the growing impact of photonics in consumer electronics ... › wp-content › uploads › 2018 ›...
TRANSCRIPT
The growing impact of
photonics in consumer
electronics and automotive
markets Markus Bilger
Viavi OSP
E-mail: [email protected]
Abstract
Photonics is experiencing a period of high
growth. The drive to ever smarter and more
automated devices results in the need for these
devices to sense their environment as well as or
preferably better than humans. Optical thin film
coatings are a key ingredient in elevating
sensory performance. The ability to see at
wavelengths and speeds beyond the human eye
is key to a span of things ranging from 3D
sensing in smartphones to Lidar systems in
automotive.
Packaging based on bioORMOCER®
Sabine Amberg-Schwab, Katharina Emmert Fraunhofer Institute for Silicate Research ISC, Neunerplatz 2, 97082 Würzburg, Germany
Non-degradable plastic packaging causes pollution, onshore and offshore. Biodegradable packaging may well be the solution. Biodegradable plastics offer a potential for creating a more sustainable society and solving global environmental waste problems.
However, the properties of the state-of-the art bio-based and biodegradable plastics often do not meet all the necessary requirements and are still inferior to those of established non-biodegradable plastic products. Therefore, biopolymers have only had limited use as high-quality packaging materials for food up to now. This is because they do not provide sufficient protection of food against penetrating oxygen and water vapor and so cannot guarantee the required shelf life.
Our idea was to improve these properties by a biodegradable coating. We have been developing high-barrier coatings based on a class of materials with glass-like structural units, so called ORMOCER® (inorganic-organic hybrid polymers) for many years. This material class exhibits excellent barrier properties against gases and vapors and thus is used as a coating for food packaging materials. However, the state-of-the-art ORMOCER® is not biodegradable. Therefore, the aim of our work was to modify these ORMOCER® to be biodegradable, and at the same time, preserve
the barrier and functional properties to provide new potential barrier materials for functional biodegradable packaging.
To realize the new biodegradable coatings, several suitable bio-based / biodegradable compounds have been identified and chemically functionalized. In this way, new bio-precursors were developed that could be used for the synthesis of functional biodegradable coatings. This was achieved by nano-chemical incorporation and chemical linkage of the chemically modified biopolymers into the ORMOCER® network (s. Fig. 1).
Both bio-based and biodegradable natural materials (chitosan, cellulose derivatives) and petroleum-based biodegradable educts (e.g. polycaprolactone-triol (PCL-T)) were used for bioORMOCER® development. In order to guarantee incorporation of the biodegradable components into the hybrid polymer network, some of these components were subjected to chemical modification. For example, the polycaprolactone derivative was functionalized with triethoxysilane groups in order to subsequently allow attachment of these biodegradable components to the oxidic inorganic network via hydrolysis and condensation reactions. The cellulose was treated similarly. In this case, attachment of biodegradable precursors to the organic network of the hybrid material was achieved by functionalization with epoxy groups. The reactive epoxy groups subsequently participated in the polymerization reactions for formation of the organic network. In contrast, chitosan required no modification because it can be linked to the organic network via some of its intrinsic amino groups.
Fig. 1. From conventional hybrid (ORMOCER®) to biodegradable hybrid materials (bioORMOCER®).
Fig. 2. Improvement of the oxygen transmission rate (OTR) through bioORMOCER®.
The resulting state-of-the art bioORMOCER®
coatings are transparent. They provide very good barriers to oxygen and water vapor and can furthermore be used as a planarization layer for substrates with rough surfaces. Figure 2 shows the results regarding the oxygen transmission rates of various bioORMOCER®
coatings. All systems lead to barriers sufficient for usage in food packaging.
The bioORMOCER® based coating materials represent a new material class. The coatings have good adhesion properties on different polymer film surfaces. The coatings are abrasion resistant and robust. The kinetics of biodegradability is temporally adaptable [1, 2].
Further investigations into the promising new material class of bioORMOCER® are currently being done within the EU-project HyperBioCoat [3]. The aim of this project is to develop new formulations of bioORMOCER®
derived from food processing by-products, which can provide the high levels of protection required for the demanding areas of food, cosmetic and medical device packaging. In this project we will first focus on the chemical modification of commercially available biopolymers, which are then covalently bound to the ORMOCER®. In theory it should be possible to apply those modifications also to extracted biopolymers from fruit, vegetable or paper pulp residues. The covalent modification of the ORMOCER® is realized either by binding of the functionalized biodegradable biopolymers to the inorganic network or to the organic network (s. Fig. 3).
In the scope of the HyperBioCoat project two different inorganic modification reactions were carried out in order to introduce triethoxysilane groups to the biopolymers. Those triethoxysilane groups then provided the means to covalently attach the biodegradable components to the oxidic inorganic
ORMOCER® network via hydrolysis and condensation reactions.
Furthermore, the organic modification of the biopolymers with amino groups allowed the bonding to the organic network of the hybrid material via reaction with ORMOCER® epoxy groups. Also, the modification with amino groups leads to products which are readily soluble in the aqueous-alcoholic ORMOCER®
solvent system.
All modification methods were first tested on the commercially available cellulose and then the results were transferred onto different hemicelluloses. The successful modification of the biopolymers was confirmed by spectroscopic methods (FT-IR, Raman, NMR), elemental analysis and/or titration in each case. Different incorporation methods of the functionalized biopolymers into the ORMOCER® system were investigated (cf. Fig. 4):
Method 1: Inorganically functionalized (hemi)celluloses were used as bio-precursors in the lacquer synthesis in exchange for part of the conventional inorganic precursors
Method 2: A basic, acidic or neutral Amino-(hemi)cellulose solution was directly added into ORMOCER® lacquers
Method 3: The aqueous Amino-(hemi)cellulose solution was utilized as a part of the ORMOCER® synthesis (hydrolysis).
Fig. 3. bioORMOCER® structure: type I (pink), type II (red, M = metal), type III (blue) and type IV (green, biodegradable).
Fig. 43. Modification reactions and incorporation of biopolymers into ORMOCER®
as part of the EU-project HyperBioCoat
The resulting coatings were evaluated regarding optics, adhesion, barrier properties and biodegradability. The best results so far were achieved with the amino-functionalized biopolymers, which were incorporated as basic additive. The films containing amino-biopolymers were generally clear and transparent, the adhesion on all tested substrates was high and first signs of biodegradation were visible after a few weeks in a test-compost. Furthermore, the barrier performances of the newly
developed bioORMOCER® are comparable to the barrier properties of the state-of-the-art ORMOCER®.
The new bioORMOCER® can provide biodegradable coatings from ecologically friendly resources and can improve biodegradable polymer films, which could in the future lead to the use of these biopolymer systems for food, cosmetic and pharmaceutical packaging.
bioORMOCER® can play a decisive part in the future design and development of innovative, more environmentally friendly food packaging materials.
Reference
[1] S. Amberg-Schwab, et al., Protection for Bioplastics, European Coatings Journal 12 (2015) , 32-36.
[2] S. Amberg-Schwab, Functional Barrier Coatings on the Basis of Hybrid Polymers, in: Handbook of Sol-Gel Science and Technology; L. Klein, M. Aparicio, Andrei Jitianu (Eds.), Springer, Berlin, 2016.
[3] http://hyperbiocoat.eu, No 720736.
Maturity of moisture barriers
for large volume production Luca Gautero1, Ewout van Vugt1, Dana
Borsa1, Edward Clerkx1
1Meyer Burger (Netherlands) B.V.
Abstract
Marketability of wearable or flexible electronics, for example organic light emitting device (OLED) displays, demands inexpensive thin film encapsulation (TFE) against moisture. Large volume manufacturing tools able to
create a reliable and cost compatible moisture
barrier are not trivial. The scope of this work is
to present large volume manufacturing of TFE
based on stacks of Microwave Plasma
enhanced chemical vapour deposition (MW-
PECVD) or spatial atomic layer deposition (s-
ALD) inorganics, and jettable or dispensable
organics.
Keywords: Encapsulation, moisture, barrier,
WVTR, flexible electronics, OLEDs
1 Introduction
Barriers, with water vapour transmission rates
(WVTR) in the range of 1E-6 g/day/m2, are
manufactured at low temperatures with contact
less techniques on laboratory scale systems
(Salem et al. 2016). Throughput and yield
might not be representative for large
production.
In Europe, within the Innovation program
Horizon 2020, effort has been addressed to the
improvement of barriers by sponsoring
multiyear projects. One concrete result of this
approach is a pilot scale production of state of
the art flexible OLED for lighting (“European
Commission : CORDIS : Projects and Results :
Bringing Flexible Organic Electronics to Pilot
Innovation Scale” n.d.).
The U.S. department of energy proposed to
foster the large volume production of solid
state lighting. Part of this program addresses
OLED lighting and therefore its encapsulation
needs (Department of Energy 2016). The cost
and performances have been given aggressive
roadmaps. Companies, like OLEDWorks and
LG Display, two of the major OLED lighting
producers by volume, are meant to live by this
roadmap in order to give clear expectations on
the competition front.
Following its mission and technological
competence, Meyer Burger (Netherlands) B.V.
has developed both sheet to sheet (S2S) and
roll to roll (R2R) equipment for the fabrication
of TFE as stacks of inorganic and organic
layers. The individual equipment tools gather
their maturity from hundreds of installations
targeting several applications (photovoltaic,
display, plastic electronics and PCB
applications) at both scientific institution and
manufacturing industry. Inorganic thin film
deposition and organic layer coating are
therefore combined into single, fully automated
cluster tools with industrial manufacturing
capabilities dedicated to TFE (“Meyer Burger
Ships an Inkjet+PECVD OLED Encapsulation
System to an Asian Customer | OLED-Info”
n.d.).
The technologic transfer of the Holst processes
technology towards a large volume
manufacturing environment resulted in two
applications: S2S with PECVD and IJP
technology and R2R with s-ALD and slot dye
coating. The transfer promoted improvements
to decrease the cost of ownership of the
technologies. The goal is to exceed throughput
above several square meter/hour.
The large volume cluster combines processes
of thin film (vacuum for PECVD and
atmospheric for s-ALD) deposition of dielectric
layers as implemented on the FLEx family
tools (both R2R and S2S) together with an
inkjet printing process as implemented on the
Pixdro family tools for the organic layers into a
single fully automated cluster.
For the case of S2S, novelties in the deposition
of the layers and their characteristics will be
presented together with a discussion on their
direct implications.
2 Experimental
One important task of the technology transfer
from laboratory to industrial scale is to identify
bottlenecks of production. These were put in
light during the design phase of the S2S TFE
cluster tool. The printing process of the organic
layer can be scaled without affecting
significantly the design or the final layer
properties. Instead, the inorganic layer, an
hydrogenated amorphous silicon nitride film
(SiNx) deposited by PECVD, needs to be
carefully designed to allow a functional process
with high throughput.
Two key requirements on the inorganic layer
have the highest priority for the integration with
a TFE production cluster: High deposition rate
and accurate control of the substrate
temperature during the whole deposition
process.
Increasing the deposition rate is
straightforward on almost any plasma based
deposition tool: increasing precursor gases
rate and pressure would guarantee more
radicals to reach the substrate per amount of
time. However, the layer quality varies
dramatically (Anders 2010).
For the TFE application the quality of the
inorganic layer is key for the overall barrier
functionality and reliability over time.
Therefore, the process window is smaller.
Inorganic layers are deposited at low
temperatures (<80 ºC) by PECVD SiNx. Its
deposition rate can be as high as 12nm/s. In
Table 1 the list of the parameters of
importance for layers deposited with deposition
rates higher than 10nm/s are shown.
Table 1: Characterization of inorganic layers
Inorganic layers are characterized by Ca-test
(Nisato et al. 2014) at accelerated conditions
(60 ºC/90 %rH, a factor of 40 is used to
translate the number to 20 ºC/50 %rH
conditions) to reveal their WVTR. Microscope
glass strips are coated to map stress. Foils are
coated to measure the film failure strain.
Silicon and glass substrates are coated with
SiNx and exposed to harsh conditions
(85ºC/85%rH) while being monitored for any
possible shift in thickness, refractive index and
haze.
3 Result and Discussion
The WVTR value guarantees good performances over a long span of time.
However, the measurement of WVTR is not affected by the known oxidizing effect of harsh condition on the surface of low temperature
SiNx layers (Chiang, Ghanayem, and Hess
1989). Therefore, we observed it indirectly and
optimized the layers to extend their lifetime.
The mechanical properties reported in this
study can be used to simulate the behaviour of
a stacked configuration. From these
calculation, it turns out that the thickness of the
flexible substrate has a large role in cracking
nucleation. It influences strongly the neutral
line, which can be far from the deposited SiNx
and therefore affect it imposing a great strain.
Layer: SiNx Value Unit Relevant Condition description
WVTR 3.5±0.17 (E-6 g/day/m2) @300nm
Stress 70±20 (MPa) @300nm
Strain <0.8 (%) @150nm
Hydrolyses rate <0.1 (nm/h) @85ºC/85%rH
Haze <0.5 - @85ºC/85%rH
4 Conclusions
Well established laboratory scale processes
able to realize highly performing TFE have
been transferred to large volume production
cluster tools thanks to adaptation and
extensions of existing tool capabilities.
Increased production speed is not affecting the
quality of the production
Acknoledgements
The authors would like to thank Pavel
Kudlacek for his support in the verification of
high speed barriers.
References
[1] Anders, André. 2010. “A Structure Zone
Diagram Including Plasma-Based
Deposition and Ion Etching.” Thin Solid
Films 518 (15): 4087–90.
https://doi.org/10/b3vv5v.
[2] Chiang, J. N., S. G. Ghanayem, and D.
W. Hess. 1989. “Low-Temperature
Hydrolysis (Oxidation) of Plasma-
Deposited Silicon Nitride Films.”
Chemistry of Materials 1 (2): 194–98.
https://doi.org/10.1021/CM00002A006.
[3] Department of Energy. 2016. “Solid-State
Lighting 2016 R&D Plan.” Solid-State
Lighting (SSL) R&D Plan.
https://www.energy.gov/sites/prod/files/20
16/06/f32/ssl_rd-plan_%20jun2016_2.pdf.
[4] “European Commission : CORDIS :
Projects and Results : Bringing Flexible
Organic Electronics to Pilot Innovation
Scale.” n.d. Accessed April 23, 2018.
https://cordis.europa.eu/project/rcn/19917
5_en.html.
[5] “Meyer Burger Ships an Inkjet+PECVD
OLED Encapsulation System to an Asian
Customer | OLED-Info.” n.d. Accessed
April 23, 2018. https://www.oled-
info.com/meyer-burger-ships-inkjetpecvd-
oled-encapsulation-system-asian-
customer.
[6] Nisato, Giovanni, Hannes Klumbies, John
Fahlteich, Lars Müller-Meskamp, Peter
van de Weijer, Piet Bouten, Christine
Boeffel, et al. 2014. “Experimental
Comparison of High-Performance Water
Vapor Permeation Measurement
Methods.” Organic Electronics 15 (12):
3746–55. https://doi.org/10/gcrnj3.
[7] Salem, A., H. B. Akkerman, P. van de
Weijer, P. C. P. Bouten, J. Shen, S. H. P.
M. de Winter, P. Kudlacek, et al. 2016.
“Thin-Film Flexible Barriers for PV
Applications and OLED Lighting.” In 2016
IEEE 43rd Photovoltaic Specialists
Conference (PVSC), 1661–63.
https://doi.org/10.1109/PVSC.2016.77499
05.
Innovative Coatings for
Advanced Applications Pavel Bartovský1, Adolfo Benedito Borrás1,
José María García Pérez1, Belén Monje
Martínez1, Amador García Sancho1
AIMPLAS, Synthesis Department, Paterna
(Valencia), Spain
Abstract
Ice accumulation is a serious concern in many
applications such as aviation, shipping,
communications, and in power generation and
transmission. There is no coating available that
prevents the build-up of ice for all cases.
However, several approaches have been
employed to reduce ice formation and
adhesion on treated metal, plastic, composite,
ceramic and glass surfaces.
Superhydrophobic coatings can increase the
contact angle of water-droplets with treated
surface and therefore reduce the accumulation
of water which can lead to ice formation.
Hereby, we present results on the
development of superhydrophobic coatings
based on polymers modified with different
types of silica nanoparticles. These
dramatically increase the contact angle
between treated surfaces and water droplets
and therefore can be used for several
applications such as naval construction, wind
turbines or antifouling coatings.
When dealing with icing, the superhydrophobic
materials are not always the best solution as
they are usually not able the prevent ice
formation and accumulation. Therefore,
increasing interest is observed in the
development of materials for anti-icing
passive and active coatings that could reduce
the icing of aircrafts, ships, rail vehicles, wind
turbines and other objects and structures
exposed to dynamic extreme environmental
conditions. To reach the objectives we have
used a combination of modified polymers with
a variety of carbon-based nanoparticles which
allowed us to control the nano-rugosity of the
treated surfaces and thus to reduce the ice
adhesion and accumulation in the case of
passive prevention and to melt the already
accumulated ice using self-heated coatings as
an active anti-icing strategy.
Keywords: Coatings, superhydrophobicity, anti-
icing, nanoparticles, polymers, composites
1 Introduction
Icing is an important problem in several sectors
from aeronautics to off-shore structures,
including power lines, wind turbines, steam
power plants and even for trucks and rail-
vehicles among others. Its effects can have
catastrophic consequences which could result
in large socioeconomical losses and fatal
accidents with loss of human lives. Icing is
particularly critical for aircrafts so several anti-
icing systems (preventing ice formation) have
been developed to avoid or reduce it. Most of
these technologies require continuous supply
of hot air, chemical products, electrical power
or breaking up ice formations, usually by
inflatable boots placed on the wing leading
edge. However, modifying the structure of the
surface to reduce or eliminate icing is a much
more attractive solution. For instance, the use
of ice-phobic coatings on top of the exposed
surfaces constitutes a strategy that has
generated promising results. On the other
hand, some of the icing affected components
can also suffer in flight particle erosion causing
coating wear and losing their anti-icing
function.
SUPERHYDROPHOBICITY and
ICEPHOBICITY (passive anti-icing)
Superhydrophobic surfaces are highly
hydrophobic and thus extremely difficult to wet.
The contact angles of water droplet exceed
150º. This is also referred to as the lotus effect,
inspired by the superhydrophobic leaves of the
lotus plant. A droplet impacting on this kind of
surfaces should fully rebound. These solutions
are useful in situations when water
accumulation can cause problems such as on
the central and rear parts of wings and helices
of wind turbines, marine platforms and many
others. However, superhydrophobic surfaces
are not the best protection against icing as we
have demonstrated in this work.
The reason is the different behaviour of water
droplets and ice crystals, particularly in a
dynamic environment. The contact angle
although critical for hydrophobicity lacks
relevance when dealing with ice crystals where
different physical models are taking place as
resumed in the figure 1:
Fig 1: Hydrophobicity and icephobicity evaluation tests
The methods of protection based on
icephobicity of the composites is a very
attractive research area and these methods
are usually referred to as PASSIVE
METHODS.
To reach the main objective, the passive
protection should meet the following criteria:
1. Reduction of ice adhesion
2. Reduction of icing/nucleation
phenomena
3. Polymer matrix need to be transformed
in icephobic material
An additional challenge is to maintain the
adhesion and mechanical properties of the
polymer.
In this work, we present results on
development on novel composite coatings
comprising a superhydrophobic matrix
reinforced with hard particles in order to protect
treated surfaces from icing. For the matrix,
already known materials such as functionalized
silicones or teflons, to which nano- or micro-
particles such as oxides, carbides, carbon
nanotubes, quasicristalline materials, etc. were
added. Low cost and appropriate deposition
techniques for large surfaces were employed
in a way so that industrial application can
become a reality.
DEVELOPED PASSIVE METHODS
• Combination of nanoparticles and low
energy polymers
• PU, polyacrylates
• Specific silica modification
• Applied as coating
• Spray techniques or paint brush, roller
application
A big challenge has been poor adhesion and
high viscosity of some composites.
COATINGS WITH THERMAL PROPERTIES –
based on Joule effect (active anti-icing)
Another complementary strategy is the use of
thermal systems based on the Joule effect. In
such cases, the polymer “non-conductive”
matrix needs to be transformed in a material
able to generate heat due to passage of
electric current through the coating and thus to
reduce ice formation and/or eliminate
accumulated ice on the treated surface.
This solution can be applied on polyurethanes,
polyesters, polyacrylates and epoxy- coatings
which can be combined with passive methods
of antiícing protection such as the use of
fluorinated polymers. The specific task of heat
generation requiers the use of highly
conductive materials such as carbon
nanotubes (CNT). For this purpose, specific
techniques of CNTs modification and
dispersion are required. For a real-life
application it is necessary to develop
formulations that can be applied using
common techniques used in coating such as
spray techniques and brush or roller
application. In this point a big chalenge is to
enhance the polymer conductivity without the
loss of coating adhesion and without increase
of the coating viscosity.
2 Experimental
2.1 Superhydrophobic coatings and
passive anti-icing coatings
2.1.1 Synthesis of silica nanoparticles with
controlled size
The synthesis of the nano-silicas was
performed in size-controlled manner using well
described reactivity of tetraethyl orthosilicate
(TEOS) which undergoes reaction with water
that can be resumed by the following
equations:
This hydrolysis reaction is an example of the
SOL-GEL process. The reaction proceeds via
a series of condensation reactions that convert
the TEOS molecule into a mineral-like solid via
the formation of Si-O-Si linkages. Rates of this
conversion are sensitive to the presence of
acids and bases, both of which serve as
catalyst. Figure 2 shows different processes
employed to synthesis monodispersed nano-
silicas in a size-controlled manner:
Fig 2: Synthesis of controlled size silica
nanoparticles – schematic representation
Employing the sol-gel method described above
we prepared different monodisperse silica
spheres with diameter ranging from 100 to
1200 nm in a controlled manner and with
reaction yields between 95-99%. Two
representative examples are shown in the
table 1.
Table 1:. Some typical examples of prepared silica nanoparticles
Material NH3
Ratio
TEOS/EtOH
Reaction
time (h)Shape
Yield
(%)
Diameter
(nm)
PRO13-0333-
03-07-b2 83 1/0,88 2 Sphere 99 1100-1200
PRO13-0333-
03-23-b2 94+surfactant 1/8 1,5 Sphere 95 180-200
Figure 3 represents an example of SEM
analysis of the prepared silicas.
Fig 3:Example of SEM images of prepared silica
nanoparticles
From the industrial point of view it is worth to
mention that the synthesis has been optimized
and scale-up has been made up to the
kilograms quantity of monodispersed
nanoparticles so it allows real-life application of
the developed process.
2.1.2 Functionalization of prepared silica
nanoparticles
To improve the hydrophobicity even more and
to modify the nano-rugosity of the coatings and
thus to enhance the passive protection against
ice formation we developed nano-silicas with
controlled size functionalized with various
silanes with different chain types following the
procedure shown below:
Some of the silanes employed for the silica
functionalization were chlorotrimethylsilane
(ClTMS), dichloromethylsilane (DCMS) and
alkylchloromethylsilane (ALKCMS). The
reaction conditions and yield are shown in
table 2.
Table 2: Functionalization of silica nanoparticles with silanes
Material Silane Reaction time (h) T (ºC) Yield (%) Hydrophobicity
PRO13-0333-03-23 70% ClTMS 24 60 72 OK
PRO13-0333-03-23 70% DCMS 24 60 85 OK
PRO13-0333-03-2370%
ALKCMS24 60 83 OK
2.1.3 Development of novel passive anti-icing
formulations
The prepared silica nanoparticles and
functionalized silicas were typically employed
in matrices based on polyurethanes and
polyacrylates. Most of them were commercially
available paintings such as the following
example whose composition has been
analysed with results shown below:
Polyurethane (commercial sample):
A component (100 g):
• 30% Butyl acetate
• 40% Charges (nano-SiO2 and nano-
TiO2
• 30% Aromatic diol (phthalic) 500 g/mol
B component (33 g):
• 100% Isocyanate bi/trifunctional NCO
19%
PU coatings were modified by dispersion of a)
silica nanoparticles prepared vía sol-gel
synthesis; b) silica nanoparticles functionalized
with alkylsilanes; c) functionalized fluorinated
polymers (3 types of polyethers), d)
perfluoroalkyl silanes, e) perfluorinated diols
among others.
In some formulations NaCl, CsF, CaCl2 and
cationic polymers were added as anti-nucleic
agents.
2.2 Active anti-icing coatings
The main problem is the functionalization of a
nonconductive polymer matrix to convert it in to
an electrical and thermal conductor. Our
approach is to focus on the modification of
polyurethanes polyacrylates and
fluoropolymers with electrical and thermal
superconductors. For this purpose, different
carbon nanotubes were employed.
Specific functionalization to synthesize a
copolymer or grafted polymer was used with
the aim to create strong F-F interactions. The
results were patented (ES 2398274 (A1) –
2013-03-15).
To quantify the heating capacity of the
developed coatings several assays have been
performed. First, the resistance was measured
and from the resistivity (Ω/cm) the values of
conductivity in S/cm were calculated to confirm
whether the coatings reach the values of
conductivity which correspond to the Joule
effect as shown in the figure 4.
Fig 4: Electric conductivity scale – Joule heating
range.
Beside the theoretical evaluation, practical
tests of the heating capacity have been
performed.
Samples covered with CNTs modified coatings
were connected to a power source maintaining
constant geometry (distance between the two
electrodes and sample size) and constant
electric current. We measured the temperature
using an IR camera as shown in the figure 5.
Table 3 shows some of the formulations tested
as possible active coatings.
Fig 5. Heating capacity of a CNT modified coating –
IR camera
Table 3: Families of active coatings based on CNTs
modified
Commercial
Material
CNTs
%
Joule
Con-
ductivity
Heat-
able
Polyacrylate
(coatings) 4%
Polyacrylate
(coatings)
4%
mod
Polyurethane
(coating) 2%
Polyurethane
(coating)
2%
mod
Capstone
(fluoropolymer) 6%
Capstone
(fluoropolymer)
6%
mod
3 Results and Discussion
3.1 Superhydrophobic and passive anti-icing coatings
From the comparison of hydrophobic and ice-
phobic properties of the developed coatings it
can be observed that the superhydrophobicity
is not the optimal solution for prevention of ice
formation and accumulation. Coatings with
high contact angle present poor ice reduction
capacity as can be seen in the table 4. On the
other hand, most of the coatings with contact
angles between 110 and 120ºC were those
with better capacity to reduce ice formation on
treated surfaces.
Table 4: Hydrophobicity vs. ice-phobic capacity
These results show that more factors should
be taken in to account while predicting anti-
icing capability. It suggests that other factors
play important role along with hydrophobicity.
Measuring contact angle is not the most
relevant method of quantification in this case.
Instead sliding angle (roll-off) should be used.
However, even this approach is only a
simplified approximation because the physio-
mechanical behaviour of an ice cube or crystal
differs significantly from that of a water droplet.
For this reason, we performed ice-adhesion
tests and ice accretion/formation tests under
controlled climatic conditions which simulate
the real dynamic environment.
The most important internal factors that
influence the ice-formation were found to be
surface roughness, nucleation elements and
surface temperature besides the environmental
factors that are all shown in figure 6.
Fig 6: Environmental factors and coating properties that influence ice formation
3.2 Anti-icing active coatings
The developed active anti-icing coatings were
tested on aluminium surfaces
in simulated real environmental conditions (air
flow, humidity, temperature). The results were
very encouraging with a temperature increase
of up to 90ºC in 10 seconds. The assay is
schematically represented in figure 7 (video
available).
Fig 7: Anti-icing coating applied treated on surface
4 Conclusion
Passive anti-icing coatings were developed
based on modified hydrophobic polymers. The
use of silica nano and microparticles resulted
in enhanced hydrophobicity and nano-rugosity
modification. Combination of silicas, low
superficial energy polymers and antinucleant
agents gave as result an improvement of anti-
icing capacity of the coatings. Improvement of
our systems requires a deep knowledge in
icing formation. Ice adhesion force, ice
nucleation, roughness and sliding angle are
powerful tools in order to predict the anti-icing
behaviour. Reduction of roughness is one of
the most promising tools to improve the
passive anti-icing properties.
Increasing the thermal conductivity of the
coatings will help us to improve the efficiency
of active systems reducing energy
consumption. Anti-icing solutions require an
overall approach including surface chemistry
and roughness and environmental conditions,
e.g. temperature, speed, and water droplets.
Acknowledgments
The authors wish to kindly thank for financial
support to the Spanish Ministry of Economy
(MINECO) and to European Commission for
the financial support.
Influence of Material Surfaces on Cell MotilityTimo Grunemann1,2, Patrick Witzel1,2, Martin
Emmert1,2, Franz L. Edel1,2, Doris Heinrich3
1 Faculty for Chemistry und Pharmacy,
Julius-Maximilians-Universität Würzburg,
Röntgenring 11, 97070 Würzburg, Germany 2 Fraunhofer Institute for Silicate Research ISC,
Neunerplatz 2, 97082 Würzburg, Germany 3 Leiden University, LION Leiden Institute of
Physics, Niels Bohrweg 2, 2333 CA Leiden,
The Netherlands * Corresponding author: Doris Heinrich,
Abstract
The extracellular matrix plays an important role for essential cell functions like adhesion, migration, proliferation, and differentiation. A clear understanding of biophysical interaction mechanisms between a cell and its environment provides a solid base for a cell-type specific design of three dimensional (3D) scaffolds in terms of surface topography and chemical surface functionalization. In this work, we analyze motion patterns of D. discoideum cells on three distinct surfaces: plain glass, nano-structured silica fiber and polydimethylsiloxane (PDMS) surfaces, utilizing a global mean-squared displacement (MSD) as well as time-resolved, local mean-squared displacement (LMSD) techniques. Global MSD analysis reveals a significantly higher cell migration activity on PDMS and nano-structured fiber surfaces compared to plain glass, yet the cell movement patterns on PDMS and silica fiber cannot be further distinguished. Further evaluation based on a LMSD approach yields a more in-depth picture of the specific surface influence on cell migration as this method enables differentiation between quasi-random and directed migration phases. We find that the nanostructure of the silica fibers induces significantly more directed migration phases compared to the glass and PDMS surfaces. Furthermore, the hydrophobic PDMS surface leads to greatly increased directed migration velocities. This analysis shows the advantage of a time-resolved LMSD analysis to reveal substantial differences in cell-material interaction. These insights will facilitate development of new biomedical materials with intrinsic properties designed to control cell migration leading to novel diagnostic and therapeutic concepts.
Keywords: Cell Motility, Material Surface, Cell-Material Interaction, Mean-squared Displacement, Live-cell Imaging
1 Introduction
Cell migration plays a vital role in a wide range of processes in the human body including wound healing [1], immune response, and embryogenesis [2,3], as well as for aberrations like cancer metastasis. As such, the investigation of cell migration behavior should be taken into account for the development of novel materials designed for direct contact with human tissue. Additional control of migration patterns could enable new applications in tissue engineering ranging from drug testing in vitro, reducing expensive clinical studies, to implants minimizing engraftment times.
In this work, we demonstrate the immense influence of different surfaces on cell migration behavior utilizing global mean-squared displacement (MSD) as well as time-resolved local mean-squared displacement (LMSD) analysis [4,5]. It is well known that cell motility is affected by several cues of the cellular surrounding, called the extracellular matrix within an organism, including surface chemistry [6], Young’s modulus [7], and topography [4,8]. It is, however, still unclear which of these external cues are dominant and overwrite other influences on cell migration behavior. To get a deeper insight, we obtained and analyzed migration data of Dictyostelium discoideum (D. discoideum) cells on three distinct surfaces. D. discoideum is a social amoeba exhibiting amoeboid migration modes comparable to stem cells or immune cells [9,10]. Strains of these cell type are often used as a model organism for chemotaxis [11], phagocytosis, and human diseases because of their completely sequenced genome and the relatively simple cell culture and handling [12]. The investigated surfaces comprised glass, as a standard and widely utilized biomedical reference, silica fibers with a nano-rough surface structure, used as wound healing inserts, and polydimethylsiloxane (PDMS) as an example for the polymeric material class and commonly used in experimental microfluidic and cellular assays.
2 Experimental
2.1 Preparation and Characterization of Silica Fibers Silica fibers were fabricated by pressure extrusion on cover glass substrates according to [8,13]. The resulting silica gel fibers’ composition
was determined to be [Si(OH)0.2O1.9]n by thermal analysis in a previous work [13] similar to glass. The fibers exhibited a dog-bone-like shape with measured fiber widths of 60 ± 20 μm and fiber heights of 24 ± 8 μm. Freshly fabricated fibers exhibited a smooth surface with no distinct topography. After contact with aqueous media nano-sized surface patterns were observed through biodegradation [13]. SEM investigations on fibers that have been stored in solution for several days revealed pits and elevations on the fiber surface with feature sizes below 200 nm [8].
2.2 PDMS Preparation The polydimethylsiloxane (PDMS) is processed as a two-component material. The precursor of Sylgard 184 (Dow Corning, USA) is mixed with the curing agent at weight ratio of 9:1. For air removal the solution was degassed at 160 mbar in a desiccator for 30 min. The resin was flood coated on a silicon wafer passivated by perfluorodecyltrichlorsilane (Sigma-Aldrich, St. Louis, Missouri, USA) and again degassed at 6×10-2 mbar with a temperature progression from room temperature to 50 °C over 30 min. The PDMS was cured at 65 °C for 6 h. Previous to the migration experiments the surface of the PDMS was activated by ozone plasma for 15 min at a power level of 70 % (Diener electronic GmbH + Co. KG, Ebhausen, Germany).
2.3 Cell Culture For the migration experiments cells of the D. discoideum strain AX2 expressing homogenously distributed green fluorescent protein (GFP) in the cytoplasm were grown under 40% confluency at 19–21 °C in petri dishes at ambient air. For cell culture, HL-5C medium with glucose (HLC0102, ForMediumTM, UK) including 20 μg mL−1 gentamycin (G-418-Biochrom A2912, Biochrom AG) was used.
2.4 Preparation and Migration Experiments Previous to the migration experiments, the cells were washed four times with phosphate buffered saline (PBS) with subsequent centrifugation at 2000 rpm for 4 minutes (Eppendorf MiniSpin®
Plus, Eppendorf AG) and resuspension of the cell pellet in PBS. Afterwards the solutions’ cell density was adjusted to achieve a surface cell density of 400,000 cells per cm2. The cells were kept 30 mins at rest after seeding to the surface. Image acquisition was performed with a Nikon Eclipse Ti fluorescence microscope (Nikon, Germany) with an Intensilight (Nikon, Germany) light source and a 20x objective (Nikon, Germany). To enable GFP imaging, an
excitation filter (F36-525 HC, AHF, Germany) ensured excitation wavelengths of 457–487 nm and detection wavelengths of 500–540 nm. The signals were detected with an EM-CCD camera (Hamamatsu, Japan). Images were acquired every 7 seconds for the glass and silica fiber experiments and every 8 seconds for the measurements on PDMS for at least 45 minutes to exclude short-time effects.
2.5 Migration analysis For migration analysis, the cell’s time dependent center-of-mass coordinates were determined by single-particle tracking based on brightness clustering. The images were edited and post-processed with the software ImageJ (National Institutes of Health, USA). The tracking was performed utilizing the ImageJ plugin “Cell Evaluator” [14]. The plugin tracks the center-of-mass of each cell yielding time dependent cell trajectories. The resulting trajectory data was analyzed by a global mean-squared displacement:
MSD: ⟨ΔR()⟩ = ⟨R (t + ) − R (t)
⟩ (1)
with R (t) as the center-of-mass position of the cell at time t and the lag time .
The analysis of the time-resolved, local mean-squared displacement (LMSD) was calculated with a MATLAB® algorithm [4,8,15,16]. This algorithm is able to reliably distinguish between two modes of amoeboid cell migration: directed cell runs and phases of quasi-random migration. For this purpose, a local mean-squared displacement (LMSD) for different lag times is calculated for every time point over a rolling window of 16 frames (corresponding to a duration of = 112 s, respectively 128 s):
LMSD: (2)
⟨ΔR(τ)⟩ = ⟨R(t + τ) − R(t)
⟩
Subsequently the LMSD data is fitted by a power law for every time point by
⟨ΔR()⟩
= A
(3)
with two chosen reference values ( = 1 μm and = 1 s), so the prefactor carries no physical dimension [17]. The exponent α characterizes the migration mode of a cell. Thus, a time point is assigned to a directed run phase if α ≈ 2. In the other case of α < 2, the time point is classified as quasi-random motion and a generalized, also called efficient, diffusion coefficient is retrieved from the prefactor A of the power law fit by
=⟨()⟩
2
=
2
(4)
with d as the number of spatial dimensions (in this work 2). is a general indicator of cell motility during random migration phases.
3 Results and Discussion
The migration behavior of D. discoideum cells is investigated on three distinctly different surfaces: glass, nano-rough silica fibers and polydimethylsiloxane (PDMS). A typical example of a D. discoideum cell migrating on the PDMS surface is shown in figure 1A. A first approach on characterizing motion behavior in general and widely used for cell migration analysis utilizes the mean-squared displacement [18–22]. The value of the MSD at a given lag time describes the motility of a cell. The lag time dependence of the MSD can be described by a power law ⟨ΔR()⟩ ∝ . The exponent characterizes the type of motion. For a linearly rising MSD ( = 1) the motion is diffusive, whereas a ballistic motion yields = 2.
Cell motion analysis by MSD reveals the lowest cell motility on glass, while the data on silica fibers and PDMS samples yield nearly identical cell motion behavior, see figure 1B. Furthermore, a lag time dependent change in the MSD behavior is observed, where the motion pattern changes from superdiffusive behavior with an exponent of = 1.75, for small lag times up to 30 seconds, to nearly diffusive motion with exponents of = 0.9 on glass and = 1.1 for the silica fiber and the PDMS surface, respectively. This transition has already been observed and is well known [18,19]. Yet the MSD analysis is quite limited in terms of revealing the underlying motion patterns on those two surface types.
Fig. 1: (A) Composite of a brightfield (greyscale) and fluorescence (green) microscopy image of a Dictyostelium discoideum cell expressing GFP-protein within the cytosol, migrating on a flat PDMS surface. The cell is shown at the start and the end point, after 2,000 s, of the cell center-of-mass migration trajectory (solid white line). (B) Global mean-squared displacement (MSD) of cell motility over lag time for the different material surfaces: glass (black), silica fiber (blue), and PDMS (red). Dashed grey lines indicate power law progressions for three α exponents: 1.75, 1.1 and 0.9.
A much deeper insight is gained by analyzing the mean-squared displacement with a local, respectively time-resolved MSD approach (LMSD). This enables the differentiation of two amoeboid cell migration modes: phases of quasi-random motion and directed runs. In figure 2A, the same cell trajectory, as displayed in figure 1A, is now distinguished into the two different motion types by color code using red and blue. The corresponding time course of the LMSD function is shown in figure 2B, revealing phases of directed runs for higher α values and quasi-random motion at lower α values.
Fig. 2: (A) Composite of a brightfield (greyscale) and fluorescence (green) microscopy image of a Dictyostelium discoideum cell expressing GFP-protein within the cytosol migrating on a flat PDMS surface. The cell is shown at the start and the end point, after 2,000 s, of the cell center-of-mass migration trajectory (see figure 1A) subdivided into parts of directed cell runs in red and phases of quasi-random cell motion in blue. (B) Plot of the local mean-squared displacement (LMSD) over experimental time and lag time . The exponent α of the power law fit for each time point is shown in colorcode.
The distributions of motion parameters from the statistical LMSD analysis of the cellular motion behavior on the different substrates – also separated into directed and random motion phases – are presented in figure 3. The general LMSD motion pattern is accurately described by the distribution of the α exponent shown in figure 3A. Here, glass and PDMS exhibit similar distributions with mean values of ⟨⟩ =1.45 and ⟨⟩ =1.47, respectively. In comparison, the mean value for migration on the silica fiber surface ⟨⟩ =1.61 is significantly higher, revealing an overall increase of directed cell motion activity. This trend is also reflected in the distributions of the state durations (figure 3B). While the second peak of the directed state
durations is stable for all surfaces, revealing an inherent underlying migration pattern, the amount of directed cell migration states is strongly surface dependent. Here, the highest amount of directed runs is found for migration on silica fiber samples (36.0 %) followed by PDMS (21.6 %) and glass (16.1 %). The nano-rough topography of the fiber induces more directed migration phases of the cells compared to the chemically similar plain surface of the glass.
The measured mean cell migration velocities (see figure 3C, black) on these three substrates confirm the trend set by the state durations with the fastest cell migration velocities on the silica fiber sample with a mean value of ⟨⟩ = 0.070 µm s-1, followed directly by PDMS with a mean velocity of ⟨⟩ = 0.066 µm s-1. The slowest migration speed is observed on the glass samples with a mean velocity of ⟨⟩ = 0.041 µm s-1. A slightly different picture is drawn by the velocities of the directed migration phases
(Figure 3C, red). The highest migration velocities in directed states are observed on the PDMS surface, 12 % faster than the migration on the fiber sample and 47 % faster than on glass. Here, the polarity of the substrates is a possible explanation. The hydrophobic surface of the PDMS compared to the very hydrophilic surface of both the silica fibers and the glass samples leads to a lower amount of cell adhesion sites and a reduced bonding strength between the cells and the substrate. This, in return, enhances the cell migration velocity [23] and leads to the observed distribution of directed cell migration velocities. The value of the effective diffusion coefficient characterizes the motility of the cells in their quasi-random motion phases. The values of these diffusion coefficients (see figure 3D) are consistent with the previously discussed influences of the surface polarity and topography on the migration behavior. The highest diffusion coefficients are observed on PDMS with ⟨⟩ =6.02 µm2 s-1 succeeded by ⟨⟩ = 5.22 µm2 s-1
obtained for the silica fiber surface. Cell migration on glass exhibits the lowest diffusion coefficients with a mean of ⟨⟩ = 2.53 µm2 s-1
confirming the trend of the directed cell migration velocities.
Fig. 3: Cell motion parameter distributions for the three distinct material surfaces: glass, silica fiber, and PDMS (A) Distribution of α exponents derived from the power law fits of the local mean-squared displacement analysis. Black: overall distribution, blue: quasi-random states, red: directed cell motion, dashed line: mean values (B) Distribution of the state durations of directed cell migrations runs, dashed line indicates mean values. The amount of directed runs compared to the overall experimental time is given in percent. (C) Instantaneous velocity distributions of the cell migration. Black: overall distribution, blue: quasi-random states, red: directed cell motion, dashed lines: mean values. (D) Distribution of the effective diffusion coefficient in the quasi-random states, dashed line indicates mean values.
Table 1: Obtained cell migration parameters from LMSD analysis of D. discoideum on three different surfaces.
Glass Fiber PDMS
Mean alpha α 1.45 1.61 1.47
% directed phases of total time 16.1 36.0 21.6
Mean velocity (µm s-1) ⟨⟩ 0.041 0.070 0.066
Mean directed velocity (µm s-1) ⟨⟩ 0.058 0.097 0.111
Mean diffusion coefficient (µm2 s-1) 2.53 5.22 6.02
Number of cells Ncells 45 25 369
Number of data points N 23.975 12.421 151.439
4 Conclusion
In this work, we elucidated cell migration behavior on three different surfaces: glass, nano-rough, biomedically used silica fibers and polymeric PDMS. Migration data was obtained utilizing Dictyostelium discoideum cells, a commonly used model organism for amoeboid migration of stem and immune cells. A time-resolved, local mean-squared displacement (LMSD) approach enabled distinguishing between different phases of cell motility: directed runs and quasi-random motion phases typical for amoeboid cell migration. We found that the nanoscale surface structured silica fibers lead to more directed phases in the cell’s migration pattern compared to both plain glass and PDMS substrates. Regarding directed motion states, we discovered that the hydrophobic PDMS yielded the highest mean cell migration speed compared to the hydrophilic substrates.
While the analysis solely based on a global MSD suggested similar migration behavior on silica fibers and PDMS, the time-resolved LMSD analysis revealed striking differences between migration on these substrates. Our results recommend a time-resolved analysis for comprehensive cell migration studies to gain a deeper understanding of the cell-material interaction. Based on these approaches, future studies will focus on cell migration, proliferation, and morphology in more complex shaped 3D environments, fabricated from novel and smart materials, mimicking real physiological environments of the human body to enhance drug testing assays and enable personalized medicine.
Acknowledgements
We acknowledge Prof. Dr. Günther Gerisch (Max-Planck Institute for Biochemistry, Germany) for providing the Dictyostelium discoideum strains and the funding from the Deutsche Forschungsgemeinschaft (grant HE5958-2-1), the Volkswagen-Foundation (grant I85100) and the Fraunhofer Attract Program for the grant “3DNanoZell”.
References
[1] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and regeneration, Nature. 453 (2008) 314–321. doi:10.1038/nature07039.
[2] C.J. Weijer, Collective cell migration in development, Journal of Cell Science. 122 (2009) 3215–3223. doi:10.1242/jcs.036517.
[3] C.J. Miller, L. Davidson, The interplay between cell signaling and mechanics in developmental processes, Nat Rev Genet. 14 (2013) 733–744. doi:10.1038/nrg3513.
[4] M. Gorelashvili, M. Emmert, K.F. Hodeck, D. Heinrich, Amoeboid migration mode adaption in quasi-3D spatial density gradients of varying lattice geometry, New J. Phys. 16 (2014) 075012. doi:10.1088/1367-2630/16/7/075012.
[5] A. Nandi, D. Heinrich, B. Lindner, Distributions of diffusion measures from a local mean-square displacement analysis, Phys. Rev. E. 86 (2012) 021926. doi:10.1103/PhysRevE.86.021926.
[6] Y. Artemenko, T.J. Lampert, P.N. Devreotes, Moving towards a paradigm: Common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes, Cell Mol Life Sci. 71 (2014) 3711–3747. doi:10.1007/s00018-014-1638-8.
[7] B. Harland, S. Walcott, S.X. Sun, Adhesion Dynamics and Durotaxis in Migrating Cells, Phys Biol. 8 (2011) 015011. doi:10.1088/1478-3975/8/1/015011.
[8] M. Emmert, P. Witzel, M. Rothenburger-Glaubitt, D. Heinrich, Nanostructured surfaces of biodegradable silica fibers enhance directed amoeboid cell migration in a microtubule-dependent process, RSC Adv. 7 (2017) 5708–5714. doi:10.1039/C6RA25739A.
[9] K. Wolf, R. Müller, S. Borgmann, E.-B. Bröcker, P. Friedl, Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases, Blood. 102 (2003) 3262–3269. doi:10.1182/blood-2002-12-3791.
[10] S. Bozzaro, The Model Organism Dictyostelium discoideum, in: Dictyostelium Discoideum Protocols, Humana Press, Totowa, NJ, 2013: pp. 17–37. doi:10.1007/978-1-62703-302-2_2.
[11] B. Meier, A. Zielinski, C. Weber, D. Arcizet, S. Youssef, T. Franosch, J.O. Radler, D. Heinrich, Chemotactic cell trapping in controlled alternating gradient fields, Proceedings of the National Academy of Sciences. 108 (2011) 11417–11422. doi:10.1073/pnas.1014853108.
[12] S.J. Annesley, P.R. Fisher, Dictyostelium discoideum a model for many reasons, Mol Cell Biochem. 329 (2009) 73–91. doi:10.1007/s11010-009-0111-8.
[13] M. Kursawe, W. Glaubitt, A. Thierauf, Biodegradable Silica Fibers from Sols, Journal of Sol-Gel Science and Technology. 13 (1998) 267–271. doi:10.1023/A:1008651505705.
[14] S. Youssef, S. Gude, J.O. Rädler, Automated tracking in live-cell time-lapse movies, Integr. Biol. 3 (2011) 1095–1101. doi:10.1039/C1IB00035G.
[15] D. Arcizet, S. Capito, M. Gorelashvili, C. Leonhardt, M. Vollmer, S. Youssef, S. Rappl, D. Heinrich, Contact-controlled amoeboid motility induces dynamic cell trapping in 3D-microstructured surfaces, Soft Matter. 8 (2012) 1473–1481. doi:10.1039/C1SM05615H.
[16] D. Arcizet, B. Meier, E. Sackmann, J.O. Rädler, D. Heinrich, Temporal Analysis of Active and Passive Transport in Living Cells, Physical Review Letters. 101 (2008). doi:10.1103/PhysRevLett.101.248103.
[17] A. Nandi, D. Heinrich, B. Lindner, Distributions of diffusion measures from a local mean-square displacement analysis, Physical Review E. 86 (2012). doi:10.1103/PhysRevE.86.021926.
[18] L. Li, S.F. Nørrelykke, E.C. Cox, Persistent Cell Motion in the Absence of External Signals: A Search Strategy for Eukaryotic Cells, PLOS ONE. 3 (2008) e2093. doi:10.1371/journal.pone.0002093.
[19] P. Dieterich, R. Klages, R. Preuss, A. Schwab, Anomalous dynamics of cell migration, Proceedings of the National Academy of Sciences. 105 (2008) 459–463. doi:10.1073/pnas.0707603105.
[20] C.P. McCann, P.W. Kriebel, C.A. Parent, W. Losert, Cell speed, persistence and information transmission during signal relay and collective migration, J Cell Sci. 123 (2010) 1724–1731. doi:10.1242/jcs.060137.
[21] P.-H. Wu, A. Giri, S.X. Sun, D. Wirtz, Three-dimensional cell migration does not follow a random walk, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 3949–3954. doi:10.1073/pnas.1318967111.
[22] P. Suraneni, B. Rubinstein, J.R. Unruh, M. Durnin, D. Hanein, R. Li, The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration, The Journal of Cell Biology. 197 (2012) 239–251. doi:10.1083/jcb.201112113.
[23] S.L. Gupton, C.M. Waterman-Storer, Spatiotemporal Feedback between Actomyosin and Focal-Adhesion Systems Optimizes Rapid Cell Migration, Cell. 125 (2006) 1361–1374. doi:10.1016/j.cell.2006.05.029.
Influence of Material Surfaces on Cell Motility Timo Grunemann1,2, Patrick Witzel1,2, Martin Emmert1,2, Franz L. Edel1,2, Doris Heinrich3
1 Faculty for Chemistry und Pharmacy, Julius-Maximilians-Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany 2 Fraunhofer Institute for Silicate Research ISC, Neunerplatz 2, 97082 Würzburg, Germany 3 Leiden University, LION Leiden Institute of Physics, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands * Corresponding author: Doris Heinrich, [email protected]
Abstract The extracellular matrix plays an important role for essential cell functions like adhesion, migration, proliferation, and differentiation. A clear understanding of biophysical interaction mechanisms between a cell and its environment provides a solid base for a cell-type specific design of three dimensional (3D) scaffolds in terms of surface topography and chemical surface functionalization. In this work, we analyze motion patterns of D. discoideum cells on three distinct surfaces: plain glass, nano-structured silica fiber and polydimethylsiloxane (PDMS) surfaces, utilizing a global mean-squared displacement (MSD) as well as time-resolved, local mean-squared displacement (LMSD) techniques. Global MSD analysis reveals a significantly higher cell migration activity on PDMS and nano-structured fiber surfaces compared to plain glass, yet the cell movement patterns on PDMS and silica fiber cannot be further distinguished. Further evaluation based on a LMSD approach yields a more in-depth picture of the specific surface influence on cell migration as this method enables differentiation between quasi-random and directed migration phases. We find that the nanostructure of the silica fibers induces significantly more directed migration phases compared to the glass and PDMS surfaces. Furthermore, the hydrophobic PDMS surface leads to greatly increased directed migration velocities. This analysis shows the advantage of a time-resolved LMSD analysis to reveal substantial differences in cell-material interaction. These insights will facilitate development of new biomedical materials with intrinsic properties designed to control cell migration leading to novel diagnostic and therapeutic concepts.
Keywords: Cell Motility, Material Surface, Cell-Material Interaction, Mean-squared Displacement, Live-cell Imaging
1 Introduction
Cell migration plays a vital role in a wide range of processes in the human body including wound healing [1], immune response, and embryogenesis [2,3], as well as for aberrations like cancer metastasis. As such, the investigation of cell migration behavior should be taken into account for the development of novel materials designed for direct contact with human tissue. Additional control of migration patterns could enable new applications in tissue engineering ranging from drug testing in vitro, reducing expensive clinical studies, to implants minimizing engraftment times.
In this work, we demonstrate the immense influence of different surfaces on cell migration behavior utilizing global mean-squared displacement (MSD) as well as time-resolved local mean-squared displacement (LMSD) analysis [4,5]. It is well known that cell motility is affected by several cues of the cellular surrounding, called the extracellular matrix within an organism, including surface chemistry [6], Young’s modulus [7], and topography [4,8]. It is, however, still unclear which of these external cues are dominant and overwrite other influences on cell migration behavior. To get a deeper insight, we obtained and analyzed migration data of Dictyostelium discoideum (D. discoideum) cells on three distinct surfaces. D. discoideum is a social amoeba exhibiting amoeboid migration modes comparable to stem cells or immune cells [9,10]. Strains of these cell type are often used as a model organism for chemotaxis [11], phagocytosis, and human diseases because of their completely sequenced genome and the relatively simple cell culture and handling [12]. The investigated surfaces comprised glass, as a standard and widely utilized biomedical reference, silica fibers with a nano-rough surface structure, used as wound healing inserts, and polydimethylsiloxane (PDMS) as an example for the polymeric material class and commonly used in experimental microfluidic and cellular assays.
2 Experimental 2.1 Preparation and Characterization of Silica Fibers Silica fibers were fabricated by pressure extrusion on cover glass substrates according to [8,13]. The resulting silica gel fibers’ composition
was determined to be [Si(OH)0.2O1.9]n by thermal analysis in a previous work [13] similar to glass. The fibers exhibited a dog-bone-like shape with measured fiber widths of 60 ± 20 μm and fiber heights of 24 ± 8 μm. Freshly fabricated fibers exhibited a smooth surface with no distinct topography. After contact with aqueous media nano-sized surface patterns were observed through biodegradation [13]. SEM investigations on fibers that have been stored in solution for several days revealed pits and elevations on the fiber surface with feature sizes below 200 nm [8].
2.2 PDMS Preparation The polydimethylsiloxane (PDMS) is processed as a two-component material. The precursor of Sylgard 184 (Dow Corning, USA) is mixed with the curing agent at weight ratio of 9:1. For air removal the solution was degassed at 160 mbar in a desiccator for 30 min. The resin was flood coated on a silicon wafer passivated by perfluorodecyltrichlorsilane (Sigma-Aldrich, St. Louis, Missouri, USA) and again degassed at 6×10-2 mbar with a temperature progression from room temperature to 50 °C over 30 min. The PDMS was cured at 65 °C for 6 h. Previous to the migration experiments the surface of the PDMS was activated by ozone plasma for 15 min at a power level of 70 % (Diener electronic GmbH + Co. KG, Ebhausen, Germany).
2.3 Cell Culture For the migration experiments cells of the D. discoideum strain AX2 expressing homogenously distributed green fluorescent protein (GFP) in the cytoplasm were grown under 40% confluency at 19–21 °C in petri dishes at ambient air. For cell culture, HL-5C medium with glucose (HLC0102, ForMediumTM, UK) including 20 μg mL−1 gentamycin (G-418-Biochrom A2912, Biochrom AG) was used.
2.4 Preparation and Migration Experiments Previous to the migration experiments, the cells were washed four times with phosphate buffered saline (PBS) with subsequent centrifugation at 2000 rpm for 4 minutes (Eppendorf MiniSpin® Plus, Eppendorf AG) and resuspension of the cell pellet in PBS. Afterwards the solutions’ cell density was adjusted to achieve a surface cell density of 400,000 cells per cm2. The cells were kept 30 mins at rest after seeding to the surface. Image acquisition was performed with a Nikon Eclipse Ti fluorescence microscope (Nikon, Germany) with an Intensilight (Nikon, Germany) light source and a 20x objective (Nikon, Germany). To enable GFP imaging, an
excitation filter (F36-525 HC, AHF, Germany) ensured excitation wavelengths of 457–487 nm and detection wavelengths of 500–540 nm. The signals were detected with an EM-CCD camera (Hamamatsu, Japan). Images were acquired every 7 seconds for the glass and silica fiber experiments and every 8 seconds for the measurements on PDMS for at least 45 minutes to exclude short-time effects.
2.5 Migration analysis For migration analysis, the cell’s time dependent center-of-mass coordinates were determined by single-particle tracking based on brightness clustering. The images were edited and post-processed with the software ImageJ (National Institutes of Health, USA). The tracking was performed utilizing the ImageJ plugin “Cell Evaluator” [14]. The plugin tracks the center-of-mass of each cell yielding time dependent cell trajectories. The resulting trajectory data was analyzed by a global mean-squared displacement:
MSD: ⟨ΔR2(𝜏)⟩ = ⟨R (t + 𝜏) − R (t)2⟩𝑡 (1)
with R (t) as the center-of-mass position of the cell at time t and the lag time 𝜏.
The analysis of the time-resolved, local mean-squared displacement (LMSD) was calculated with a MATLAB® algorithm [4,8,15,16]. This algorithm is able to reliably distinguish between two modes of amoeboid cell migration: directed cell runs and phases of quasi-random migration. For this purpose, a local mean-squared displacement (LMSD) for different lag times 𝜏 is calculated for every time point 𝑡𝑖 over a rolling window of 16 frames (corresponding to a duration of 𝑇 = 112 s, respectively 128 s):
LMSD: (2)
⟨ΔR2(τ)⟩i = ⟨R(ti + τ) − R(ti)2⟩ti−
T2 < ti < ti+
T2
Subsequently the LMSD data is fitted by a power law for every time point 𝑡𝑖 by
⟨ΔR2(𝜏)⟩𝑖 𝑙2
= A𝑖 𝜏𝜏0𝛼𝑖
(3)
with two chosen reference values (𝑙 = 1 μm and 𝜏0 = 1 s), so the prefactor 𝐴𝑖 carries no physical dimension [17]. The exponent α characterizes the migration mode of a cell. Thus, a time point is assigned to a directed run phase if α ≈ 2. In the other case of α < 2, the time point is classified as quasi-random motion and a generalized, also called efficient, diffusion coefficient 𝐷eff is retrieved from the prefactor A of the power law fit by
𝐷 =⟨𝛥𝛥2(𝜏)⟩
2𝑑𝜏0=
𝐴𝑙2
2𝑑𝜏0 (4)
with d as the number of spatial dimensions (in this work 2). 𝐷eff is a general indicator of cell motility during random migration phases.
3 Results and Discussion
The migration behavior of D. discoideum cells is investigated on three distinctly different surfaces: glass, nano-rough silica fibers and polydimethylsiloxane (PDMS). A typical example of a D. discoideum cell migrating on the PDMS surface is shown in figure 1A. A first approach on characterizing motion behavior in general and widely used for cell migration analysis utilizes the mean-squared displacement [18–22]. The value of the MSD at a given lag time describes the motility of a cell. The lag time dependence of the MSD can be described by a power law ⟨ΔR2(𝜏)⟩ ∝ 𝜏𝛼. The exponent 𝛼 characterizes the type of motion. For a linearly rising MSD (𝛼 = 1) the motion is diffusive, whereas a ballistic motion yields 𝛼 = 2.
Cell motion analysis by MSD reveals the lowest cell motility on glass, while the data on silica fibers and PDMS samples yield nearly identical cell motion behavior, see figure 1B. Furthermore, a lag time dependent change in the MSD behavior is observed, where the motion pattern changes from superdiffusive behavior with an exponent of 𝛼 = 1.75, for small lag times up to 30 seconds, to nearly diffusive motion with exponents of 𝛼 = 0.9 on glass and 𝛼 = 1.1 for the silica fiber and the PDMS surface, respectively. This transition has already been observed and is well known [18,19]. Yet the MSD analysis is quite limited in terms of revealing the underlying motion patterns on those two surface types.
Fig. 1: (A) Composite of a brightfield (greyscale) and fluorescence (green) microscopy image of a Dictyostelium discoideum cell expressing GFP-protein within the cytosol, migrating on a flat PDMS surface. The cell is shown at the start and the end point, after 2,000 s, of the cell center-of-mass migration trajectory (solid white line). (B) Global mean-squared displacement (MSD) of cell motility over lag time 𝜏 for the different material surfaces: glass (black), silica fiber (blue), and PDMS (red). Dashed grey lines indicate power law progressions for three α exponents: 1.75, 1.1 and 0.9.
A much deeper insight is gained by analyzing the mean-squared displacement with a local, respectively time-resolved MSD approach (LMSD). This enables the differentiation of two amoeboid cell migration modes: phases of quasi-random motion and directed runs. In figure 2A, the same cell trajectory, as displayed in figure 1A, is now distinguished into the two different motion types by color code using red and blue. The corresponding time course of the LMSD function is shown in figure 2B, revealing phases of directed runs for higher α values and quasi-random motion at lower α values.
Fig. 2: (A) Composite of a brightfield (greyscale) and fluorescence (green) microscopy image of a Dictyostelium discoideum cell expressing GFP-protein within the cytosol migrating on a flat PDMS surface. The cell is shown at the start and the end point, after 2,000 s, of the cell center-of-mass migration trajectory (see figure 1A) subdivided into parts of directed cell runs in red and phases of quasi-random cell motion in blue. (B) Plot of the local mean-squared displacement (LMSD) over experimental time 𝑡 and lag time 𝜏. The exponent α of the power law fit for each time point is shown in colorcode.
The distributions of motion parameters from the statistical LMSD analysis of the cellular motion behavior on the different substrates – also separated into directed and random motion phases – are presented in figure 3. The general LMSD motion pattern is accurately described by the distribution of the α exponent shown in figure 3A. Here, glass and PDMS exhibit similar 𝛼 distributions with mean values of ⟨𝛼⟩ =1.45 and ⟨𝛼⟩ =1.47, respectively. In comparison, the mean value for migration on the silica fiber surface ⟨𝛼⟩ =1.61 is significantly higher, revealing an overall increase of directed cell motion activity. This trend is also reflected in the distributions of the state durations (figure 3B). While the second peak of the directed state
durations is stable for all surfaces, revealing an inherent underlying migration pattern, the amount of directed cell migration states is strongly surface dependent. Here, the highest amount of directed runs is found for migration on silica fiber samples (36.0 %) followed by PDMS (21.6 %) and glass (16.1 %). The nano-rough topography of the fiber induces more directed migration phases of the cells compared to the chemically similar plain surface of the glass.
The measured mean cell migration velocities (see figure 3C, black) on these three substrates confirm the trend set by the state durations with the fastest cell migration velocities on the silica fiber sample with a mean value of ⟨𝑣⟩ = 0.070 µm s-1, followed directly by PDMS with a mean velocity of ⟨𝑣⟩ = 0.066 µm s-1. The slowest migration speed is observed on the glass samples with a mean velocity of ⟨𝑣⟩ = 0.041 µm s-1. A slightly different picture is drawn by the velocities of the directed migration phases 𝑣dir (Figure 3C, red). The highest migration velocities in directed states are observed on the PDMS surface, 12 % faster than the migration on the fiber sample and 47 % faster than on glass. Here, the polarity of the substrates is a possible explanation. The hydrophobic surface of the PDMS compared to the very hydrophilic surface of both the silica fibers and the glass samples leads to a lower amount of cell adhesion sites and a reduced bonding strength between the cells and the substrate. This, in return, enhances the cell migration velocity [23] and leads to the observed distribution of directed cell migration velocities. The value of the effective diffusion coefficient 𝐷eff characterizes the motility of the cells in their quasi-random motion phases. The values of these diffusion coefficients (see figure 3D) are consistent with the previously discussed influences of the surface polarity and topography on the migration behavior. The highest diffusion coefficients are observed on PDMS with ⟨𝐷eff⟩ = 6.02 µm2 s-1 succeeded by ⟨𝐷eff⟩ = 5.22 µm2 s-1 obtained for the silica fiber surface. Cell migration on glass exhibits the lowest diffusion coefficients with a mean of ⟨𝐷eff⟩ = 2.53 µm2 s-1 confirming the trend of the directed cell migration velocities.
Fig. 3: Cell motion parameter distributions for the three distinct material surfaces: glass, silica fiber, and PDMS (A) Distribution of α exponents derived from the power law fits of the local mean-squared displacement analysis. Black: overall distribution, blue: quasi-random states, red: directed cell motion, dashed line: mean values (B) Distribution of the state durations of directed cell migrations runs, dashed line indicates mean values. The amount of directed runs compared to the overall experimental time is given in percent. (C) Instantaneous velocity distributions of the cell migration. Black: overall distribution, blue: quasi-random states, red: directed cell motion, dashed lines: mean values. (D) Distribution of the effective diffusion coefficient in the quasi-random states, dashed line indicates mean values.
Table 1: Obtained cell migration parameters from LMSD analysis of D. discoideum on three different surfaces.
Glass Fiber PDMS
Mean alpha α 1.45 1.61 1.47
% directed phases of total time 16.1 36.0 21.6
Mean velocity (µm s-1) ⟨𝑣⟩ 0.041 0.070 0.066
Mean directed velocity (µm s-1) ⟨𝑣dir⟩ 0.058 0.097 0.111
Mean diffusion coefficient (µm2 s-1) 𝐷eff 2.53 5.22 6.02
Number of cells Ncells 45 25 369
Number of data points N 23.975 12.421 151.439
4 Conclusion
In this work, we elucidated cell migration behavior on three different surfaces: glass, nano-rough, biomedically used silica fibers and polymeric PDMS. Migration data was obtained utilizing Dictyostelium discoideum cells, a commonly used model organism for amoeboid migration of stem and immune cells. A time-resolved, local mean-squared displacement (LMSD) approach enabled distinguishing between different phases of cell motility: directed runs and quasi-random motion phases typical for amoeboid cell migration. We found that the nanoscale surface structured silica fibers lead to more directed phases in the cell’s migration pattern compared to both plain glass and PDMS substrates. Regarding directed motion states, we discovered that the hydrophobic PDMS yielded the highest mean cell migration speed compared to the hydrophilic substrates.
While the analysis solely based on a global MSD suggested similar migration behavior on silica fibers and PDMS, the time-resolved LMSD analysis revealed striking differences between migration on these substrates. Our results recommend a time-resolved analysis for comprehensive cell migration studies to gain a deeper understanding of the cell-material interaction. Based on these approaches, future studies will focus on cell migration, proliferation, and morphology in more complex shaped 3D environments, fabricated from novel and smart materials, mimicking real physiological environments of the human body to enhance drug testing assays and enable personalized medicine.
Acknowledgements
We acknowledge Prof. Dr. Günther Gerisch (Max-Planck Institute for Biochemistry, Germany) for providing the Dictyostelium discoideum strains and the funding from the Deutsche Forschungsgemeinschaft (grant HE5958-2-1), the Volkswagen-Foundation (grant I85100) and the Fraunhofer Attract Program for the grant “3DNanoZell”.
References
[1] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and regeneration, Nature. 453 (2008) 314–321. doi:10.1038/nature07039.
[2] C.J. Weijer, Collective cell migration in development, Journal of Cell Science. 122 (2009) 3215–3223. doi:10.1242/jcs.036517.
[3] C.J. Miller, L. Davidson, The interplay between cell signaling and mechanics in developmental processes, Nat Rev Genet. 14 (2013) 733–744. doi:10.1038/nrg3513.
[4] M. Gorelashvili, M. Emmert, K.F. Hodeck, D. Heinrich, Amoeboid migration mode adaption in quasi-3D spatial density gradients of varying lattice geometry, New J. Phys. 16 (2014) 075012. doi:10.1088/1367-2630/16/7/075012.
[5] A. Nandi, D. Heinrich, B. Lindner, Distributions of diffusion measures from a local mean-square displacement analysis, Phys. Rev. E. 86 (2012) 021926. doi:10.1103/PhysRevE.86.021926.
[6] Y. Artemenko, T.J. Lampert, P.N. Devreotes, Moving towards a paradigm: Common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes, Cell Mol Life Sci. 71 (2014) 3711–3747. doi:10.1007/s00018-014-1638-8.
[7] B. Harland, S. Walcott, S.X. Sun, Adhesion Dynamics and Durotaxis in Migrating Cells, Phys Biol. 8 (2011) 015011. doi:10.1088/1478-3975/8/1/015011.
[8] M. Emmert, P. Witzel, M. Rothenburger-Glaubitt, D. Heinrich, Nanostructured surfaces of biodegradable silica fibers enhance directed amoeboid cell migration in a microtubule-dependent process, RSC Adv. 7 (2017) 5708–5714. doi:10.1039/C6RA25739A.
[9] K. Wolf, R. Müller, S. Borgmann, E.-B. Bröcker, P. Friedl, Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases, Blood. 102 (2003) 3262–3269. doi:10.1182/blood-2002-12-3791.
[10] S. Bozzaro, The Model Organism Dictyostelium discoideum, in: Dictyostelium Discoideum Protocols, Humana Press, Totowa, NJ, 2013: pp. 17–37. doi:10.1007/978-1-62703-302-2_2.
[11] B. Meier, A. Zielinski, C. Weber, D. Arcizet, S. Youssef, T. Franosch, J.O. Radler, D. Heinrich, Chemotactic cell trapping in controlled alternating gradient fields, Proceedings of the National Academy of Sciences. 108 (2011) 11417–11422. doi:10.1073/pnas.1014853108.
[12] S.J. Annesley, P.R. Fisher, Dictyostelium discoideum a model for many reasons, Mol Cell Biochem. 329 (2009) 73–91. doi:10.1007/s11010-009-0111-8.
[13] M. Kursawe, W. Glaubitt, A. Thierauf, Biodegradable Silica Fibers from Sols, Journal of Sol-Gel Science and Technology. 13 (1998) 267–271. doi:10.1023/A:1008651505705.
[14] S. Youssef, S. Gude, J.O. Rädler, Automated tracking in live-cell time-lapse movies, Integr. Biol. 3 (2011) 1095–1101. doi:10.1039/C1IB00035G.
[15] D. Arcizet, S. Capito, M. Gorelashvili, C. Leonhardt, M. Vollmer, S. Youssef, S. Rappl, D. Heinrich, Contact-controlled amoeboid motility induces dynamic cell trapping in 3D-microstructured surfaces, Soft Matter. 8 (2012) 1473–1481. doi:10.1039/C1SM05615H.
[16] D. Arcizet, B. Meier, E. Sackmann, J.O. Rädler, D. Heinrich, Temporal Analysis of Active and Passive Transport in Living Cells, Physical Review Letters. 101 (2008). doi:10.1103/PhysRevLett.101.248103.
[17] A. Nandi, D. Heinrich, B. Lindner, Distributions of diffusion measures from a local mean-square displacement analysis, Physical Review E. 86 (2012). doi:10.1103/PhysRevE.86.021926.
[18] L. Li, S.F. Nørrelykke, E.C. Cox, Persistent Cell Motion in the Absence of External Signals: A Search Strategy for Eukaryotic Cells, PLOS ONE. 3 (2008) e2093. doi:10.1371/journal.pone.0002093.
[19] P. Dieterich, R. Klages, R. Preuss, A. Schwab, Anomalous dynamics of cell migration, Proceedings of the National Academy of Sciences. 105 (2008) 459–463. doi:10.1073/pnas.0707603105.
[20] C.P. McCann, P.W. Kriebel, C.A. Parent, W. Losert, Cell speed, persistence and information transmission during signal relay and collective migration, J Cell Sci. 123 (2010) 1724–1731. doi:10.1242/jcs.060137.
[21] P.-H. Wu, A. Giri, S.X. Sun, D. Wirtz, Three-dimensional cell migration does not follow a random walk, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 3949–3954. doi:10.1073/pnas.1318967111.
[22] P. Suraneni, B. Rubinstein, J.R. Unruh, M. Durnin, D. Hanein, R. Li, The Arp2/3 complex is required for
lamellipodia extension and directional fibroblast cell migration, The Journal of Cell Biology. 197 (2012) 239–251. doi:10.1083/jcb.201112113.
[23] S.L. Gupton, C.M. Waterman-Storer, Spatiotemporal Feedback between Actomyosin and Focal-Adhesion Systems Optimizes Rapid Cell Migration, Cell. 125 (2006) 1361–1374. doi:10.1016/j.cell.2006.05.029.
Circulating tumor cells (CTC) filtration of non-fluorescent membrane FilterMasaru Hori1, Naoto Kihara1,2, Kenji Ishikawa1,
Hidefumi Odaka1,2, Daisuke, Onoshima1, and
Yoshinobu Baba1
1 Nagoya University, Furo-cho, Chikusa, Nagoya,
Aichi, 464-8601 Japan2 Asahi Glass Co.,Ltd. Yokohama 221-8755 Japan
Abstract
Rapidly increasing demands for trapping of circulating tumor cells (CTCs) from a whole human blood, it is necessary to develop methods that biological cells are filtrated rapidly and precisely for the medical diagnostics and therapies. The commercial polymer membrane filters are in general opaque and have been fabricated by opening randomly pores for the filtration with their size. To diagnose capturing cells rapidly and precisely, the targeting cells are separated based on the mechanism of size-exclusion and cytoskeletal deformability, and then they are usually stained by fluorescent molecules of the cell-surface expression markers. The membrane filter with through-holes was fabricated by using the photolithographic patterning and dry etching method. The holes were precisely aligned more than 300,000 with a diameter of 1-μm-scale in a typical area of a diameter of 13 mm. Non-fluorescent polymer membrane is used in order for the fluorescent detection of rare CTCs in the human blood. Here, advanced technology of the non-fluorescent filter membranes with precisely aligned through-holes will be shown.
Keywords: Membrane filter; Circulating tumor cells (CTC); Non-fluorescent polymer membrane
1 Introduction
Membrane filters have been commonly used for separation purposes in the analytical biochemistry. The commercial polymer membrane filters are in general opaque and have been fabricated by opening randomly pores for the filtration with their size.
Rapidly increasing demands for trapping of circulating tumor cells (CTCs) from a whole human blood, it is necessary to develop methods that biological cells are filtrated rapidly and precisely for the medical diagnostics and therapies. The membrane filters are candidates
for cell-separation on the mechanism of size-exclusion. To diagnose capturing cells rapidly and precisely, the targeting cells are usually stained by fluorescent molecules of the cell-surface expression markers.[1]
The size-exclusive microfiltration device made of polyethylene terephthalate (PET) for the CTCs were successfully developed by our group.[2] This has been fabricated by using photolithography-patterned metallic masks and plasma etching. The PET filter could isolate cells precisely with size exclusions, however suffers the auto-fluorescence in the microscopy. The materials used for filter are issued on auto-fluorescence of polydimethylsiloxane (PDMS),[3] polycarbonate (PC),[4-6] and the photoresist.[14] The metals such as Ni[7-9] and Pd [10] provide only opaque membranes. In addition, the track etching method fabricates randomly pores, in which connected plural holes unfortunately act to be relatively large holes.[6,11]
Ethylene-tetrafluoroethylene (ETFE) is a copolymer, which indicates the excellent chemical durability for both acid and alkali, and transmittance to the visible light.[12,13] and mediates the auto-fluorescence problem. This is also a high heat-resistant polymer compared to PET and PC. In use of ETFE, the large thermal expansion of ETFE is taken into account for plasma etching. We have optimized a fabrication method and etch-mask material.
Here we report an optically transparent ETFE membrane-filter with several tens of millions of bored-through holes. This has been fabricated by using photolithography-patterned metallic masks and plasma etching. The ETFE membrane filter was fabricated with high etching rate (1.35 μm/min), has been realized with high etching selectivity of Ti mask and precisely aligned 380,000 holes. The holes with each diameter of 7 μm, were precisely fabricated. This filter can be used for the filtration of the CTCs in the whole blood.
2 ETFE membrane filter
2.1 Materials and methods
Figure 1 shows fabrication scheme of ETFE membrane filters. A silicon wafer with a diameter of 150 mm was used as a substrate. To paste an ETFE film (AGC Asahi Glass) with a thickness of 25 μm to the Si wafer, a double-coated polyimide tape with a square of 100 mm, one side of weak adhesive force and the other side of strong
adhesive force was pasted on the Si substrate by a hand roller. Subsequently, the ETFE film was pasted on the front-side face of the double-coated adhesive tape.
A Ti film was deposited with a thickness of 150 nm on the ETFE surface by a DC magnetron sputtering apparatus. During the sputtering, an ultra-high purity Ar gas was introduced and the pressure was kept at 0.7 Pa. metallic Ti has good resistances in thermal crack due to very large coefficients of ETFE for thermal expansion.
Photolithographic patterns were prepared using a positive tone photoresist resin. The photomask patterns were a diameter of 7 μm arrayed with a fixed distance of 20 μm in an area with a diameter of 13 mm. The exposed patterns were then developed. The 0.38 millions of hole patterns were successfully formed. Then, the resist patterns were transferred to the Ti film by plasma etching using Cl2 gas. A commercial inductively coupled plasma (ICP) etcher was used for Ti etching. RF power was applied to an antenna coil. Simultaneously, RF bias power was applied to a wafer stage of the ICP etcher. The pressure was fixed at 1 Pa.
Through-holes etching processes were performed using O2 plasma at 0.5 and 4 Pa. The patterns on the Ti-mask were transferred to the ETFE film by plasma etching using pure O2 gas. A commercial ICP etcher was also used. The temperature of cooling water of the stage was 12 °C. The back side of the Si substrate was filled with a helium gas with a pressure of 1000 Pa for temperature control.
After the ETFE etching, the Ti film was removed by exposure of Cl2 plasma. The bored ETFE film was peeled off by using a tweezer, from the front side of the double-coated adhesive polyimide tape on the Si substrate.
Fig. 1: Fabrication scheme of an ETFE membrane filter.[14,15]
2.2 Results and discussionEtching condition was optimized by these parameters. The antenna power was fixed at 200 W. The bias power, flow rate of, process pressure and etching area were varied 50-250 W,
20-100 sccm, 0.5-4.0 Pa and 1.3-5.3 cm2, respectively.
The etch rates of both ETFE and Ti increased similarly with the increase of the substrate bias power, while the selectivity of ETFE over Ti remained was a constant of 670. The etch rates increased in the ion-enhanced physical sputtering manner.
For the pure Ti in the O2 plasma, the physical sputtering determined the selectivity of ETFE over Ti. Large etching rates enhanced chemically by fluorine. The ETFE-etch byproducts remediated the Ti etch rates. The selectivity for the Ti mask decreased with the increase of processing pressure and the ETFE-exposed etch area and the decrease of O2 flow.
In order to clarify why the Ti etch rate increased, the as-etched Ti surface was analyzed using XPS and fluoride species such as Ti-F and C-F were detected. The result indicated that etch-byproducts of ETFE in O2 plasma reacted with the Ti surface. The fluorides were considered to generate volatiles such as TiF4. In order to remain the Ti mask after etching process, high selectivity or thick Ti mask was required. Since thick Ti mask cause cracks and worse productivity, high selectivity was required. High selectivity was obtained lower pressure and larger O2 flow condition. Lower fluorine partial pressure condition improved selectivity. As a consequence, the 150 nm Ti mask for the ETFE etch processes enabled us to fabricate the through-hole membranes on the 25 μm thick ETFE film (Fig. 2).
Fig. 2: Fabricated ETFE membrane filter. (a) Top view and (b) cross-sectional view.[15]
ETFE etching byproducts generated inside the hole adhere to the sidewall, leading to adhesion of a thick film. As the pressure increased, a gas residence time was 8 times longer 0.70 s at 4 Pa than 0.09 s at 0.5 Pa. The mean free path of oxygen was 21.1 mm for 0.5 Pa and 2.6 mm for 4 Pa. Composition of the sidewall of the film was
analyzed by XPS. The holes were covered by a thin layer comprising CF2 and CH2 which is the composition of ETFE.[16] Under the high pressure condition, the sputtering rate was low because the amount of gas in the chamber was large, and the oxygen ions accelerated by the bias lost energy due to collision with the gas before reaching the Ti surface. The hole-shape on the front side was a circle reflecting the pattern of the photomask regardless of the processing pressure. The hole-shape on the back side was smooth circular like the surface under the high pressure 4 Pa condition. In contrast, at low pressure 0.5 Pa sample, the roughness on the inner wall increased as the etching proceeded.
As a mechanism of occurrence of sidewall roughness of the inner wall is considered: (1) something adheres to the inner wall surface of the ETFE and becomes an etching mask, (2) reflecting roughness of the Ti mask. Ti originated from the Ti mask was detected, caused by metallic fine particles sputtered from the etching chamber walls serve as an etching mask.[17] Ti fine particles adhere to the inner wall of the hole. The amount of Ti adhering to the inner wall (Ti/F or Ti/C) was significantly higher in the low pressure condition than that of in the high pressure condition.
The reason why the shape of the back surface is different while both Ti adhere at low pressure condition 0.4 Pa and high pressure condition 4 Pa is considered as follows. At the low pressure condition of 0.5 Pa, the scattering of incident ions at sidewall is small because the mean free path of oxygen is as long as 21.1 mm, and the shape of the Ti film adhered near the entrance of the hole is magnified and projected. In contrast, in the high pressure condition 4 Pa, the mean free path of oxygen is as short as 2.2 mm and there are many scatterings, so it is presumed that a smooth circular shape was obtained without reflecting the shape of the Ti film adhered near the hole inlet. In addition, the surface roughness generated in the resist is transferred to the Ti mask, which is enlarged and projected to cause sidewall roughness.
Lastly, in the fabricated ETFE membrane filters, the holes were straight shape and precisely aligned 380,000 holes with a diameter of 7 μm covered an area of a diameter of 13mm. Etch selectivity of the Ti mask played important roles for the fabrication of the through-holes. Inappropriate etching condition caused disappearing of Ti mask after processing, and rough ETFE surface was obtained. Although
lower process pressure condition improves the selectivity of Ti mask and suppresses sidewall film deposition and delamination, the shape of the back side hole was rough. Lower process pressure condition in order to prevent the sidewall deposition from becoming contamination at the genetic analysis was chosen in this study.
3 CTC entrapment
The non-small lung cancer cell line NCI-H358 was cultured in RPMI 1640 medium containing 2 mML-glutamine, 10% (v/v) FBS, and 1% (v/v) penicillin/ streptomycin solution for 3−4 days at 37 °C in a humidified atmosphere containing 5% CO2. Immediately before each experiment, confluent cells were trypsinized and resuspended in PBS.
The Normal human blood samples were collected from healthy donors at the Nagoya University. Samples were collected in a collection tube with EDTA to prevent coagulation and used within 12 h.
The fabricated ETFE filter membrane was put in a commercial filter holder, and input syringe was set on upper side of the filter holder. Waste syringe set on commercial syringe pump and lower side of the filter holder were connected by a commercial tube.
Auto-fluorescent and optical absorption of ETFE films are low at wavelength from 340 to 370 nm in the laser excitation wavelength and from 430 to 480 nm in the DAPI region, as compared with PET films.
The blood sample (7.5 mL) was diluted two times with PBS (7.5 mL). Then, H358 cell suspension of 1000 cells was spiked in the blood sample. The blood sample was added to the reservoir. Subsequently, negative pressure was applied to the cell suspension with a syringe pump. The sample was passed through the filter at a flow rate of 1 mL/min for 15 min. To remove blood cells that remained on the filter, PBS was added to the reservoir and passed through the filter at a flow rate of 1 mL/min for 5 min.
Cell staining solution was introduced into the reservoir and passed through the filter with a syringe pump after washing. To identify CTCs and leukocytes, 200 μL of cell staining solution containing 1 μg/mL Hoechst 33342, a cocktail of PE-labeled anti-EpCAM antibodies and an anti-CD45 antibody was passed through the filter at a flow rate of 4 μL/min for 50 min. Finally, the filter was washed with 3 mL of PBS to remove excess dye. Fluorescence images were obtained
with a fluorescence microscope integrated with DAPI, TRITC, Cy5 filter sets.
Separation test of CTC model cells spiked into whole blood was performed using our fabricated microfiltration system. Tens of billions of blood cells in 15 mL sample were able to pass through the filter without clogging, and CTC model cells were trapped on the filter. The trapping efficiency of CTC model cells was over 96% (Fig. 3). In contrast, almost all leukocytes were depleted, partly because leukocytes include cells that differ in size and deformability. Our system will have potential as a tool for isolating CTCs from whole blood with high efficiency and selectivity.
Fig. 3: (a) Size-based CTC isolation system. (b) Fluorescent image and (c) trapping efficiency of CTC model cells and Leukocytes.[15]
4 Conclusions
Optically transparent and extremely low auto-fluorescent ETFE membrane filters with several tens of millions of through-holes were developed by using the photolithography and the plasma etching. Etch-byproducts of ETFE in O2
plasma reacted with the Ti surface. At low pressure, lower partial pressure of fluorine with a large O2 flow improved selectivity of etching of the Ti mask. The low pressure condition suppressed sidewall film deposition and delamination. The shape of the back side hole was rough at low pressure condition. Very high trapping efficiency of over 96% was obtained at separation test of CTC model cells spiked into whole blood using our fabricated microfiltration system. The method of fabricating device for size-base capture of rare cells in blood such as CTCs was established in this study.
Acknowledgements
This research was in part supported by the
Center of Innovation Program at Nagoya University (Nagoya University-COI) from the Japan Science and Technology Agency (JST).
References
[1] V. Parks, C. D. Koopman, and Z. Werb, Science 341, 1186 (2013).
[2] D. Kuboyama, D. Onoshima, N. Kihara, H. Tanaka, T. Hase, H. Yukawa, H. Ishikawa, H. Odaka, Y. Hasegawa, M. Hori, and Y. Baba, Proc. Micro Total Analysis Systems, (Georgia, USA, 2017), p. 882.
[3] X. Fan, C. Jia, J. Yang, G. Li, H. Mao, Q. Jin, and J. Zhao, Biosensor. Bioelectron. 71, 380 (2015).
[4] M. Ilie, V. Hofman, E. Long-Mira, E. Selva, J.-M. Vignaud, B. Padovani, J. Mouroux, C.-H. Marquette, and P. Hofman, PLoS ONE 9, 10, e111597 (2014).
[5] A. Lee, J. Park, M. Lim, V. Sunkara, S. Y. Kim, G. H. Kim, M.-H. Kim, and Y.-K. Cho, Anal. Chem. 86, 11349 (2014).
[6] P. Rostagno, J. L. Moll, J. C. Bisconte, and C. Caldani, Anticancer Res. 17, 2481 (1997).
[7] M. Hosokawa, T. Hayata, Y. Fukuda, A. Arakaki, T. Yoshino, T. Tanaka, and T. Matsunaga, Anal. Chem. 82, 6629 (2010).
[8] M. Hosokawa, T. Yoshikawa, R. Negishi, T. Yoshino, Y. Koh, H. Kenmotsu, T. Naito, T. Takahashi, N. Yamamoto, Y. Kikuhara, H. Kanbara, T. Tanaka, K. Yamaguchi, and T. Matsunaga, Anal. Chem. 85, 5692 (2013).
[9] D.Choi, G. Yoon, J. W. Park, C. Ihm, D. Lee and J. Yoon. J. Micromech. Microeng. 25 105007 (2015).
[10] A. Yusa, M. Toneri, T. Masuda, S. Ito, S. Yamamoto, M. Okochi, N. Kondo, H. Iwata, Y. Yatabe, Y. Ichinosawa, S. Kinuta, E. Kondo, H. Honda, F. Arai, and H. Nakanishi, PLoS ONE 9, 2, e88821 (2014).
[11] O. Lara, X. D. Tong, M. Zborowski, and J. J. Chalmers, Exp. Hematol. 32, 891 (2004).
[12] H. Teng, Appl. Sci. 2, 496 (2012).
[13] D. W. Smith, S. T. Iacono, and S. S. Iyer, Handbook of Fluoropolymer Science and Technology (Wiley, New York, 2014).
[14] N. Kihara, H. Odaka, D. Kuboyama, D. Onoshima, K. Ishikawa, Y. Baba, and M. Hori, Jpn. J. Appl. Phys.57, 037001 (2018)
[15] N. Kihara, D. Kuboyama, D. Onoshima, K. Ishikawa, R. Koguchi, H. Tanaka, N. Ozawa, T. Hase, H. Yukawa, H. Odaka, Y. Hasegawa, Y. Baba, and M. Hori, Jpn. J. Appl. Phys. (2018) in press.
[16] Y. Yamamoto, S. Higashi, and K. Yamamoto, Surf. Interface Anal. 40, 1631 (2008).
[17] H. Nabesawa, T. Hitobo, S. Wakabayashi, T. Asaji, and T. Abe, Sens. Actuators, B 132, 637 (2008).
Coating technology for locally varying optical function on 2d and 3d elementsD. Gloess*, H. Bartzsch, T. Goschurny, A. Drescher, U. Hartung, P. FrachFraunhofer-Institut für Organische Elektronik, Elektronenstrahl- und Plasmatechnik FEP, Winterbergstraße 28, 01277 Dresden, GERMANY *[email protected]
Keywords: pulse magnetron sputtering, optical filters, lateral thickness gradients, freeform
In this paper, a coating technology will be presented that allows achieving a locally varying optical function on 2d and 3d elements. This can be used, for example, to compensate layer thickness errors occurring during the deposition on tilted surfaces of larger 3d optical components. Other applications can be coatings for lateral varying light extraction of large-area waveguides in displays, wavefront correction or variable optical filters for hyper spectral cameras. In the coating plant PreSensLine at FEP (by Von Ardenne GmbH, s. Fig. 1) deposition rates in the range of 20 to 50 nm∙m/min allow efficient fabrication of optical layer systems for use in IR, VIS and UV [1].
Fig. 1: Deposition equipment for large area precision coatings at FEP: PreSensLine.
In this coating plant, various lateral gradient layer systems were fabricated. A special pulse parameter variation with pulse unit UBS-C2 (by FEP in cooperation with dresden elektronik ingenieurtechnik GmbH), adapted to the substrate movement (rotation/translation) with the precision drive (by LSA GmbH) allows to precisely adjust the deposition rate in dependence on the substrate position. On this basis, lateral varying layer thicknesses can be deposited on 2d substrates (Figs. 2-4). Therefore, uniform coatings of 3d substrates can be realized, among other things.
Fig. 2: Layer thickness distribution of a linear gradient of 300 nm across 400 mm substrate width deposited with PreSensLine.
Fig. 3: Sample photo and layer thickness distribution of a non-linear lateral thickness gradient layer of Nb2O5.
Fig. 4: Sample photo and layer thickness distribution of a 2D gradient coating deposited in PreSensLine: saddle shaped layer of Nb2O5.
In case of layer deposition on lenses, instead of an inhomogeneous layer thickness distribution with constant translation speed, a homogeneous distribution can be realized on the curved surface. This requires a superposition of substrate rotation with a precise controlled variation of the translation velocity. With the presented technology, for flat as well as for spheric, aspheric or freeform surfaces, exceptional layer thickness profiles can be realized to adapt layer systems to special requirements.
Acknowledgement
Part of the results within project funded by the European regional development fund (ERDF) and the Free State of Saxony.
References
[1] P. Frach, D. Gloess, T. Goschurny, A. Drescher, U. Hartung, H. Bartzsch, A. Heisig, H. Grune, L. Leischnig, S. Leischnig, C. Bundesmann, "Large area precision optical coatings by pulse magnetron sputtering", Proc. SPIE 10181, Advanced Optics for Defense Applications: UV through LWIR II, 101810K (11 May 2017); doi: 10.1117/12.2262541.
Plastic lens that has the effect of
reducing visible light uniformly at
each wavelength Ryosuke Suzuki1, Hirotoshi Takahashi1
1 TOKAI OPTICAL CO., LTD. R&D Department, Okazaki-city, Aichi, Japan
Abstract
In general, anti-glare spectacle lenses are produced
by dyeing their plastic substrates with some
dyestuffs. Because the dyestuffs have steep
absorption peaks at each specific wavelength, even
the lenses dyed gray also do not reduce visible light
uniformly at each wavelength in strict sense.
Therefore the color of vision with wearing the lenses
seems different from the actual color. On the other
hand, the neutral-density filter that is one of camera
filters has the effect of reducing visible light almost
uniformly.
In this research, we tried to make the anti-glare
spectacle lens that has the effect of reducing visible
light almost uniformly by using the multilayer coating
of the neutral density filter. Unsaturated nickel oxide
NiOx (x=0~1) was used as the absorbent layer. We
controlled uniformity of the transmittance by
controlling oxidized state of the NiOx layer. The
transmittance of visible light was reduced and
uniformed at each wavelength in the lens. The
spectral luminance meter was used for confirming the
change of color with the lens.
Keywords: plastic, lens, neutral density (ND) filter,
spectacle lens, luminance,
1 Introduction
In general, anti-glare spectacle lenses are produced
by dyeing their plastic substrates with some
dyestuffs. The color of the lens is determined by
choosing dyestuffs, and the transmittance of the lens
is determined by the amount of dyestuffs.
Compared to other colors, lenses dyed gray reduce
the color change of vision with wearing the lenses.
However, because the dyestuffs have steep
absorption peaks at each specific wavelength, even
the lenses dyed gray also do not reduce visible light
uniformly at each wavelength in a strict sense.
Therefore, the color of vision with wearing the lenses
seems different from the actual color.
On the other hand, the neutral density (ND) filter that
is one of camera filters has the effect of reducing
visible light almost uniformly. In many ND filters, the
effect is caused by the multilayer coating with optical
absorbent layers. Unsaturated metal oxides are used
as the absorbing layer [1]. They are produced by
depositing the metal with flowing oxygen gas. The
optical absorption property is controlled by the
deposition condition and optimized for the property of
the ND filter. The multilayer coating of the ND filer
also has the effect of the antireflective coating in
visible light [2].
In this research, we tried to make the anti-glare
spectacle lens that has the effect of reducing visible
light almost uniformly using the multilayer coating of
the ND filter.
2 Experimental
2.1 Sample
We prepared the spectacle lens plastic substrate that
refractive index was 1.76. The substrate was first
coated with the buffer layer and second coated with
scratch-resistance layer. These layers were coated
by the dipping method. The buffer layer had the
refractive index of 1.67 and the thickness of about 1.0
μm. The scratch-resistance layer had the refractive
index of 1.60 and the thickness of about 2.5 μm.
The ND multilayer coatings were produced by a
vacuum deposition method. The unsaturated nickel
oxide NiOx (x=0~1) layer was used as the optical
absorbent layer. The NiOx layer was produced by
depositing the nickel with flowing 10 sccm oxygen
gas. The deposition rate of the NiOx layer was 0.3
nm/s. The refractive index and the extinction
coefficient of the NiOx layer are shown in Fig.1. The
multilayer was consisted of NiOx, Al2O3, and SiO2
doped with a small amount of Al2O3. The materials
were heated by the electron beam. The deposition
rate of the Al2O3 layer and the SiO2 layer was 1 nm/s.
The starting pressure of the deposition was 1.0×10-3
Pa. The preset temperature of the process chamber
was 70 °C. The ND multilayer was coated on the
concave surface of the lens substrate. The convex
surface of that was coated with the antireflective
coating consisted of SiO2 and ZrO2. These
multilayers were coated on the scratch resistance
layer.
Layer MaterialThickness
[nm]Material
Thickness
[nm]Material
Thickness
[nm]
1 Al2O3 35 Al2O3 30 Al2O3 30
2 SiO2+Al2O3 30 NiOx 4.2 NiOx 4.5
3 NiOx 3 SiO2+Al2O3 35 SiO2+Al2O3 60
4 SiO2+Al2O3 75 NiOx 4.2 NiOx 5.5
5 - - SiO2+Al2O3 60 SiO2+Al2O3 50
6 - - - - NiOx 5.5
7 - - - - SiO2+Al2O3 70
Air - - - - - -
T=80% T=50% T=25%
Fig. 1: Refractive index and extinction coefficient of the
NiOx layer
Three types of the ND multilayer that transmittance
properties were about 80%, 50%, and 25% were
designed and produced. These multilayers also had
the effect of the antireflective coating in visible light.
Their film constructions are shown in Table 1. The
transmittance properties of the lenses are shown in
Fig. 2. The reflectance properties of the ND
multilayers are shown in Fig. 3.
Table 1: Film constructions of the ND multilayers.
Fig. 2: Transmittance properties of the lenses.
Fig. 3: Reflectance properties of the ND multilayers.
2.2 Evaluation method
2.2.1 Color measurement
A spectral luminance meter was used for confirming
the change of color with the sample lens. In the
darkroom, the plates painted in bright colors was
placed 3 m away from the spectral luminance meter.
The colors of the plate were red, blue, green, and
yellow. The object color of the plate lighted with the
daylight white fluorescent lamp was measured. The
measurement was performed with putting the sample
lens in front of the spectral luminance meter’s lens.
The sample lenses had no power. The measurement
also was performed without putting the sample lens.
The results were plotted to L*a*b* chromaticity
diagram.
2.2.2 Weathering and adherence test
The sample lens was weathered 240 hours by a
sunshine carbon arc weather meter. The adherence
test was carried out every 60 hours of the weathering
test in the following steps. The cross-cut of 100 cells
were made on the coatings with a cutter knife. The
distance of the cut was 1mm. A cellophane tape was
applied and removed rapidly five times. The number
of the peeled cells was counted.
2.2.3 Constant temperature and humidity test
The sample lens was put seven days in an
environment of with constant temperature and
humidity control. The temperature was 60 °C and the
humidity was 95%. The transmittance was measured
before and after the test.
T=80% T=50% T=25%
0h 0 (No Peeling) 0 (No Peeling) 0 (No Peeling)
60h 0 (No Peeling) 0 (No Peeling) 0 (No Peeling)
120h 0 (No Peeling) 0 (No Peeling) 0 (No Peeling)
180h 0 (No Peeling) 0 (No Peeling) 0 (No Peeling)
240h 0 (No Peeling) 0 (No Peeling) 0 (No Peeling)
Color Lens L* a* b* C* h ⊿h
--- 79,72 -5,87 74,83 75,06 94,49 ---
T=80% 74,43 -5,42 71,29 71,50 94,35 -0,14
T=50% 60,79 -4,58 59,25 59,43 94,42 -0,07
T=25% 44,95 -3,36 46,83 46,95 94,11 -0,38
--- 51,40 -27,32 13,01 30,26 154,54 ---
T=80% 47,78 -25,37 12,36 28,22 154,03 -0,51
T=50% 39,00 -21,57 10,66 24,06 153,70 -0,84
T=25% 27,04 -16,78 7,01 18,18 157,33 2,79
--- 51,77 -8,20 -28,75 29,89 254,07 ---
T=80% 47,95 -8,23 -25,95 27,22 252,41 -1,66
T=50% 38,80 -7,07 -21,81 22,93 252,04 -2,03
T=25% 27,01 -4,21 -19,50 19,95 257,81 3,74
--- 40,52 40,84 23,77 47,25 30,20 ---
T=80% 37,72 37,94 22,27 44,00 30,41 0,21
T=50% 29,98 32,15 18,74 37,21 30,24 0,04
T=25% 20,03 26,20 13,68 29,56 27,56 -2,64
Red
Blue
Green
Yellow
3 Results and Discussion
3.1 Color measurement
The results of the measurement are shown in Table 2
and plotted to L*a*b* chromaticity diagram in Fig. 4.
The straight lines in Fig.4 are drawn from the origin to
the each point that is measured without the sample
lenses. There are the points near the line
corresponding to the each color when they are
measured with the sample lenses. It is suggested that
there are little change in the color of vision with
wearing the sample lenses, because hue is denoted
the direction from the origin to the point in the
diagram. Hue is shown as hue angle (h) that is given
as h = tan-1(b*/a*). The absolute values of h change
are lower than five in each color measured with the
lenses.
The point tends to be nearer the origin when the
transmittance is lower. The distance between a point
and the origin denotes Chroma (C*). C* becomes
lower when the transmittance becomes lower.
Lightness (L*) changes similar to C*.
Table 2: Results of the color measurement
Fig. 4: L*a*b* chromaticity diagram plotted the results of
the color measurement
3.2 Weathering and adherence test
The results of the test are shown in Table 3. The
peeling of the coating was not found. It is thought that
the ND multilayers have good adhesion to the lens
substrate.
Table 3: Results of the adherence test
3.3 Constant temperature and humidity test
The transmittances of the T=25% lens before and
after the test are shown in Fig.5. There was little
change in the transmittance after the test. It is
thought that the NiOx layers are not affected in the
environment of the test.
Fig. 5: Transmittance change of the T=25% lens through
the constant temperature and humidity test
4 Conclusion
The neutral density multilayer coating that had the
optical absorbing layer was deposited on the
spectacle lens substrate. The spectral luminance
meter was used for confirming the change of color
with the lens. Because change of the hue angle is
little in each color measured with the lens, it is
suggested the lens have the anti-glare effect and
change the color of vision little. The coatings have the
basic durability applied the spectacle lens.
References
[1] R.Suzuki, H.Takahashi, Japanese Unexamined
Patent Application Publication No.2017-151219
[2] M.Ikeya, M.Sugiura, Japanese Patent
No.5066644
Heterogeneous microoptical
structures with a precision
below 1 µm Sönke Steenhusena, Frank Burmeisterb,
Matteo Großa, Gerhard Domanna, Ruth
Houbertzc, Stefan Nolteb,d
a Fraunhofer ISC, Neunerplatz 2, 97082
Würzburg, Germany b Fraunhofer IOF, 07745 Jena, Germany c Multiphoton Optics GmbH, Friedrich-Bergius-
Ring 15, 97076 Würzburg, Germany d Institute of Applied Physics, Abbe Center of
Photonics, Friedrich-Schiller-Universität Jena,
07743 Jena, Germany
Abstract
We demonstrate the fabrication of microoptical
elements made from hybrid polymers using
two-photon polymerization (2PP). To overcome
the throughput limitations of 2PP we use
galvanometric mirrors and propose a hatching
strategy for rotationally symmetric objects
which allows a significant process acceleration
to tolerable fabrication times while preserving
the surface accuracy of the fabricated
elements. Using the new strategy and
optimized processing parameters we
demonstrate the fabrication of an aspheric
microlens for diffraction limited focusing which
could be fabricated in less than 1.5 minutes.
Thorough topographic characterization with
atomic force microscopy (AFM) and laser
scanning microscopy (LSM) reveal excellent
agreement of the fabricated surfaces with the
theoretical design with a surface accuracy
below 100 nm. Our approach enables the
generation of arrays of custom shaped lenses
and large arrangements of freeform
microoptical elements within a few hours.
1 Introduction
The application of microlenses in e.g. micro-
imaging, beam-shaping and integrated optics is
a crucial factor for increased performance and
further miniaturization of devices and setups
ranging from the laboratory scale to consumer
products. Aspheric and freeform surfaces might
further advance the application of microlenses
in optical systems. However, as conventional
technology platforms for the fabrication of
microlenses are limited to spherical surfaces, a
demand for 3D fabrication technologies arises.
Two-photon polymerization (2PP) has proven
to be a versatile tool for the fabrication of
complex three-dimensional structures in
several photoresists [1–3]. In this technique,
femtosecond laser pulses are tightly focused
into the resist triggering two-photon absorption
in the focal volume of the employed
microscope objective. Thus, the
photopolymerization, i.e. solidification of the
resist, is confined strongly and it is possible to
create almost arbitrary shapes in a single
process step by simply scanning the focus in
3D space. Its flexibility and scalability pave the
way for 2PP in several fields of research:
Alongside applications in biomedicine like drug
delivery [4] or tissue scaffolds [5], 2PP enables
the fabrication of very sophisticated devices
with interesting physical properties like carpet
cloaks [6] and mechanical metamaterials [7,8].
In addition, 2PP allows permanent modification
of the polymers' refractive index which can be
exploited for optical waveguides [9,10]. In
contrast to other state of the art techniques
such as photoresist reflow [11], e-beam
lithography [12], focused ion beam milling [13],
selective chemical etching [14], and gray-tone
lithography [15], 2PP is an additive
manufacturing technology, which is almost free
of any constraints. Consequently, the
generation of microlenses is a very promising
area which can benefit from 2PP. This was
demonstrated impressively by single
microlenses or microlens arrays directly
attached to a substrate [16] and by very
complex freeform lens assemblies on optical
fibers [17–19].
However, despite its 3D capability 2PP is a
comparably slow process as the solidification of
the photopolymer is accomplished in a serial
(or point-to-point) manner with sub-µm volume
pixels (voxels). Employing higher velocities of
the positioning system or using low numerical
aperture (NA) focusing optics to increase the
polymerization rate (polymerized volume per
time) results in a trade-off between fabrication
duration and surface accuracy. Furthermore,
for the creation of functional optical elements
with 2PP, it is necessary to utilize a photoresist,
which reveals superior optical quality as well as
mechanical stability and that must be
polymerizable using femtosecond laser pulses.
In this paper we address these requirements by
utilizing a special ORMOCER®, which was
designed for optical applications and which has
low losses at telecom and datacom
wavelengths. To cope with the trade-off
between fabrication duration and surface
accuracy in 2PP, we developed and optimized
a hatching strategy for rotationally symmetric
microlenses. Combined with a systematic
investigation of the influence of fabrication
parameters on the lens' topography, this
strategy provides a significant acceleration for
the fabrication of highly accurate microlenses
with low surface roughness. This is
demonstrated by the fabrication of an aspheric
microlens. Additionally, we demonstrate the
fabrication of different microoptical elements
using galvanometric mirrors for rapid
positioning of the focal volume.
We believe that our approach with the special
choice of material, structuring hardware and
hatching strategy can spur the use of 2PP
written structures in future microoptical
applications like micro-imaging, beam-shaping
and optical tweezers.
2 Experimental details
2.1 Patterning setup for two-photon
polymerization
The setup for the two-photon fabrication of
microoptical elements is based on a 1030 nm,
10 MHz femtosecond oscillator (Amplitude
Systems t-pulse 200) as a laser source [20,21].
Pulses are frequency doubled to 515 nm and
focused into the sample using a microscope
objective with a NA of 1.4 (Zeiss Plan
Apochromat). Adjustment of pulse energy is
accomplished with a combination of polarizing
beamsplitter and halfwave plate mounted on a
computer controlled rotary stage (Aerotech
ADR-160). In order to monitor the structuring
process in-situ, we use a dichroic mirror, red
light illumination and a CCD camera. The
sample is mounted on a three axis positioning
system (Aerotech ABL1000) providing
velocities up to 30 mm/s with sub-micron
accuracy. The total travel of the axis system is
15 x 15 x 10 cm3 allowing for the patterning of
up to six inch wafers. Rapid beam positioning
inside the field of view of the microscope
objective is accomplished by galvanometric
mirrors (Lightfab GmbH). Fast switching of the
laser by an acousto-optical modulator (AOM) is
necessary to ensure synchronization to the
position of the axis system or the galvanometric
mirrors, respectively. This allows a highly
accurate switching exactly at the position
where the laser is supposed to be "on" or "off"
on a timescale of microseconds. Additionally,
the 2PP system is equipped with an autofocus
system for automatic substrate detection. A
second cw laser with a wavelength of 635 nm
is focused into the sample. Due to its low
intensities the detection laser does not interact
with the polymer. The reflected intensity
detected with a photodiode has a maximum for
the position of the focus at the interface
between substrate and polymer.
2.2 Materials
As a material for the fabrication of microlenses
we use an acrylate ORMOCER® (abbreviated
OC-V) formulated with 3 wt.-% of Irgacure®
OXE02 as the radical photoinitiator [21]. Like
other ORMOCER®s it is synthesized from
alcoxysilane precursors which undergo
hydrolysis and polycondensation reactions and
result in an organically functionalized inorganic
network (still liquid resin). In general, the
inorganic network of the ORMOCER® is
responsible for the material’s glass-like
properties i.e. excellent chemical, thermal and
mechanical stability as well as low shrinkage
and low optical absorption. With respect to
these properties ORMOCER®s predominantly
yield purely organic photoresists, which makes
them ideal candidates for 2PP fabrication with
high surface accuracy. Regarding the organic
functionalization, photochemically or thermally
polymerizable moieties allow for the
solidification and thus patterning of
ORMOCER®s analogously to conventional
(organic) polymers. The properties of
ORMOCER®s can be tailored to the needs of
the target application by the choice of
precursors, synthesis condition and processing
conditions, respectively. Details on the
synthesis and properties of the class of
ORMOCER® materials can be found in the
literature [22,23].
The formulation of OC-V + Irgacure® OXE02
exhibits strong interaction with the employed
515 nm laser pulses, which is a requirement for
patterning with high velocities [20]. At first, this
is a consequence of the larger overlap of the
initiator's linear absorption spectrum with the
employed laser wavelength compared to
800 nm illumination. Two-photon absorption
(TPA) occurring at 515/2 nm = 267.5 nm is
located in a regime of high linear absorption
while TPA at 800/2 nm = 400 nm has very
small overlap as the absorption of most
photoinitiators vanishes towards visible
wavelengths. Secondly, in previous z-scan
studies [24] it was found, that Irgacure®
OXE02 has a TPA cross section twice as high
as Irgacure® 369, which is a conventionally
employed photoinitiator [21].
For 2PP fabrication a droplet of OC-V +
Irgacure® OXE02 is casted in between a
sandwich of two microscope coverslips which
are separated by a 100 µm thick spacer. The
cover slip facing to the focusing optics is the
substrate. After 2PP the sample is rinsed using
a 1:1 solution of isopropyl alcohol and MIBK
(methyl isobutyl ketone).
2.3 Structure characterization
Surface profiles and roughness of the
fabricated elements were characterized using
atomic force microscopy (AFM, WITec Alpha
300) in tapping mode. Additionally, laser
scanning microscopy (LSM, Keyence VK-X210)
was used to obtain surface profiles semi-
automatically and faster than by using AFM.
Using a benchmark microlens, it was verified
that both AFM and LSM deliver equivalent
results.
The focal intensity distributions of the
fabricated microlenses were characterized by
scanning a microscope objective in 3D across
the focal region and monitoring the detected
intensity with a photomultiplier. For this the
microlenses were illuminated by a collimated
laser with a wavelength of 532 nm. Further
characterization was carried out by scanning
electron microscopy (SEM, Zeiss Supra).
3. Results and Discussion
3.1 Optimization of the hatching
strategy
To improve the fabrication times for aspheric
microlenses while maintaining high accuracy a
new hatching strategy for 2PP fabrication is
necessary. We propose a combination of
annular hatching [25] and shell hatching. This
means that only the shell of the structure is
solidified by 2PP using adjacent circular
motions which form the surface of the lens.
This is particularly useful for rotationally
symmetric lenses. After 2PP and solvent wash,
the liquid core of the lens is treated by UV flood
exposure for five minutes. In comparison to full
volume or XYZ hatching (inscribing stacks of
rods), this yields a process acceleration of
approximately three orders of magnitude. To
demonstrate this strategy Figure 1 (a-d) shows
transmission microscopy images of fabricated
microlenses.
Fig.: 1: Optimization of the hatching strategy for
manufacturing rotationally symmetric lenses. (a)
Non-optimized strategy. (b) Acceleration and
deceleration distances. (c) Randomly distributed
starting points. (d) Combination of acceleration and
deceleration distances + randomly distributed
starting points.
In Figure 1 (a) structural quality is affected,
because a line evolves on the surface at the
starting and ending positions of the circular
hatching traces as already observed by
Malinauskas et al. [26]. This is caused by two
effects: First, the limited acceleration of the
positioning system has to be taken into
account. Due to larger dwell times at the
starting and ending positions of the ring
compared to the ring itself, an elevation
evolves at the surface. To compensate this, we
introduced acceleration and deceleration
distances (where the laser is off) to the
hatching traces as depicted in Figure 1 (b). At a
closer look, the surface quality is better but still
not seamless, due to a small overlap of
beginning and ending of a hatching trace.
Since this is difficult to eliminate, we distributed
the starting points randomly over the surface as
depicted in Figure 1 (c). As it can be seen
there, a random distribution itself does not
avoid surface deterioration. Only the
combination of randomly distributed
beginning/ending points with tangentially
orientated acceleration distances, as shown in
Figure 1 (d), enables smooth surface
fabrication. Please note that in the hatching
strategy optimization the hatching distance is
too large and individual circular traces are
visible on the surface of the lens.
3.2 Fabrication of an aspheric lens
To demonstrate the potential of using 2PP with
optimized hatching strategy and fabrication
parameters we defined a target design for an
aspheric lens with a diameter d = 50 µm, a
height
h = 10 µm, and a focal distance f = 50 µm. The
design was carried out using commercial
raytracing Software (Zemax). The aspherical
surface is described by Equation 1, with c
being the curvature (1/r) and k the conic
constant.
() =
1 + 1(1 + )(1)
With fixed parameters for d, h, and f
optimization of the surface function assuming a
refractive index
n = 1.502 (determined by Abbe refractometry
on UV-treated OC-V layers) resulted in c = -
39.811 and
k = -2.257. According to the design this surface
focuses incoming light (λdesign = 532 nm) to a
diffraction limited spot.
For a diffraction-limited focal spot of the
microlens, the surface needs to have a peak-
to-valley error smaller than λ/4 (Rayleigh
criterion) and a RMS error smaller than λ/10
(Marechal criterion). To investigate if these
criteria can be met we fabricated lenses with a
writing velocity v = 500 µm/s and a hatching
distance ∆x = 0.1 µm. As the lens' surface has
a varying slope over the cross section, the
ellipticity of the voxel might influence the
surface formation despite the constant ring
separation (=∆x) along the surface. For this
reason, an array of microlenses according to
the design was fabricated. In this array the
average laser power was varied starting from
P = 1000 µW to P = 1800 µW with an
increment of ∆P = 100 µW. Additionally, the
starting z-position, a, with respect to the
substrate was varied by using the autofocus
system. This was done to compensate axial
offsets of the patterning laser and the detection
laser and to determine the optimum z-position
of the lens on the substrate. The parameter a
was varied starting from a = -2.0 µm to
a = 0.0 µm with an increment of ∆a = 0.25 µm
(negative values correspond to a position
deeper inside the photopolymer).
The topography of all fabricated lenses was
analyzed by performing a two-dimensional
surface fit according to Equation (1). We found
the best fit parameters c = -38.67983 and k = -
2.22479 for the lens fabricated with
P = 1500 µW and a = -0.25 µm. The mean
deviation of both parameters from the design is
only 3.18 %. This deviation from the design
parameters might originate from a slight tilt of
the substrate during the characterization of the
topography or the little polymerization
shrinkage. In addition, the employed increment
with respect to P and a might have been too
large, resulting in a (still) non-optimized
combination of voxel size and
distance to substrate.
Figure 2 depicts a cross-section through the
fabricated lens (upper part; inset: 2D surface
representation).
Fig. 2: Characterization of the fabricated aspheric
microlens: cross-section of the best matching
experimental data (black line) and fitted data
(equation 1) blue line.
The good agreement with the design is
indicated by the fit function according to
Equation (1) (solid blue line). In the lower part
of Figure 2 the deviations of the experimental
data from the design can be seen.
It is obvious, that the experimental data match
the aspheric design with maximal deviations of
200 nm, which are particularly prominent in the
center of the microlens. This might be due to
smaller annular radii in the center, which lead
to higher angular accelerations of the axis
system and thus larger positioning errors.
Furthermore, the time for the creation of a
single ring decreases the closer the laser spot
is to the center of the element. That is
why chemical interactions of radicals produced
along the rings might be stronger in the center
of the element and influence structure
formation. This is
a typical phenomenon also present in the
literature [26]. Furthermore, deviations are
comparably strong at the margins of the lens.
This might be due to substrate-induced effects
such as surface tension when the fabricated
voxels are close the substrate. Despite both
above mentioned criteria not being met, the
presented result is still excellent compared to
deviations published in the literature with sub 1
µm accuracy [17].
Both main sources of surface deviation from
the target design are also visible in Figure 3 (a)
which shows a color-coded two-dimensional
plot of the deviations.
Fig. 3: Characterization of the fabricated aspheric
microlenses: (a) 2D color representation of surface
deviations from the design. (b) microcope image of
best matching lens (P = 1500 mW, a = -0.25 mm).
(c) SEM image of lens array.
The RMS deviations are less than 76 nm,
which is λ/7 and to our knowledge a significant
improvement for 2PP fabricated microlenses.
However, ring shaped surface distortions which
might stem from laser power instabilities can
clearly be seen in Figure 3 (a) [27]. These
surface distortions might scatter incoming light
and consequently corrupt the imaging quality of
the fabricated lens. An optical image of an
individual lens (v = 500 µm/s, P = 1500 µW,
a = -0.25 µm, ∆x = 0.1 µm) is shown in Figure 3
(b).
Finally, the optimized hatching strategy in
combination with the capability of the axis
system for large area and high velocity
fabrication together with the highly efficient
material combination enable a reduction of the
fabrication time of a single lens to 1.5 minutes
allowing the fabrication of arrays in just a few
hours. This is demonstrated in Figure 3 (c)
where an array of 25 x 25 microlenses was
fabricated.
For preliminary optical characterization the
focus of the fabricated lens was mapped by
taking 128 axial slices with a resolution of 256 x
256 pixels. The resulting geometry which was
scanned had a volume of 10 x 10 x 25 µm³ (XYZ) around the focus.
Fig. 4: Focal slices of the fabricated microlens.
Fig. 5: Results of optical characterization for beam width, w(z), and maximum intensity I0(z) and focal intensity I
(r, z = 0). (a) On-axis intensity and beam width as a function of z. (b) Focal intensity.
Figure 4 depicts a selection of images of these
focal slices starting from index 24 which
corresponds to
-8 µm to index 122 (11.3 µm) with an increment
of 2 slices (positive values correspond to
increasing distance from the lens' surfaces). It
can be seen that the incoming light is focused
to a small spot, which has highest intensity at
slice index 66 close to the center of the
mapped volume. The absolute position of the
focal spot is 50 µm away from the lens apex.
This agrees very well with the design focal
length.
However, it can also be seen, that a second
peak with smaller intensity occurs between
slices 108 and 120. This is a typical indication
for aberrations caused by erroneous surface
shapes. To obtain a more detailed insight into
the formation of the focal volume, we analyzed
the radial intensity distribution in the focal plane
I(r,z = 0) depicted in Figure 5 (a) and the
average focal width (1/e²) and the on-axis
intensity for each of the focal slices shown in
Figure 5 (b).
In both plots dots represent the measured data
and solid lines simulated data. From
Figure 5 (a) a beam width of w0 = 650 nm could
be derived. On the other hand, the simulated
beam width is only 503 nm. The source of this
deviation might be the characterization device
itself: The small focal intensity distribution is
sampled by a high NA microscope objective
which has a comparable focal geometry. Thus,
the detected signal is a convolution of both
focal geometries and might not represent the
microlenses focal distribution as desired. This
can only be accomplished by (theoretically)
employing a Dirac-δ-shaped sampling function.
This effect can be estimated by assuming two
Gaussian intensity distributions. The focal
geometry of the microscope objective can be
described by its focal width according to
0.61 λ / NA = 361 nm. The resulting width of a
convolution of two Gaussian signals is given by
= +
. The resulting signal width
would be 619 nm, which is close to the
measured beam width. The effect of sampling
with a finite NA is also visible in I(z) in
Figure 5 (b) which is significantly broader than
the simulated on-axis intensity distribution. For
this reason, in future the optical properties of
2PP fabricated microlenses will be analyzed
using different methods e.g. by measurement
of the MTP (modulation transfer function).
Despite the described drawbacks of the
characterization method, it is still obvious that
the focusing behavior is not ideal as the second
maximum of I(z) is also visible in Figure 5 (b).
The cause of this maximum needs to be
investigated more detailed in the future.
3.3 Heterogeneous microoptical
elements
As 2PP is a freeform technology, the
fabrication of microoptical elements is not
limited to a single design that is fabricated at
predetermined spots, particularly regular
arrays, on a substrate. On the contrary, 2PP
allows the generation of different shapes and
sizes of elements directly located next to each
other. This is depicted in Figure 6.
Fig. 6: Different microoptical elements fabricated on
the same substrate.
Here, a pyramid, a prism, a hemisphere, and a
cone were placed directly next to each other.
The elements were fabricated by using the
galvanometric mirrors with a scanning velocity
of 10 mm/s and an average laser power of
3 mW. In this case the entire volume filled with
a line distance of 0.1 µm in the lateral (XY)
plane and the slice distance in the axial
direction (Z) was 0.1 µm as well. It can clearly
be seen, that the four designs with a height
between 30 and 40 µm could be fabricated
precisely. The edges of the prism and the
pyramid are sharp, the surfaces are flat without
any grating-like structure and the curved
surface on the hemisphere and the cone are
very smooth.
3.4 Custom arrangements of
microoptical elements
The rapid fabrication of refractive microoptical
elements using galvanometric mirrors and/or
shell hatching enables the fabrication of large
arrangements of these elements on a cm scale.
This is demonstrated in Figure 7 with an
arrangement of 13,600 small total internal
reflection mirrors (TIRs) forming the logo of the
ICCG 12.
Fig.7: Arrangement of microoptical elements. (a)
Design of the total internal reflection mirror. (b)
Microscope image of a small section. (c) Photograph
of the entire arrangement under blue light
illumination.
Figure 7 (a) depicts the computer design with a
size of 25 µm in each direction (XYZ). A
microscope image of a small section is shown
in Figure 7 (b). Finally, in Figure 7 (c) the entire
arrangement can be seen under blue light
illumination. The individual elements were
fabricated with a scanning velocity of 10 mm/s
and an average laser power of 3.5 mW. Axial
and lateral lines distances were 0.25 µm. Such
arrangements or also regular arrays could be
used as light outcoupling layers because they
can redirect incoming light into a direction
determined by the slope of the elements by
means of total internal reflection. In addition to
the significant acceleration of the 2PP
processing, these arrangements can also be
replicated easily. First experiments were
conducted in which a PDMS mold was
fabricated using the 2PP-written pattern as a
master structure. Then this mold was used to
fabricate an exact copy of the arrangement by
UV-assisted nanoimprint lithography. Details on
this process will be published in the future.
4 Conclusion and Outlook
Strategies for fast and accurate fabrication of
complex refractive microoptical elements using
2PP were developed and experimentally
demonstrated. In particular, it was shown that
differently shaped elements can be fabricated
on a single substrate.
By employing a shell hatching strategy in which
only the surface is fabricated by 2PP and the
liquid core is UV-treated after 2PP fabrication,
we could reduce the fabrication duration of an
individual microlens to 1.5 minutes. Despite the
acceleration, a surface accuracy below 100 nm
could be obtained by optimizing the annular
hatching (i.e. randomly distributed starting
points, acceleration distances) and analyzing
the impact of the relevant parameters such as
hatching distance, laser power, and starting z-
position offset. This enables the fabrication of
custom designed microlens-arrays for real
world applications. As a demonstrator a
diffraction limited microlens was designed and
fabricated taking the above mentioned hatching
considerations into account. Thorough
topographical characterization reveals good
agreement between design and fabricated
structure. However, measurements of the focal
intensity distribution of the fabricated
microlenses show differences from simulations,
indicating aberrations caused by surface
deviations. This will be investigated in more
detail in the future.
The advance in process acceleration has to be
accompanied by further material research as
the ORMOCER®'s polymerization rate has to
keep pace with increasing scanning speeds.
Acknowledgements
The authors acknowledge the financial support
by the German Research Foundation (DFG,
grants HO 2475/3-1 and TU 92/19-1). We also
thank our coworkers in Würzburg and Jena for
their support and fruitful discussions.
References
[1] J. Serbin, A. Egbert, A. Ostendorf, B.N.
Chichkov, R. Houbertz, G. Domann, J.
Schulz, C. Cronauer, L. Frohlich, M.
Popall, Femtosecond laser-induced two-
photon polymerization of inorganic-
organic hybrid materials for applications
in photonics, Opt. Lett. 28 (5) (2003)
301–303.
[2] M. Farsari, M. Vamvakaki, B.N.
Chichkov, Multiphoton polymerization of
hybrid materials, J. Optics-UK 12 (12)
(2010) 124001.
[3] S. Kawata, H.B. Sun, T. Tanaka, K.
Takada, Finer features for functional
microdevices, Nature 412 (6848) (2001)
697–698.
[4] A. Ovsianikov, B. Chichkov, P. Mente,
N.A. Monteiro-Riviere, A. Doraiswamy,
R.J. Narayan, Two photon
polymerization of polymer-ceramic hybrid
materials for transdermal drug delivery,
Int. J. Appl. Ceram. Tec. 4 (1) (2007) 22–
29.
[5] T. Weiss, G. Hildebrand, R. Schade, K.
Liefeith, Two-Photon polymerization for
microfabrication of three-dimensional
scaffolds for tissue engineering
application, Eng. Life Sci. 9 (5) (2009)
384–390.
[6] J. Fischer, T. Ergin, M. Wegener, Three-
dimensional polarization-independent
visible-frequency carpet invisibility cloak,
Opt. Lett. 36 (11) (2011) 2059–2061.
[7] T. Bückmann, M. Thiel, M. Kadic, R.
Schittny, M. Wegener, An elasto-
mechanical unfeelability cloak made of
pentamode metamaterials, Nat.
Commun. 5 (2014) 4130.
[8] T. Frenzel, M. Kadic, M. Wegener,
Three-dimensional mechanical
metamaterials with a twist, Science 358
(6366) (2017) 1072–1074.
[9] R. Houbertz, V. Satzinger, V. Schmid, W.
Leeb, G. Langer, Optoelectronic printed
circuit board: 3D structures written by
two-photon absorption, Proc. SPIE
70530B (2008).
[10] G. Langer, V. Satzinger, V. Schmidt, G.
Schmid, W.R. Leeb, PCB with fully
integrated optical interconnects, Proc.
SPIE 794408 (2011).
[11] H. Yang, C.-K. Chao, M.-K. Wei, C.-P.
Lin, High fill-factor microlens array mold
insert fabrication using a thermal reflow
process, J. Micromech. Microeng. 14 (8)
(2004) 1197.
[12] C. Vieu, F. Carcenac, A. Pépin, Y. Chen,
M. Mejias, A. Lebib, L. Manin-Ferlazzo,
L. Couraud, H. Launois, Electron beam
lithography: resolution limits and
applications, Appl. Surf. Sci. 164 (1–4)
(2000) 111–117.
[13] F. Schiappelli, R. Kumar, M. Prasciolu,
D. Cojoc, S. Cabrini, M. de Vittorio, G.
Visimberga, A. Gerardino, V. Degiorgio,
E. Di Fabrizio, Efficient fiber-to-
waveguide coupling by a lens on the end
of the optical fiber fabricated by focused
ion beam milling, Microelectron. Eng.
73–74 (0) (2004) 397–404.
[14] K.M. Tan, M. Mazilu, T.H. Chow, W.M.
Lee, K. Taguchi, B.K. Ng, W. Sibbett,
C.S. Herrington, C.T.A. Brown, K.
Dholakia, In-fiber common-path optical
coherence tomography using a conical-
tip fiber, Opt. Express 17 (4) (2009)
2375–2384.
[15] J. Yao, Z. Cui, F. Gao, Y. Zhang, Y. Guo,
C. Du, H. Zeng, C. Qiu, Refractive micro
lens array made of dichromate gelatin
with gray-tone photolithography,
Microelectron. Eng. 57–58 (2001) 729–
735.
[16] M. Malinauskas, A. Zukauskas, V.
Purlys, K. Belazaras, A. Momot, D.
Paipulas, R. Gadonas, A. Piskarskas, H.
Gilbergs, A. Gaidukeviciute, I. Sakellari,
M. Farsari, S. Juodkazis, Femtosecond
laser polymerization of hybrid/integrated
micro-optical elements and their
characterization, J. Optics-UK 12 (12)
(2010) 124010.
[17] T. Gissibl, S. Thiele, A. Herkommer, H.
Giessen, Sub-micrometre accurate free-
form optics by three-dimensional printing
on single-mode fibres, Nat. Commun. 7
(2016) 11763.
[18] T. Gissibl, S. Thiele, A. Herkommer, H.
Giessen, Two-photon direct laser writing
of ultracompact multi-lens objectives,
Nat. Photonics 10 (8) (2016) 554–560.
[19] P.-I. Dietrich, M. Blaicher, I. Reuter, M.
Billah, T. Hoose, A. Hofmann, C. Caer,
R. Dangel, B. Offrein, U. Troppenz, M.
Moehrle, W. Freude, C. Koos, In situ 3D
nanoprinting of free-form coupling
elements for hybrid photonic integration,
Nat. Photonics 12 (4) (2018) 241–247.
[20] S. Steenhusen, T. Stichel, R. Houbertz,
G. Sextl, Multi-photon polymerization of
inorganic-organic hybrid polymers using
visible or IR ultrafast laser pulses for
optical or optoelectronic devices, Proc.
SPIE 7591 (2010) 759114.
[21] R. Houbertz, S. Steenhusen, T. Stichel,
G. Sextl, Two-Photon Polymerization of
Inorganic-Organic Hybrid Polymers as
Scalable Technology Using Ultra-Short
Laser Pulses, in: F. J. Duarte (Ed.),
Coherence and Ultrashort Pulse Laser
Emission, Rijeka , 2010, pp. 583–608.
[22] C. Sanchez, P. Belleville, M. Popall, L.
Nicole, Applications of advanced hybrid
organic-inorganic nanomaterials: from
laboratory to market, Chem. Soc. Rev 40
(2) (2011) 696–753.
[23] K.H. Haas, H. Wolter, Properties of
Polymer-Inorganic Composites, in: K. H.
J. Buschow, R. W. Cahn,M. C. Flemings,
B. Ilschner, E. J. Kramer, S. Mahajan, S.,
P. Veyssière (Eds.), Oxford, 2001,
pp. 7584–7594.
[24] A. Ajami, W. Husinsky, R. Liska, N.
Pucher, Two-photon absorption cross
section measurements of various two-
photon initiators for ultrashort laser
radiation applying the Z-scan technique,
J. Opt. Soc. Am. B 27 (11) (2010) 2290–
2297.
[25] R. Guo, S. Xiao, X. Zhai, J. Li, A. Xia, W.
Huang, Micro lens fabrication by means
of femtosecond two photon
photopolymerization, Opt. Express 14 (2)
(2006) 810–816.
[26] M. Malinauskas, H. Gilbergs, A.
Zukauskas, V. Purlys, D. Paipulas, R.
Gadonas, A femtosecond laser-induced
two-photon photopolymerization
technique for structuring microlenses, J
Optics-UK 12 (3) (2010) 035204.
[27] D. Wu, S.-Z. Wu, L.-G. Niu, Q.-D. Chen,
R. Wang, J.-F. Song, H.-H. Fang, H.-B.
Sun, High numerical aperture microlens
arrays of close packing, Appl. Phys. Lett
97 (3) (2010) 31109.
Synthesis and Characterization
of Polyurethane Acrylate
Encapsulation Materials for
Organic Photonic Systems Oguzhan Cimen1,2, Canan Varlikli31 Ege University, Solar Energy Institute, Izmir, Turkey 2 Gizem Seramik Frit ve Glazür San. ve Tic. A.Ş, Sakarya,Turkey 3,Izmir Institute of Technology, Department of Photonic, Izmir, Turkey
Abstract
In this study we have designed and synthesized a new polyurethane acrylate derivative to be used in encapsulation of organic photonic devices. Polyurethane acrylate type of polymers have to potential of utilization as UV-curable materials under ambient conditions. Herein, the synthetic steps and structural characterization (FTIR, TGA and 1H NMR, 13C NMR) results of one of the derivatives is presented.
Keywords: polyurethane, acrylate, co-polymer, organic photonics
1 Introduction
Organic photonic systems, such as, organic light
emitting diodes and organic photovoltaic devices
are in high demand due to their lower production
costs, and higher flexibility compared to their
inorganic counterparts. [1]
Organic photonic devices do consist of organic materials and metal electrodes. The oxidation of metal electrodes due to moisture and oxygen directly affects the lifetime of the complete device. For this reason, the primary challenge is to improve the efficient moisture barrier layers which protect the device from moisture and oxygen. [2-3]
Increasing the lifetime of organic photonic systems by preserving moisture and oxygen is a promising industrial R & D topic.
Herein, the synthetic steps and structural characterization (FTIR, TGA and 1H NMR, 13C NMR) results of a polyuretan acrylate co-polymer derivative are presented.
2 Experimental
2.1 Synthesis of model compound
2-(((isocyanatomethyl)3,5,5-trimethylcyclohexyl)carbomylethyl methacrylate (U-AC)
58.57 g of isophorone diisocyanate, 0.4 g DBTL and 0.3 g hydroquinone were added to a three-neck flask with a mechanical stirrer under nitrogen atmosphere. 100 g of 2-hydroxyethyl acrylate were then dropwise added to the system in 1 h. The reaction mixture was stirred at 35 ºC in water bath for 30 min.
2-((3-((3-((3-hydroxy-2-(hydroxymethyl)-2-methylpropanoyl)oxy)-2-(((2-(hydroxymethyl)-2-methylbutanoyl)oxy)methyl)-2-methylpropanoyl)oxy)-2,2-dimethylpropoxy)carbonyl)-2-methylpropane-1,3-diyl bis(3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate) (HBPP)
Bis-MPA (0.6mol, 80.48 g), NPG (0.2 mol, 20.83 g) and p-TSA (0.4 g) were mixed in a three-necked flask equipped with mechanical stirrer. The flask was placed in an oil bath previously heated to 150 ºC. The reaction was left under N2
atmosphere for 2 hs. Then Bis-MPA (0.6 mol, 80.48 g) and p-TSA (0,405 g) were added and the mixture was allowed to stir for 2 h. The reaction was followed via FTIR spectra.
2-(10-(((3-hydroxy-2-(hydroxymethyl)-2-methylpropanoyl)oxy)methyl)-6-(hydroxymethyl)-1-(5-(((2-(methacryloyloxy)ethoxy)carbonyl)amino)-1,3,3-trimethylcyclohexyl)-6,10,14,14-tetramethyl-3,7,11-trioxo-4,8,12,16-tetraoxa-2-azaheptadecan-17-oyl)-2-methylpropane-1,3-diyl bis(3-hydroxy-2-(hydroxymethyl)-2-methyl propanoate (HBPU-AC)
HBPP (111.53 g, 0.1 mol) was dissolved in DMF at 90 ºC. UAC (22.23 g, 0.1 mol) was added to a flask with mechanical stirrer under N2
atmosphere. The flask was placed in an oil bath previously heated to 90 ºC. After that HBPP was added and allowed to mix for 3.5 h. The product was precipitated with water and filtered. The filtrate was separated in ethyl acetate with a separating funnel and the ethyl acetate was removed using a rotary evaporator.
3 Results and Discussion
3.1 Synthesis of HBPU-AC oligomer
HBPU-AC oligomers synthesis via a three step procedure as shown in the following Scheme. First, urethane monoacrylate (IPDI-HEMA) was obtained from the reaction between the secondary cycloaliphatic NCO group of IPDI and the OH group of HEMA using DBTDL as catalyst. Second, The addition polymerization of NPG and MPA was carried out at 150 ° C with p-TSA catalyst. Third, the UAC reacted with HBPP at 90 ºC for 3.5 h, and resulted in HBPU-AC oligomer.
Scheme. Synthesis route for urethane acrylate (HBPU-AC)
Figures 1a and 1b show the FTIR spectra of the UAC and HBPP, resepctively, and the spectra of HBPU-AC are shown in Figure 1c.
Fig. 1. a) FTIR spectrum of UAC, b) FTIR spectrum of HBPP, c) FTIR spectrum of HBPU-AC.
The NCO peak stretching absorption appears at 2250 cm-1 and the NH hydrogen bond peak at 3350 cm-1 for UAC (Fig. 1a). As the reaction continues, the carboxylic acids carbonyl peak disapeared (1696 cm-1), the ester carbonyl peak appeared (1730 cm-1) (Fig. 1b) and finally the NCO peak stretching absorption at 2250 cm-1
disappeared (Fig. 1c).
The 1H-NMR spectrum of HBPU-AC is shown in Fig. 2. The peaks at 7.4 ppm and 7.0 ppm clearlyconfirm the existence of the N-H bond. The peaks at 6.0 ppm and 5.6 ppm can be attributed to the acrylate group in the HBPU-AC. There is a broad peak at about 4.3 ppm, which is an overlap peak associated with the methylene groups bond with oxygen in the ester group. The analysis above confirms the formation of HBPU-AC oligomer, which is in accordance with the results of FTIR.
Fig.2 a) 1H NMR spectra of HBPU-AC, b) 13C NMR spectra of HBPU-AC.
a)
b)
c)
a)
b)
The TGA spectrum of HBPU-AC is shown in Fig 3. The decomposition temperature for the synthesized material is 180 °C. 50% decomposition was observed at 320 °C.
Fig. 3. TGA curve of HBPU-AC.
4 Conclusion
In this work, results related to the synthesis and characterization of an urethane acrylate oligomer for the development of UV curable coatings for organic photonic systems have been reported.
Acknowledgements
Financial support from the Gizem Seramik Frit ve Glazür San. ve Tic. A.Ş R&D Center.
References
[1] N.Grossiord, J.M. Kroon, R. Andriessen, P.W.M. Bloom, Org. Electron. 13 (2012) 432.
[2] S. Majee, M.F. Cerquira, D. Tondelier, etc. Progress in Org. Electron. 80 (2015) 27-32
[3] R. S. Kumar, M. Auch, E.Ou, G.Ewald, C.S.Jin, Thin Solid Flms 417 (2002) 120-126