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Multiprotein Printing Light-induced molecular adsorption of

P.-O. Strale, A. Azioune, G. Bugnicourt,
Y. Lecomte, M. Chahid,
V. Studer* ...................................... X–XX
Multiprotein Printing by Light-Induced
Molecular Adsorption
proteins (LIMAP) allows for quantitative
sub-micrometer-resolution printing of
multiple biomolecules. Surface-bound
gradients are patterned within minutes
over an entire glass cover-slip. LIMAP is
used to perform selective immuno-assays,
to dynamically control the adhesion of
individual cells, and to achieve hierarchi-
cal co-cultures instrumental for tissue
engineering.

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Multiprotein Printing by Light-Induced Molecular
Adsorption
Pierre-Olivier Strale, Ammar Azioune, Ghislain Bugnicourt, Yohan Lecomte,
Makhlad Chahid, and Vincent Studer*
Molecular printing, which involves the controlled deposi-
tion of molecules on a substrate at the micrometer scale, has
developed tremendously in the past few years. Micropatterned
substrates are now routinely used for biological research.[1]
For these applications, printed patterns of biomolecules aim
at mimicking the complexity of the in vivo microenvironment
of cells. Micrometer-scale gradients of multiple proteins are in
turn highly sought after. However, fast and reliable micropat-
terning methods are still limited to only one type of molecule
per pattern and do not allow for gradient patterning.
Well-established protein patterning methods either rely on
a slow serial writing process[2–4] or are based on parallelized
photolithographic techniques.[5,6] In the latter case, photo-
masks[7] or elastomer stamps are required, but cumbersome
mask alignment procedures[8] under biomolecule friendly wet
environments usually impair the ability to generate multipro-
tein patterns.[5] More recently, maskless projection lithography
systems based on spatial light modulators such as digital
micromirror devices (DMD) have been introduced for protein
printing.[9,10] They combine the high throughput of wide-field
photolithographic methods with the versatility of direct writing
approaches. Moreover DMDs can be used to generate grayscale
patterns of light. Recently they have been combined with a pho-
tobleaching-based grafting chemistry to enable single protein
gradient patterning.[10] Maskless projection lithography systems
thus have the potential to overcome registration issues when
multiple biomolecules are printed sequentially, yet they usually
suffer from lower contrast and resolution when compared to
laser scanning schemes.[11] Maskless projection systems require
a well-collimated light source to reach micrometer resolution
on the entire field of view. In practice, with current UV illumi-
nation sources, only a few mW mm−2 of UV power are available
thus requiring a very light sensitive grafting photochemistry.
Dr. P.-O. Strale, Dr. A. Azioune, Dr. G. Bugnicourt,
Y. Lecomte, Dr. M. Chahid, Dr. V. Studer
Interdisciplinary Institute for Neuroscience
University of Bordeaux
F-33077 Bordeaux, France
E-mail: vincent.studer@u-bordeaux.fr
Dr. P.-O. Strale, Dr. A. Azioune, Dr. G. Bugnicourt,
Y. Lecomte, Dr. M. Chahid, Dr. V. Studer
CNRS UMR 5297
F-33077 Bordeaux, France
Dr A. Azioune
Ecole Nationale Supérieure de Biotechnologie
Université Ali Mendjeli
BP E66 25100 Constantine, Algeria
DOI: 10.1002/adma.201504154
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Finally, the sequential printing of multiple molecules requires
an efficient antifouling background compatible with several
steps of grafting and rinsing. Poly(ethylene glycol) (PEG) immo-
bilized on surfaces is by far the preferred strategy for blocking
protein adsorption.[12] In subtractive patterning, this strongly
antifouling coating is locally removed to allow adsorption of
proteins. Compared to additive patterning methods based on
photoactivatable surface bound linkers,[13,14] subtractive tech-
niques usually exhibit more specific binding but require high
energies incompatible with maskless projection systems.[15]
Here we describe how to perform multiprotein micro-
patterning using light-induced molecular adsorption (LIMAP).
LIMAP is based on a water-soluble photoinitiator which is
able to tune the antifouling property of polymer brushes when
exposed to near UV light by a photoscission mechanism[16]
(Figure S1 and S2, Supporting Information). Very interestingly
after incubation of a protein solution onto the exposed pattern,
the density of adsorbed molecules scales almost linearly with
the dose of UV light (Figure 1 and Figure S1 and S2, Supporting
Information). Of note, the adsorption kinetics of the proteins on
the exposed pattern can be well-fitted with a Langmuir model[17]
(Figure S3, Supporting Information). The adsorbed protein
density depends linearly on the concentration of photoinitiator
(Figure S4, Supporting Information) and depends strongly on
the protein concentration (Figure S5, Supporting Information).
The low required dose of about 100 mJ mm−2 in the near UV
(375 nm) is well adapted to wide-field illumination schemes.
We used a high contrast and high resolution wide-field DMD-
based projection system coupled to a conventional epifluores-
cence microscope to generate arbitrary grayscale patterns of
UV light (Figure 1D). Consequently, micrometer-scale adhe-
sive patterns, onto which proteins can adsorb, are printed on
an antifouling PEG background within seconds. The very low
background out of these patterns allows for the sequential
printing of multiple proteins (Figure 1A,B). Patterns ranging
from 500 nm (Figure S6, Supporting Information) to 1 mm
can be printed in one step. Our maskless illumination scheme
prevents registration issues when multiple biomolecules are
to be patterned sequentially. All the illumination and incuba-
tion steps are performed in a liquid environment in a micro-
well with a few microliters of reagents (Figure 1E). For a given
fluorescent protein, after calibration of the UV dose-dependent
adsorption (Figure 1C and Figure S6, Supporting Informa-
tion), controlled gradients of arbitrary shape can be patterned.
As a demonstration, we provide an epifluorescence image of a
pattern of purified Green Fluorescent Protein (GFP) showing
three gradients with a logarithmic, linear, and exponential
shape (Figure 2A). For each gradient, the theoretical and the
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Figure 1. A) Schematics of the LIMAP patterning principle. (1) A solution of Pll-g-Peg is deposited on an air-plasma-activated glass cover-slip. Positively
charged Pll-g-Peg molecules adsorb to the negatively charged substrate and form a dense antifouling polymer brush. (2) After rinsing of the Pll-g-Peg
solution, a solution of photoinitiator is added. (3) A first pattern of UV light is projected onto the surface. The light-activated photoinitiator molecules
locally cleave the PEG chains. In turn the substrate becomes adhesive for the first incubated proteins (4). This process can be repeated to create
another pattern of a second protein (4-5-6). B) Two-color epifluorescence microscopy image of a pattern composed of GFP (green) and mCherry (red).
On the outer square, both proteins are patterned. Scale bar: 50 µm. C) Quantification of the density of immobilized mEOS2 proteins plotted against
the UV dose in mJ mm−2. D) Schematic of the optical arrangement for DMD-based-UV-patterned illumination. L1, L2: f = 20 mm lens (UV Fused
Silica Bi-Convex). L3, L4: f = 150 mm lens (UV Fused Silica Bi-Convex). L5: f = 200 mm lens (UV Fused Silica Bi-Convex). AD: Aperture diaphragm.
M1, M2: UV dielectric mirror mounted on a kinematic mount in a periscope arrangement. RHD: Rotating holographic diffuser (diffuser angle 10°, rota-
tion speed 1000 rpm). DMD: Digital Micromirror Device. DM: Single band dichroic mirror (cutoff wavelength: 405 nm) placed in the filter turret of the
epifluorescence microscope. Obj: Microscope objective. E) Photograph of the sample holder placed on top of the microscope objective. The sample
consists in a 22 mm × 22 mm glass cover-slip with nine silicone microwells. Each microwell is filled with a droplet of 5 µL of reagent.

experimental fluorescence intensity profile are plotted (Figure 2B)
and are in good agreement. Interestingly, this process can be
repeated sequentially for the superimposed patterning of two
different proteins. A first linear gradient of GFP was patterned
according to the previously described protocol. A second oppo-
site gradient of the protein mCherry was printed on top of the
first pattern with a good agreement with the expected linear
theoretical profiles (Figure 2C). As a demonstration of the
robustness of LIMA protein printing, we reproduced “the birth
of Venus” of Botticelli by superimposing complex patterned
gradients of three different fluorescent molecules (Figure 2D).
To our knowledge, with a resolution of about 50 000 dpi and
a printing speed of about 0.1 mm2 s−1, the “color” molecular
printing capabilities of LIMAP overcome by far the existing
methods[18,19] (Table S1, Supporting Information). Furthermore,
LIMAP is a generic method compatible with all the substrates
commonly used in cell biology (glass, plastic, and elastomer)
(Figure S7, Supporting Information), and with a wide range of
biomolecules without any chemical modification. The orthog-
onal micropatterning of multiple functional biomolecules on a
surface is a challenging task.[18] A major difficulty is to avoid
nonspecific binding while retaining the functionality of the
immobilized proteins. We used LIMAP to pattern two different
antibodies at different locations of the same field of view. After
an incubation step of a mixture of their fluorescently labeled
ligands, we observed their expected segregation (Figure 3A).

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between streptavidin and biotin (Figure 3B,
right panel). At such low density, the single-
molecule co-localization reveals unambigu-
ously the functional interactions of the two
partners. In both cases, more than 40% of the
ligands co-localize with their patterned target
revealing that a significant fraction of the pat-
terned molecules is functional. Altogether,
these results show that LIMAP is an efficient
method to dynamically recruit biologically
active ligands onto prepatterned targets. We
further explored this potentiality in the field
of cell biology where protein patterning has
become a well-established tool to control
cell adhesion.[20] Here we provide a generic
approach to trigger cellular response on
demand by specifically recruiting biologically
active molecules at well-defined locations
and densities. We printed “Yin Yang” shape
micropatterns of fibronectin and strepta-
vidin and fibroblastic cells were seeded. After
20 min, non-adherent cells were washed
away, revealing the specific adhesion of indi-
vidual cells on the fibronectin-coated “Yin.”
Then biotinylated fibronectin was added to
the cell culture medium, triggering lamel-
lipodium formation and eventually cel-
lular spreading over the full Yin Yang circle
Figure 2. Demonstration of the performances of the LIMAP patterning method. A) Epifluo- (Figure 3C and Movie S1, Supporting Infor-
rescence microscopy image of a logarithmic (top), linear (middle), and exponential (bottom) mation). Building on the multiscale capa-
printed gradients of GFP. B) Fluorescence intensity profile (red squares) of each patterned gra-
bilities of LIMAP, we performed a similar
dient of (A). The theoretical expected profile is plotted in blue. C) Top: Two-color fluorescence
microscopy image of a pattern combining two opposite linear gradients of proteins: GFP in experiment on larger “Yin Yang” patterns. A
green and mCherry in red. Scale bar: 50 µm. Bottom: Fluorescence intensity profile of each first population of confluent fibroblastic cells
linear gradient pattern. D) Three-color fluorescence microscopy image of a “color” micrometer- adhered specifically to the fibronectin-coated
scale reproduction of the Birth of Venus by Boticelli. This complex micropattern combines Yin pattern. After incubation of biotinylated
three different fluorescent molecules (GFP, Pll-g-PEG-TRITC, and Neutravidin-Ato647) which fibronectin a second population of the same
have been printed sequentially at the same location of a glass cover-slip. Scale bar: 50 µm. The
cell type was subsequently able to adhere on
reproduction of the image of “Botticelli, NASCITA di VENERE”, Galeria Uffizi is on concession
of the Italian Ministry of Cultural Heritage and Activities and Tourism. Subsequent reproduc- the Yang pattern (Figure S8, Supporting Infor-
tion or duplication by any means is prohibited. mation). More interestingly, using the same
robust protocol, we were able to hierarchi-
This experiment demonstrates the multicomponent patterning cally pattern two different cell types. S180 cells stably expressing
of functional biomolecules by LIMAP. Due to the very low back- E-Cad GFP were allowed to adhere to the FN-coated Yin, and
ground provided by PEG-coated surfaces, LIMAP allows for the after biotinylated fibronectin incubation, mouse embryonic
immobilization of proteins at a controlled and very low density fibroblast (MEF) cells specifically spread on the Yang pattern.
compatible with single-molecule localization (Figure 1C). We Even on such an intricate pattern with cellular scale details, the
show that orthogonal patterning is efficient even at the single- two cell populations were properly segregated (Figure 3D).
molecule level: a two-protein Yin Yang pattern has been real- These results demonstrate unmatched orthogonal cell printing
ized by sequential UV illumination and incubation of GFP capabilities, which turn LIMAP into a very powerful method for
and Alexa-568-labeled secondary antibody. A digital quantifica- multiscale co-culture with straightforward applications in tissue
tion showed that more than 80% of the individual molecules engineering. LIMAP provides a generic method for fast and high
are localized on their dedicated pattern (Figure 3B, left panel). resolution patterning of multiple biomolecules. The range of appli-
Such patterns of individual proteins are of great interest to cations extends from the single molecule up to the multicellular
test biomolecular interactions. Indeed classical biochemistry scale with an exquisite control over local protein density. Micro-
approaches to study molecular interactions are time-consuming patterns of individual fluorescent molecules provide a promising
and require high amounts of proteins. LIMAP allows to study in tool for the high throughput quantitative study of bio-molecular
the minute time scale interactions at the single-molecule level interactions. At larger scale, the ability to rapidly generate complex
using tens of nanograms of proteins. We provide as a proof and functional protein landscapes will enable large-scale studies
of concept two examples of interactions between an antibody of the cell–cell and cell–matrix interactions with potential applica-
(anti-GFP) and its target (GFP) (Figure 3B, middle panel) and tions to biomedical research and tissue engineering.

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Figure 3. Application of the multiscale patterning capabilities of the LIMAP method. A) Dual immuno binding assay. A mixed solution of Cy3-labeled
Fc fragment and Cy5-labeled Streptavidin was incubated over a “Yin” micropattern of anti-Fc and “Yang” of Pll-Peg-biotin. The orthogonal binding of
each ligand on its specific patterned target was observed by epifluorescence microscopy (Cy3 in green and Cy5 in red); scale bar: 100 µm. B) Orthog-
onal micrometer-scale patterning of individual functional proteins observed by fluorescence TIRF microscopy. Left: A two-protein Yin Yang pattern
has been realized by sequential UV illumination and incubation of GFP (in green) and Alexa-568-labeled secondary antibody (in red). Each protein
is present at more than 80% in its dedicated pattern. Scale bar: 20 µm. Middle: Single-molecule co-localization assay of patterned GFP molecules
and Alexa-647-labeled anti-GFP. Scale bar: 10 µm. More than 60% of the detected antibodies co-localize with a GFP molecule. Right: Single-molecule
co-localization assay of patterned FITC-labeled streptavidin and Alexa-647-biotin. Scale bar: 10 µm. More than 40% of the detected individual biotin
molecule co-localizes with a streptavidin molecule. C) Orthogonal patterning of adhesion proteins as a tool to dynamically control single cell spreading.
Left: Two-color epifluorescence microscopy image of a two-protein Yin Yang pattern composed of Cy3-labeled Fibronectin (red) and Alexa-488-labeled
streptavidin (green). DIC microscopy image of single MEF cell adhering to Fibronectin “Yin” pattern (scale bar: 20 µm). After addition of biotinylated
Fibronectin, the cell starts to spread over the “Yang” pattern and finally engulfs the nonadhesive disk (yellow star) and covers the entire pattern.
D) LIMAP orthogonal patterning of adhesion proteins as a tool to perform co-culture experiments. Left panel: Two-color epifluorescence microscopy
image of a two-protein Yin Yang pattern composed of Cy3-labeled Fibronectin (red) and Alexa-488-labeled streptavidin (green). The first population
of S180 cells expressing E-Cadherin GFP was first seeded. After incubation with biotinylated FN, MEF was allowed to adhere. Middle panel: Epifluo-
rescence microscopy image showing S180 cells stably expressing E-cadherin GFP adhering only to the fibronectin “Yin” pattern. Right panel: Phase
contrast images showing S180 cells and MEF adhering to the FN-coated Yin and biotinylated FN-coated Yang, respectively. Scale bar: 100 µm.

Experimental Section
Substrate Preparation: All our experiments on glass substrates were
performed on cleanroom cleaned 22 mm × 22 mm NEXTERION Cover-slip,
#1.5H Glass D263 (SCHOTT Technical Glass Solutions, Jena, Germany).
Glass cover-slips were first activated by a 5 min air plasma treatment
(Harrick Plasma, Ithaca, NY, USA). A 20 mm × 20 mm, 250 µm thick
piece of Poly(dimethylsiloxane) (PDMS), with four or nine 3 mm × 3 mm
square holes was cut in a transparent silicone film (Figure 1E) (BISCO
HT-6240, Rogers Corporation Carol Stream, IL, USA) with a Craft Robo
Pro vinyl cutter (Graphtec, Irvine, CA, USA) and placed on the cover-
slip (Figure 1E). In each 3 mm × 3 mm well, 5 µL of a 0.1 mg mL−1 Pll-
g-PEG (PLL(20)-g[3.5]-PEG(5), SuSoS AG, Dübendorf, Switzerland)
solution in phosphate buffered saline (PBS) was incubated for 1 h.
LIMAP experiments on biotinylated Pll-g-PEG were performed as above by
using PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-biotin(20%), (SuSoS AG, Dübendorf,
Switzerland) instead of Pll-g-PEG (Figure S2, Supporting Information).
Experiments on PDMS substrates were performed using PDMS-
coated glass cover-slips (IBIDI µ-Dish 35 mm High ESS, 15 kPa). The
dry PDMS surface was activated with deep UV using a low-pressure
mercury lamp (Heraeus, Noblelight GmbH, NIQ 60/35 XL Longlife,
185 and 254 nm, quartz tube 60W) at a 5 cm distance (13 mW cm−2)
during 5 min. A 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at
10 × 10−3 M and N-hydroxysuccinimide at 7.5 × 10−3 M solution in MES and
NaCl buffer was incubated in each of the four wells (fabricated according
to the protocol for glass substrates above) at room temperature during
15 min and then rinsed with PBS. In each 3 mm × 3 mm well, 5 µL of a
0.1 mg mL−1 Pll-g-PEG (PLL(20)-g[3.5]-PEG(5), SuSoS AG, Dübendorf,
Switzerland) solution in PBS was incubated for 3 h.[21]
Experiments on plastic substrates were performed using polystyrene
flat-bottom untreated 96-well plates (Evergreen, Los Angeles, CA, USA).
Each well was treated with a 2 g L−1 solution of Pluronics F127 (Sigma–
Aldrich) for 1 h and rinsed with PBS without drying the surface.
Wide-Field Maskless UV Projection System: The optical projection
system is based on a standard epifluorescence inverted microscope
(Nikon Ti-E, Nikon Instruments, Champigny-sur-Marne, France) coupled
to a Digital Light Processing device (Texas-Instrument DLP Discovery
4100 UV) including a DMD to generate spatially modulated excitation
patterns. The latter consists in a 1920 × 1080 array of micromirrors of
size 10.8 µm × 10.8 µm each that can independently switch between

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two stable orientations: −12° or +12° regarding the DMD surface. The
maximum frequency, around 10 kHz, of each independent mirror (in
full chip mode) allows generating single independent binary patterns
at high rates. The DMD window is Ar-coated for a best transmission
region between 320 and 400 nm (UV). One of the main critical aspects
of this experimental setup relies on a correct alignment of the DMD
with the optical path defined by the microscope system (Figure 1D).
A UV continuous-wave laser (iBeam smart–Compact Diode Laser,
lambda = 375 nm, 70 mW maximum output power, Toptica Photonics
AG, Graefelfing, Germany) produced a collimated laser beam focused
by a first 20 mm lens (UV Fused Silica Bi-Convex, Thorlabs, Newton NJ,
USA) on a holographic UV rotating diffuser (UV holographic diffuser,
angle 10°, 50 mm diameter, Edmund Optics, York, UK) to produce
a speckle-free homogenous light distribution. At the other side of the
diffuser, (according to the divergence angle of the diffuser) another
lens was placed (identical to the first one) to expand the laser beam
into a 2 cm diameter collimated and homogenous beam. The latter
was oriented toward the DMD at an angle of incidence corresponding
to twice the tilting angle of the DMD mirrors (≈24°) using a pair of
UV-coated dielectric mirrors (BB2-E01, Thorlabs). Depending on the
computer controlled and independent orientation of each micromirror,
the spatially light modulated image was carried to the back illumination
path of the microscope through a 4f imaging system relay (f1, f2, and f3
lenses, f1 = f2 = 150 mm UV Fused Silica Bi-Convex, and f3 = 200 mm
lens tube UV Fused Silica Bi-Convex, Thorlabs, Newton NJ, USA), which
did not change the magnification of the image. An aperture diaphragm
was centered in the focal plane of f1 and f2 to isolate the central order
of diffraction of the DMD. The mirror plane of the DMD was conjugated
with the image plane of the microscope after reflection on a UV dichroic
mirror (405 nm single-edge laser-flat dichroic beamsplitter, Semrock,
Idex Corporation Lake Forest, Illinois USA) placed in the filter turret of the
microscope. The size of each mirror of the DMD was 10.8 × 10.8 µm, the
equivalent mirror size in the image plane was equal to the 10.8 µm per
M, where M is the magnification of the microscope objective: 10× (Nikon
Plan Fluor), 20× (Nikon Plan Apo Vc), and 100× (Plan Apo Tirf 1.49)
for our experiments. All UV illumination patterns were projected as
758 × 758 pixels binary images. In this condition, and independently from
M, the field of the DMD UV illumination matched the field of view of our
512 × 512 pixels EMCCD imaging camera (Evolve 512, Photometrics,
Tucson AZ, USA). The real size of the projected UV full field pattern
in the sample plane of the microscope thus depended only on M,
and was equal to 820 µm × 820 µm for M = 10×, 410 µm × 410 µm
for M = 20×, and 82 µm × 82 µm for M = 100×. Eight-bit grayscale
illumination patterns were projected as a sequence of 255 binary images
generated from an original 8-bit uncompressed 758 × 758 pixels TIFF
image. This image sequence was generated so that each pixel of the
original image with a grayscale level of 0 < n < 255 was “on” for the n
first images of the sequence and “off” for the 255-n following patterns.
In turn, a grayscale level n converted into a fraction of the total exposure
time of the sequence. The illumination power in the entire field of
view of the microscope for a full white (n = 255), 758 × 758 pattern
was measured for each microscope objective and was 7.8, 6.47, and
1.89 mW for the 10×, 20×, and 100× objective, respectively, at full laser
power (70 mW). The illumination dose could thus be controlled for each
pixel of the field of view independently.
Protein Patterning: For all the LIMAP experiments, 5 µL of a solution
of photoinitiator (4-benzoylbenzyl-trimethylammonium chloride,
custom synthesis by Sigma-Aldrich outsourced to SinoChem, China)
was incubated in each microwell (Figure 1E) for 1 min at 50 × 10−3 M in
PBS before UV exposure of the pattern. The UV-activated photoinitiator
molecules locally cleaved the PEG chains (Figure S1 and S2, Supporting
Information) in a UV dose- and photoinitiator-concentration-dependent
manner (Figure 1C and Figure S5, Supporting Information). After
extensive rinsing with PBS, the microwell was filled with 5 µL of protein
solution in PBS which adsorbed to the UV-exposed areas.
Gradient Patterning: To investigate the UV dose dependence of the
patterned protein density, an 8-bit grayscale pattern of 9 squares was
projected by the DMD. Each gray level was converted into a percentage
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of the total exposure time. In turn, for a total exposure time of 15 s and
for a surface power (in the image plane) of 9.4 mW mm−2, the maximum
UV dose in the upper left square of the pattern was 140 mJ mm−2.
The center square corresponds to a gray level of zero and is a direct
measure of unspecific protein binding. For each square, the number of
mEOS2 protein was counted by single-molecule localization microscopy
using PALMTracer.[22] A PALM reconstruction of the mEOS2 pattern is
represented on Figure S4 in the Supporting Information. The plot of the
mEOS2 density as a function of UV dose is shown on Figure 1C. This
relation between protein density and UV dose (and thus gray scale level
of each pixel of the DMD pattern) can be used to obtain any grayscale
pattern of the same fluorescent protein. When patterning a grayscale
image on a substrate, the relation between the gray level and its intensity
on the resulting fluorescence image is not strictly linear but can be
calibrated (Figure 1C) for a given protein and substrate. Therefore, the
experiments requiring a precise control of the adsorbed protein density
(Figure 2A–C) were performed after applying a correction function to the
initial gray levels in order to account for the true dose response of the
LIMAP process.
One-Protein Gradients: The three gradients shown in Figure 2A
(exponential, linear, and logarithmic) are projected simultaneously with
a 20× objective and a UV dose of 140 mJ mm−2 for the brightest pixels.
After extensive washes (PBS), purified GFP (10 µg mL−1, kind gift from
Matthieu Sainlos, IINS CNRS Bordeaux) was allowed to adsorb for
2 min at room temperature.
Two-Protein Crossed Gradients: The first gradient was obtained
by LIMAP of purified GFP (10 µg mL−1 in PBS, for 2 min) with a
20× objective and a UV dose of 140 mJ mm−2 for the brightest pixels.
Then PLL-g-PEG (5 min, 1 mg mL−1 in PBS) was incubated in order
to block further adsorption on unoccupied sites. The second gradient
was obtained by LIMAP of purified mCherry (10 µg mL−1, kind gift from
Matthieu Sainlos, IINS CNRS Bordeaux) with a 20× objective and a UV
dose of 140 mJ mm−2 for the brightest pixels.
“Birth of Venus” Reproduction: The initial colors of the painting
were separated in three 8-bit grayscale images (red, green, blue) and
patterned consecutively by LIMAP with a 20× objective, following similar
steps as above. The first “blue” pattern was obtained by LIMAP of
purified GFP (100 µg mL−1 in PBS, for 2 min) with a 20× objective and
a UV dose of 40 mJ mm−2 for the brightest pixels. The second “green”
pattern was obtained by LIMAP of TRITC-labeled Pll-g-PEG (PLL(20)-
g[3.5]-PEG(2)-TRIC, SuSoS AG, Dübendorf, Switzerland) 100 µg mL−1
in PBS, for 2 min) with a 20× objective and a UV dose of 80 mJ mm−2
for the brightest pixels. The third “red” pattern was obtained by LIMAP
of Atto-647-labeled neutravidin (kind gift from Matthieu Sainlos, IINS
CNRS Bordeaux) (100 µg mL−1 in PBS, for 2 min) with a 20× objective
and a UV dose of 120 mJ mm−2 for the brightest pixels. As described
for the two proteins gradient above, PLL-g-PEG (5 min, 1 mg mL−1 in
PBS) was incubated in order to block further adsorption on unoccupied
sites between each LIMAP step. Of note, the reconstructed image
used the conventional red–green–blue colors instead of realistic colors
corresponding to the dyes, and all fluorescence images used were taken
at the same location of the sample after the three patterning steps.
Dual Immuno Assay: The “Yin” (defined hereafter as the right part of
the YinYang) micropattern was projected through a 20× objective (UV
dose of 50 mJ mm−2). Biotinylated Pll-PEG (0.1 mg mL−1) was then
incubated for 1 min. The “Yang” (defined hereafter as the left part of the
YinYang) micropattern was projected through the same objective without
moving the sample (UV doses of 100 mJ mm−2). Anti-Fc antibody (Goat
Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch,
20 µg mL−1) was incubated for 3 min. A mixed solution of Cy3-labeled
human Fc fragment (1 µg mL−1, kind gift from Olivier Thoumine,
IINS CNRS Bordeaux) and Cy5-labeled Streptavidin (1 µg mL−1, Life
Technologies) in PBS with 2% bovine serum albumin (BSA) (Sigma–
Aldrich) and 0.1% Tween 20 (Sigma–Aldrich) was incubated for 10 min.
Single-Molecule “Yin Yang”: The “Yin” and “Yang” micropatterns
were projected through a 100× objective (UV doses of 133 mJ mm−2
and 533 mJ mm−2, respectively). Purified GFP was incubated for
3 min at 300 ng mL−1; goat antirat 568 was incubated for 3 min
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at 100 ng mL−1. TIRF microscopy images were acquired with an
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automated epifluorescence microscope (Nikon TI-E) with a motorized
TIRF illuminator coupled to a laser bench (Roper Scientific France,
Evry, France) with three lasers (wavelengths 488, 561 and 647 nm)
and a 100× NA 1.49 objective. For each type of fluorophore, a stream
of images were acquired (exposure time 20 ms, 30 frames per second)
until complete photobleaching with an EMCCD camera (Evolve 512,
Photometrics, Tucson AZ, USA). Single molecules were detected and
localized on a max projection of the entire stream of images using the
WaveTracer module (Molecular Devices) with a combination of wavelet-
based localization and anisotropic Gaussian fitting methods.[22]
Single-Molecule Co-localization Experiments: Micropatterns were
projected through a 100× objective (175 and 42.5 mJ mm−2 for the “cross”
and the “9 squares” patterns, respectively). For the “cross” pattern,
purified GFP was incubated for 1 min at 15 ng mL−1 and Alexa-647-
labeled anti-GFP (Invitrogen, A-31852 Life Technologies) was incubated
for 5 min at 1 µg mL−1. For the “9 squares” pattern, biotinylated Pll-PEG
(PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-biotin(20%), SuSoS AG, Dübendorf,
Switzerland) was incubated for 3 min at 100 ng mL−1, Alexa-488-labeled
Streptavidin (Life Technologies, Saint Aubin, France) was incubated
for 3 min at 100 ng mL−1 and Alexa-647-labeled biotin (PV6032, Life
Technologies, Saint Aubin, France) at 100 × 10−9 M. Single molecules
were detected and localized as above. Molecules at a distance of lower
than 160 nm were considered as co-localized.
Unicellular “Yin Yang”: The “Yin” and “Yang” micropatterns were
projected through a 20× objective (100 mJ mm−2). The cells were
prepared as explained above. Cells adhering to the “Yin” FN-coated
structure were imaged at 37 °C, 5% CO2 at four images per minute by
differential interference contrast microscopy 60× or by phase contrast
microscopy at 20×. Then biotinylated FN (200 µg mL−1 in Dulbecco’s
modified Eagle medium (DMEM)) was added allowing the cells to
spread on the “Yin Yang.” Live-cell imaging was performed as described
above.
Multicellular “Yin Yang”: A “Yin” UV micropattern was projected
on Pll-g-Peg treated glass cover-slips through a 10× objective
(172mJ mm−2). Fibronectin (100 µg mL−1) was then added and allowed
to adsorb for 15 min at room temperature. After extensive washes, a
“Yang” UV micropattern was projected (172 mJ mm−2). Pll-Peg-Biotin
(100 µg mL−1) was incubated for 3 min before Alexa-561-labeled
streptavidin (Life Technologies) (100 µg mL−1) was added. After
washes, the wells were filled with DMEM and the sample was put in
a humidified incubator (37 °C, 5% CO2). S180 mouse sarcoma cells
stably expressing E-Cadherin GFP (kind gift from Virgile Viasnoff, MBI,
University of Singapore) or MEF (kind gift from Gregory Gianonne
IINS CNRS Bordeaux) (Figure S8, Supporting Information) were grown
under standard conditions. Cells were detached by a 0.05% trypsin–
ethylenediaminetetraacetic acid (EDTA) solution, resuspended in
complete culture medium before centrifugation (5 min, 1000 rpm).
The pellet was resuspended in DMEM and the cell suspension was put
in the incubator for 15 min before deposition on the micropatterned
wells. After 20 min, low adherent cells were washed away, revealing
the specific adhesion of S180 cells (or MEF) on the FN-coated “Yin.”
The medium was then removed and biotinylated FN (100 µg mL−1 in
DMEM) was incubated for 15 min. MEFs were then seeded (the same
detachment procedure as above) and extensive washes were done
after 20 min revealing the proper adhesion of S180 cells (or MEF) and
MEF, respectively, on the Yin and Yang structures. Live-cell imaging
was performed on an inverted epifluorescence microscope (Nikon
Ti-E, Nikon Instruments, Champigny-sur-Marne, France) placed in
an incubator (The Box, Life Imaging Services, Basel, Switzerland) to
control temperature (37 °C), humidity (100%), and CO2 (5%).
AFM Imaging: Atomic force microscopy imaging was performed in
liquid in peak force mode with a Bruker Fastscan AFM and a SNL-10
AFM tip. The scan size is 20 µm × 20 µm with a scan rate of 1 Hz. Peak-
force setpoint 100 pN.
6 wileyonlinelibrary.com © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgments
P.-O.S., A.A., and G.B. contributed equally to this work. This work was
supported by funding from the French “Ministère de l’Enseignement
Supérieur et de la Recherche” through the program Investissement
d’Avenir Vibbnano. The authors thank Mathieu Sainlos for insightful
discussions and for kindly providing them with purified fluorescent
proteins. The authors are grateful to Anne Bourdoncle (IECB, Bordeaux),
Jean-Pierre Aimé (CBMN CNRS Bordeaux), and Jean-Paul Salvetat
(CRPP, CNRS Bordeaux) for sharing their equipment and for their kind
assistance for AFM Imaging experiments. The authors thank Jean-
Louis Viovy (Institut Curie, Paris) for his very inspiring comments and
suggestions concerning their work. The authors acknowledge Corey
Butler for his careful reading of the manuscript. The authors thank
Nikon Instruments France for loaning some of their equipment.
Received: August 25, 2015
Revised: September 29, 2015
Published online:
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DOI: 10.1002/adma.201504154