarylethene
chromophore has been shown to be extremely stable under
radical polymerization conditions.[47] After the polymerization
reaction, photoswitch 2oc behaves similar to the phenylderivative
1oc due to the negligible influence of the alkyl-substituents in the
para-position of the phenyl groups. The photophysical properties
of photoswitch 1oc are illustrated in Table S1 of the Supporting
Information. The open and closed forms can be interconverted by
irradiation with suitable wavelengths (for more details see Figure
S1). The irradiation with UV-light mainly results in the formation of
the closed form, while the irradiation with the visible light recovers
the open form. The low off-switching (cycloreversion) quantum
yield of the benzothiophene-1,1-dioxide-based diarylethene
photoswitches is essential for PALM imaging. It allows for the
collection of a high number of photons necessary for an accurate
localization of the position[48] of single photoswitches before they
switch back to the dark state. 2oc can be imaged with only one
excitation wavelength as also shown for 1oc by Irie and
coworkers.[49] This is of great practical advantage in various
super-resolution fluorescence microscopy applications.
Here, we introduced
a
photochromic diarylethene
photoswitch as cross-linker into a microgel for superresolution
imaging. Not only has this dye never been used to address
polymer networks and gels before, but we also show for the first
time that cross-linker densities and distributions can be visualized
with unprecedented precision using photoactivation localization
microscopy (PALM) as nanoscopy method. We demonstrate the
high potential of this approach for imaging of the heterogeneous
cross-linker distribution in pNIPAM-based microgels as one of the
most prominent microgels and, in general, polymer network
system.
Diarylethenes, apart from their high potential in data storage,
chemical sensing,[40-41] and their possibility to induce switching in
other chromophores,[42] have recently found application as a
universal tool for super-resolution imaging.[43-45] They possess
spectrally well-separated on- and off-states, high absorption
coefficients, high fluorescence quantum yield of the on-state, high
photostability, a low quantum yield for the off-switching, and a
reasonable quantum yield for on-switching.
Figure 2. Synthesis of the pNIPAM-based microgels under investigation.
Imaging of cross-links with our novel diarylethene derivative 2oc
was performed on microgels, as they are highly relevant swollen
polymer networks with a heterogeneous crosslinking density as
determined by scattering techniques.[21] pNIPAM-based
microgels were synthesized by free radical precipitation
polymerization with N,Nꞌ-methylenebis(acrylamide) (BIS) and 2oc
as cross-linkers as shown in Figure 2. The amount of our DAE
cross-linker 2oc with respect to BIS was 4 mol% in order not to
modify the properties of the microgels significantly from the well-
known and well-characterized microgels cross-linked with BIS.
After synthesis, the microgels were purified via dialysis and
centrifugation in order to minimize the amount of free dye to a
non-significant amount. The detailed procedure can be found in
the Supporting Information.
Figure 1. Chemical structures of the open and the closed form of photoswitches
1oc and 2oc; absorption spectra of the closed forms 1c (blue solid line) and 2c
(red solid line), and the corresponding fluorescence spectra of 1c (blue dashed
line) and 2c (red dashed line), respectively. All spectra were recorded in 1,4-
dioxane.
Superresolved PALM imaging of the microgels containing 2oc
cross-linkers was performed on
a widefield fluorescence
microscope, the details of which can be found in the supporting
information. 3D localization was obtained by astigmatism. For this
purpose a cylindrical lens with 1000 mm focal length was placed
at an appropriate position in the emission path, resulting in z-
position dependent elongation of the single emitter point spread
function along two axes. The excitation wavelength and power
was set to 488 nm and approx. 6.25 kW cm-2, respectively. These
conditions directly resulted in appropriate blinking conditions for
PALM. It should be mentioned at this point that, in contrast to
We synthesized photoswitches 1oc and 2oc (Figure 1) by a
Suzuki–Miyaura coupling reaction using the 6,6ʹ-diiodo derivative
of 1,2-bis(2-ethyl-1,1-dioxidobenzothiophene-3-yl)perfluorocyclo-
pentene and corresponding boronic acids according to the
procedure previously described by Irie and coworkers (for more
details see the Supporting Information).[46] The absorption and
fluorescence spectra of the corresponding closed forms 1c and
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typically dSTORM measurements,[50] no additives to induce
blinking were required. Movies of the blinking events with 10 ms
integration time were recorded on a EMCCD camera (iXon Ultra
897) and analyzed with ThunderSTORM software.[51]
Superposition of single 3D localizations leads to a super-resolved
image of the cross-links in microgels as shown in Figure 3. Every
dot in this figure corresponds to a single localization event. As an
additional way to illustrate the cross-linker density, the dots were
color-coded according to the number of neighboring positions
within a radius of 30 nm. The resulting reconstructed images of
the microgels exhibit a dense, highly cross-linked center and less
cross-linked outer boundaries. In the center, a high amount of
localizations is surrounded by 20 or more localizations within
30 nm neighborhood, whereas single localizations can be found
in the outer periphery of the microgel.
center where also the polymer density is at its maximum.
Proceeding out of the center, the polymer density remains still
rather constant until ca. 80 nm distance from the microgel center,
whereas the relative density of cross-linker 2 already dropped off
to ca. 50% at this distance. In the outer microgel regions, i.e. the
fuzzy region with dangling chains, both polymer and cross-linker
2 density are relatively low. Such a detailed comparison of the
differences between polymer density and cross-linker density has
never been performed before since it was technically not possible
to disentangle both values, as our current approach allows for. It
should be noted here that any modification of a cross-linker can
have significant influence on polymerization kinetics and,
therefore, the way how it is distributed.[52] This is true for
fluorescent cross-linkers introduced during polymerization or
subsequent chemical reaction to a cross-linking group. Typical
cross-linkers such as BIS, however, do not allow for super-
resolved imaging neither in fluorescence nor in electron
microscopy. As a consequence, our approach presented here is
the only possibility to visualize single cross-links and future work
will concentrate on combining the fluorescent cross-linker with
typical ones in order to determine how polymerization conditions
and the properties of the cross-linkers determines their
distribution in the final polymer networks.
Figure 3. 3D distributions of the cross-linker positions of an individual microgel
in their hydrated, swollen state and the corresponding 2D projections to three
different planes. Each point represents a localization of a diarylethene cross-
linker. The color scale indicates the number of localizations within a radius of 30
nm, i.e. the cross-linker density. The grey circle illustrates the hydrodynamic
radius rh as determined by DLS (see Figure S2). A 3D animation can be found
in the Supporting Material.
Beyond
heterogeneous appearance,
a
visualization of the cross-linkers and their
thorough analysis of their
a
distributions is of high theoretical as well as practical importance.
In particular, for pNIPAM microgels, it has been found by different
scattering methods that the microgel density decreases from the
center towards the periphery.[21] Additional indication by super-
resolved dSTORM imaging with freely diffusing dyes was recently
published by Bergmann et al.[39] In contrast, we directly observed
the positions of cross-links, and can correlate their appearance to
the local microgel density. The latter was determined by static
light scattering (SLS). As shown in the upper graph of Fig. 4, the
static light scattering data can be fitted with the fuzzy sphere
model by Stieger[21] (for details of the parameter see the
Supporting Information). In the lower graph of Fig. 4 the resulting
polymer density is compared with the one obtained by
superresolution microscopy. The radial distributions of the
polymer density and cross-linker 2 are significantly different. In
contrast to the polymer density, which can be well described as a
fuzzy sphere, the radial density of cross-linker 2 levels off more
rapidly and, within 30 to 150 nm, in an almost linear way. The
highest density of cross-linker 2 can be found in the microgel
Figure 4. Upper graph: Static light scattering data of the p(NIPAM-co-BIS-co-
DAE)-microgel (black circles). The data were fitted with a fuzzy sphere model
(green line, for more details see Supporting Information). Lower graph: Radial
distribution of polymer density as obtained by the fit of the SLS-data (open blue
diamonds) and radial distribution of DAE-cross-linkers obtained from PALM 3D-
images (full orange circles), averaged over 20 microgels. Error bars indicate the
standard deviation of the density obtained from single microgel images. All
radial distributions were normalized by their value in the center.
In summary, we developed a diarylethene photoswitch with two
styrene groups which can act as cross-linker. This allows for a
visualization and analysis of the cross-linker distribution with
localization-based super-resolution fluorescence microscopy
down to the nanoscale without the need for any additives that
affect pH and ionic strength. We demonstrated the power of this
approach with microgels which are known to exhibit
inhomogeneous polymer density, but for which the cross-linker
density has never been experimentally analyzed. It was shown
that both, polymer and cross-linker density decreases when
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Acknowledgements
We thank the Deutsche Forschungsgemeinschaft (DFG) for
support within the projects C5 and A3 of the SFB 985 “Functional
Microgels and Microgel Systems”.
Keywords: photoswitch • super-resolution microscopy • soft
matter • microgels • cross-linker
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Entry for the Table of Contents (Please choose one layout)
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Nanoscopy on polymer cross-links:
A cross-linking photoswitch allows for
the visualization of cross-linker
positions with super-resolution
fluorescence microscopy giving
unprecedented insights into the
heterogeneity of polymer networks
which are essential for a fundamental
understanding and application of
cross-linked polymer structures.
Author(s), Corresponding Author(s)*
Page No. – Page No.
Nanoscopic visualization of cross-
linking density in polymer networks
with diarylethene photoswitches
Layout 2:
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Author(s), Corresponding Author(s)*
((Insert TOC Graphic here))
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Title
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