Supplemental Material
In-situ guidance of individual neuronal processes by wet femtosecond-laser
processing of self-assembled monolayers
Hideaki Yamamoto, Kazunori Okano, Takanori Demura, Yoichiroh Hosokawa, Hiroshi
Masuhara, Takashi Tanii, and Shun Nakamura
Figure S1
FIG. S1. (a) Neurons cultured for 24 h after scanning laser in the absence of laminin-1. No neurite
elongation was observed along the laser-scanning line (indicated by a black arrow). (b) A schematic
illustration of the pattern. Solid black lines and circles are modified with DETA SAM, and gray
background with ODS SAM. Laser-scanning lines are indicated with dotted lines. Scale bar: 30 m.
Fabrication process of the DETA/ODS SAM template
DETA and ODS SAMs were patterned on a Pyrex glass substrate (15 mm x 15 mm x 0.5 mm) by
electron-beam (EB) lithography. The glass slide was first cleaned in piranha solution (H2SO4:H2O2 =
4:1) and ammonia/hydrogen peroxide mixture (NH4OH:H2O2:H2O = 1:1:7, 80 oC) for 5 min each.
An SiO2 layer was then deposited onto the substrate by plasma chemical vapor deposition (gas:
Tetraethyl orthosilicate (TEOS) 4.0 sccm and O2 500.0 sccm, pressure: 53.0 Pa, temperature: 300 oC,
RF power: 80 W, time: 5 min). (Fig. S2: Step 1) Note that although we routinely cover the substrate
surface with TEOS-derived SiO2 layer to reduce scratch marks (1-4 nm in depth) on the glass slide,
this process is very likely to be omissible without affecting the results of cell patterning and that of
the fs-laser processing.
Next the TEOS-covered glass substrate was modified with ODS SAM. (Fig. S2: Step 2) The
substrate was placed upside-down in a 65 ml Teflon jar containing 350 l ODS, and the jar was
heated for 3 h at 110 oC. After the deposition, the substrate was sonicated in chloroform and ethanol
for 10 min each. The substrate was then immersed in 1% bovine serum albumin solution for 30 min
at room temperature to render the surface hydrophilic.
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A positive-tone EB resist, ZEP-520A (Nippon ZEON; 60% (v/v) dilution in ZEP-A), was spincoated on the substrate surface at 300 rpm for 3 sec and at 4000 rpm for 120 sec, and the preexposure bake was performed at 225 oC for 5 min. Then a charge-dissipating agent, ESPACER
300Z (Showa Denko) was spin-coated at 1500 rpm for 35 sec and at 5500 rpm for 25 sec. (Fig. S2:
Step 3) EB lithography (accelerating voltage: 20 kV, beam current: 1 nA, dose: 40 C cm-2) was
performed on a Hitachi S-4200SE scanning electron microscope equipped with Tokyo Technology
Beam Draw system. After the EB lithography, ESPACER was removed by rinsing the substrate in
H2O, and the sample was subsequently developled in ZED-N50 for 3 min and rinced in ZMD-B
(both from Nippon Zeon) for 30 sec. After development, the sample was immersed in buffered
hydrofluoric acid (HF:NH4F:H2O = 1:4:180) for 30 sec to remove ODS molecules and expose
TEOS-SiO2 film in the unmasked region. (Fig. S2: Step 4)
Then the DETA SAM was deposited placing the sample upside-down in a Teflon jar containing
300 l of the silane and heating the jar in an oven for 2 h at 100 oC. (Fig. S2: Step 5) After the SAM
deposition, the substrate was sonicated in toluene for 10 min, tetrahydrofurane for 10 min, ethanol
for 5 min, and water for 5 min, in this order to remove residual ZEP520A layer and non-specifically
adsorbed DETA. (Fig. S2: Step 6)
FIG. S2. Fabrication process of the DETA/ODS SAM template.
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Setup of the fs-laser processing system
Fs-laser (wavelength: 800 nm, pulse duration: 120 fs, repetition rate: 1 kHz, power: 0.5 mW) from
regeneratively amplified fs Ti:Sapphire laser system (Hurricane, Spectra-Physics) was directed into
an inverted microscope (IX-71, Olympus) and focused with a 20x bright-field objective lens (N.A.:
0.46). The focused laser beam was scanned across the ODS region by moving a motorized xy-stage
of the microscope (scanning velocity: 10 m s-1). Width of line modified by laser scanning was 2.1 ±
0.1 m as determined by AFM imaging. Laser spot was approached to the tip of neurites until the
separation was approximately 1-2 m.
Fluorescence imaging of laminin adsorbed on the laser-irradiated region
Laminin-1 (Sigma, L2020; 1 mg ml-1 in Tris buffered NaCl) was buffer-exchanged into phosphate
buffered saline (PBS(-)) using size-exclusion chromatography (GE Healthcare, HiTrap Desalting).
Protein concentration of laminin-1/PBS(-) was measured spectrophotometrically by evaluating UV
absorbance at 280 nm (Shimadzu, BioSpec-mini) and was determined to be 0.36 mg ml-1. The
protein was then labelled with NHS-Fluorescein (Thermo Scientific, 46410) and was purified by
size-exclusion chromatography. Concentration and the degree of labelling of the dye-labelled protein
were 90 g ml-1 and 9.3, respectively. The protein was finally diluted in PBS(-) to a concentration of
10 g ml-1.
Fluorescence images were obtained using confocal microscopy system (Olympus, FV300)
equipped on the inverted microscope (IX-71, Olympus). A diode-pumped solid-state laser (Shanghai
Dream Lasers Technology, SDL-473-030T, 473 nm) was used as an excitation light source. The
laser was scanned in a two-dimensions (single-section mode), and fluorescence signal (510-570 nm)
was imaged to detect Fluorescein.
An ODS SAM-modified glass slide was immersed in culture medium for 3 h, the fs-laser was
irradiated in a 30 x 30 m2 region, and the sample was rinsed in PBS(-). Fluorescein-labelled
laminin-1 (10 g ml-1) was spotted on the sample, and after a 4 h incubation, the sample was rinsed
and immersed in PBS, and was imaged under the confocal microscope.
Discussion: Limiting factors of the success rate
Success rate of the in-situ guidance is currently estimated to be 14% (n = 16 out of 115 scan lines)
and requires further improvement. The following two factors are seemingly restricting the
probability, namely, a fluctuation of surface modification with laser-irradiation and a laser-induced
damage to the cell or the neurite.
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The first point was examined by evaluating neurite extension on laser-scanning lines which were
prepared prior to cell seeding. This enabled us to estimate the sole fluctuation of successful surface
modification and eliminate the effect of laser-induced cell damage on the success rate. The
DETA/ODS SAM template was immersed in the complete culture medium for 3 h, and the laser was
scanned on the ODS region so as to extend an existing DETA line or connect two DETA lines in the
presence of 10 g ml-1 laminin. After laser scanning (laser power: 0.5 mW, scan speed: 10 m s-1),
the sample was stored in a CO2 incubator over-night. Then the whole medium was exchanged to
remove unattached laminin, and primary chick neuron was seeded as described before. After two
days of culture, the cells were observed by phase-contrast microscopy. Of 120 laser-scanning lines
prepared, neurite elongation toward laser-scanning lines was observed on 26 lines. Among these
lines, neurite extension was observed on 8 lines, whereas on the remaining 18 lines, neurites
extended to the terminal of the DETA line but did not extend further onto the laser-scanning line.
(Fig. S3) Hence the percentage of successful surface modification was calculated to be 31% (= 8/26),
showing that success probability of the current protocol does not reach 100% even in the absence of
laser-induced damage to cultured cells.
The second point was examined by evaluating how laser irradiation to the tip of their neurites
influences the survival rate of the neurons. Here the survival rate was determined by detachment of
the cells from the adhesion sites 24 h after laser irradiation. When a cell was subjected to one or
more laser scans the survival rate was estimated to be 47% (n = 27 out of 57 cells), whereas the rate
was 93% (n = 37 out of 40 cells) for cells without any laser irradiation. Moreover, the amount of
laser irradiation seems to influence subsequent development of the cells, as shown in Fig. S4.
The amount of protein adsorption to the laser-irradiated region depends severely on depth of the
laser focal point within a range of several microns as well as the laser power,1 and this is suspected
to be the primary cause of the fluctuation of the successful surface modification with laser irradiation.
We expect to circumvent the fluctuation by using of other adhesion molecules with higher affinity to
neurites. Suspected causes of the laser-induced cell damage are mechanical damage (due to shortlifetime cavitation bubbles2 which are unobservable with video-rate cameras), photochemical
damage, and chemical damage (due to a generation of reactive oxygen species and subsequent
disruption of plasma membrane2,3). Depth and lateral positioning of the laser focal point as well as
the laser power is the most direct parameters that influence these effects, and hence further
optimization of the laser-irradiation condition is essential.
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FIG. S3. (a) A representative image of a neurite growing on a laser-scanning line which was
processed prior to cell seeding (indicated by a black arrow). White arrow and black arrowhead
indicate a neuron adhered on a laser-scanning line and a scanning line which failed to support
neurite growth, respectively. (b) A schematic illustration of the pattern. Solid black lines and circles
are modified with DETA SAM, and gray background with ODS SAM. Laser-scanning lines are
indicated with dotted lines. Scale bar: 50 m.
FIG. S4. Dependence of survival rate on the number of laser irradiations per cell. Survival rate
proportionally decreases as the number of laser irradiation for neurite guidance increase.
References
[1] A. Matsui, K. Okano, Y. Maezawa, M. Matsubara, H. Yamamoto, Y. Hosokawa, F.-J. Kao, and
H. Masuhara, unpublished.
[2] A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, Appl. Phys. B 81, 1015 (2005).
[3] U. K. Tirlapur, K. König, C. Peuckert, R. Krieg, and K.-J. Halbhuber, Exp. Cell Res. 263, 88
(2001).
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