Supplementary Information for
In-channel integration of designable microoptical devices using flat
scaffold-supported femtosecond-laser microfabrication for coupling-free
optofluidic cell counting
Dong Wu1, Jian Xu1, Li-Gang Niu2, Si-Zhu Wu1,2, Katsumi Midorikawa1, and Koji Sugioka*1
1
Laser Technology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2
State Key Laboratory on Integrated Optoelectronics, College of Electronic Science and
Engineering, Jilin University, 2699 Qianjin Street, Changchun 130012, China
*e-mail: ksugioka@riken.jp
Table of Contents
 Figure S1 ～ Figure S9 (P2～P13)
 Table S1 (P14)
 Captions of Supplementary Movie 1 and Movie 2 (P15)
1
Fig. S1. The schematic fabrication process of 2D Fresnel zone plates (FZP) and 3D microlens
arrays (MLA) by flat-scaffold supported hybrid femtosecond laser microfabrication (FSSHFLM) method in channel and on surface. The main fabrication process contains polymer
coating, prebaking, SA-TPP, developing, and drying. The prebaking time (18 h) and the laser
power (100 W) in channel are bigger than the ones (1 h, 50 W) on surface.
2
(a) Top-view: Flat scaffold
FZP
Flat-scaffold FZP
＋
=
Side-view: Flat scaffold
2 m
FZP
(b) Top-view: Flat scaffold
Flat-scaffold FZP
1 m
＋
=
Single microlens
＋
20 m
Single microlens
＋
(c) Top-view: Flat scaffold
10 m
Microlens array
＋
Side-view: Flat scaffold
5 m
Flat-scaffold microlens
20 m
Side-view: Flat scaffold
280 m
3 m
=
20 m
1 m
87.6 m
87.6 m
87.6 m
Flat-scaffold microlens
=
11 m
Flat-scaffold microlens array
280 m
280 m
=
Microlens array
10 m
＋
Flat-scaffold microlens array
15 m
=
Fig. S2. The schematic design and fabrication of flat-scaffold supported 2D FZP and 3D
MLA. The shape and size of flat-scaffold are designed according to the size and structure of
optical devices on it. Usually, the lateral size of flat-scaffold is the same as the optical devices.
The data program for creating scaffold is integrated with that for microoptical device. So, in
the laser writing step, both of the scaffold and microoptical devices are fabricated by singlestep laser scanning. The scaffold fabrication doesn’t increase the complexity of the whole
laser fabrication process.
3
Fig. S3. The schematic fabrication of flat-scaffold supported a 3D MLA in microchannel. The
flat-scaffold is scanned from the inner part of glass below the bottom surface of channel to the
inner part of channel. Usually, the lateral size and pitch of laser scanning for constructing the
flat-scaffold were the same as the optical devices. For fabrication of the flat-scaffold as well
as microoptical devices, smaller scanning pitch (200 nm) was adopted to ensure high surface
smoothness (average roughness~2.5 nm) as compared with 500 nm for filter/mixer fabrication
used in our previous work.
4
Fig. S4. Comparison of cell detection by femtosecond-laser written optical waveguides and
optofluidic microlens. Compared with femtosecond-laser written optical waveguide (a), the
optofluidic microlens cell detection method (b) provides three advantages. The first one is that
this method is simple because it doesn’t need complex and precise fiber coupling system. The
second one is that the microlens can focus white light and it doesn’t need laser. Third,
polymer microlens can be realized in microchannel with various materials (PDMS, polymer,
glass), because the resin is afterward filled into the channel for laser microfabrication and
subsequently washed out by the developer. On the other hand, the optical waveguide relies on
the refractive index change of glass substrate of microfluidics induced by femtosecond laser,
so that it cannot be realized by a different kind of material from the substrate.
5
Fig. S5. For the fabricated microchannels, slight curvature on the bottom of the channel
happened due to the combined effects of HF etching and high temperature annealing. To
eliminate the effects, we design FZP with certain thickness, e.g., 3 m because the surface
irregularities are about 2 m according to our measurement. Too thin (<1m) FZP will lead
to missing of some parts of FZP. In the meanwhile, too thick (>5m) will lead to the floating
or collapse of polymer rings of FZP, especially for outer rings with smaller widths (~3.7m).
6
Fig. S6. The calculation of the focal length of 2D FZP in microchannel with liquid
environment.
Firstly, according to diffraction theory, the focal length of FZP in ethanol is
f eth 
neth r12

(1)
Due to the refraction of the ethanol/glass and glass/air interfaces, the focal length is changed
from “O1” to “O”.
tan eth  r12 / feth
(2)
According to refraction formula at the interface of ethanol/glass,
neth  sin  eth  ng  sin  g
(3)
 g  arc sin(neth  sin  eth / ng ) (4)
According to the formula of rectangular triangle,
tan  g  (d1  d 2 ) / h2  (r12  h1  tan  eth  d 2 ) / h2 (5)
We can get
d 2  r12  h1  tan  eth  tan g  h2
(6)
Then, according to refraction formula at the interface of glass/air,
7
nair  sin  air  ng  sin  g (7)
air  arcsin(ng  sin g / nair ) (8)
By the equation of (6) and (8), we can get
h3  d2 / tan air
(9)
Finally, we can obtain the focal length
f  h1  h2  h3
(10)
For red light (632 nm), we can calculate feth  1.379 mm, eth  3.634 ,  g  3.266 ,
d 2  75.800 m, air  4.952 , h3  0.875 mm, so the focal length of red light is f red  1.075
mm.
For yellow light (570 nm), we can calculate feth  1.529 mm, eth  3.278 ,  g  2.947 ,
d 2  76.956 m, air  4.467 , h3  0.985 mm, so the focal length of yellow light is
f yellow  1.185 mm.
For green light (522 nm), we can calculate feth  1.670 mm, eth  3.002 ,  g  2.699 ,
d 2  77.854 m, air  4.091 , h3  1.089 mm, so the focal length of green light is
f green  1.289 mm.
The diffraction efficiency of FZP is defined as the ratio of the power collected to the
primary focal spot to the total incidence. The measured efficiency of this amplitude FZP in
microchannel with ethanol environment is about 2%, which is comparable to glass-based
amplitude FZP by high-power femtosecond laser irradiation of silica 1. According to the
diffractive theory, the theoretical maximum value of 1/  2  10.1% . The possible reason is
that the thickness of the fabricated polymer zones in microchannel has not been precisely
controlled. The efficiency of FZP can be further enhanced by designing phase-type and multilevel FZP with precise thickness.
1
W. Watanabe, D. Kuroda, K. Itoh, J. Nishii, Opt. Express, 10, 978 (2002).
8
Fig. S7. The point spread function of the focal spot produced by the microlens and the
calculation of the focal length of the microlens in 3D embedded microchip by aplanatic
9
principle and refraction theory. The light is focused by the microlens and passes two different
media (ethanol and glass). The refraction at the interface of ethanol/glass happens. The
original focal spot at position “O1” is changed to the position “O”. Firstly, we calculated the
position “O1” by the aplanatic principle. Then, according to the refraction theory, we can get
the position “O” and the focal length. According to the aplanatic principle, the formula is
showed as follows;
h  nsu8  (h1  h  h2 )  neth  r 2  (h1  h2 )2  neth
Given h=10, h1=60, nsu8=1.58, neth=1.362, r=20, we can get
h2 
neth 2 (r 2  h 2 )  h 2 nsu8 2
 h1  h  64.15 m
2hneth (nsu8  neth )
tan  eth 
r
20

 0.1752
h1  h2 114.15
eth  arctan 0.1752  9.9378
d1  h2  tan eth =64.15  0.1752=11.239 m
According
g  arcsin(
to
the
refraction
theory,
neth  sin  eth  ng  sin  g
,
we
can
get
neth
 sin eth )  8.9255
ng
h3  d1 / tan 2  11.239 / tan 8.9255  71.562 m
Finally, we can obtain the focal length f  h1  h3  131.562 m, which is well agreed with the
measured value of 135±5 m.
10
In air, according to the aplanatic principle, the formula is expressed as follows;
h  nsu8  ( f  h)  r 2  f 2
Given h=10, nsu8=1.58, r=20, we can get
f 
(r 2  h 2 )  h 2 nsu8 2
 h  32 m. This is well agreed
2h(nsu8  1)
with the measured value of 35±5 m.
11
Fig. S8. (a) The time-dependent intensity variation of the focal spot of microlens. Ten
intensity drops mean that ten cells pass through above the microlens. The magnitude of
intensity drop has some variation (0.2～0.8). This is probably ascribed to the cells passing
through different lateral position above the microlens. (b) Namely, cells passing the center of
the lens bring about the largest drop while the drops become smaller as they are off from the
center.
12
Fig. S9. (a-b) The common filter usually is deformed and falls down due to its bad stability.
With 2 mins ultrasonic bathing in acetone, the filter fell down. (c-d) The “W”-shape can
significantly increase its stability (Supplementary Figure S6) and avoid deformation or
breakdown during the developing and characterization process. Even with 30 mins ultrasonic
bathing in acetone, the W-shape filter remained unchanged.
13
Methods
(FSSHFLM)
3D
optofluidic
chip
References
This work
 UV, Soft lithography: 2D optical  FSS-HFLM —integrating 3D
devices[4-8]
polymer optical devices
 Colloidal sphere, interference
into
3D
channel
by
lithography: specific periodic
optimally fabricating a layer
3D optical microstructures[9,10]
of polymer scaffold and
 TPP: 3D polymer optical device
eliminating
the
slightly
on flat surface [14,15]
unflattened
internal
channel surface.
 Most works are 2D optofluidic  3D
programmable
device; few works are 3D
optofluidic
microchips:
optical
devices
into
2D
3D+3D — 3D designable
microchannels (2D+2D [4-8],
microoptical devices into
2D+3D [9,10])
3D glass microchannels
 Disadvantages: low flexibility
and weak designability
 Waveguide detection: cell  Multifunctions:
detection [22-24]
microfocusing,
 Disadvantages:
complex
microimaging,
cell
coupling system and only
detection/ counting
suitable for glass microchip
 White light, couplingfree, wide compatibility
Table 1 Systemic comparison between previous progress and this work from three aspects:
Function
and
application
method, device, and function/application. Most of previous works focused on the integration
of 2D optical components or specific periodic 3D optical devices into 2D microchannels,
which lead to low flexibility and weak designability. FLAE of glass can realize some
functional microcomponents, but this method suffered from low precision. TPP has fabricated
3D polymer optical devices on flat surface, but it remains challenging to be performed in
microchannel. In this work, high quality integration of various 2D-3D microoptical devices
with 3D microfluidics in a precise and flexible manner was realized for the first time by
developing a method of FSS-HFLM. Two kinds of typical diffractive and refractive devices —
2D FZP and 3D MLA were integrated into 3D microchannels. They showed excellent optical
properties, e.g., multicolor spots (red/yellow/green) of FZP, elongated (>3 times) focal length
of MLA, and clear imaging of “RIKEN”, and further demonstrated optofluidic application for
coupling-free white-light cell counting with as much as 93% efficiency.
14
Supplementary Movie 1. The biocompatibility test of the 3D integrated optofluidic
microchip. After filling the optofluidic microchip with mass of cells, we can find that cells
can survive and freely move, which indicate that the 3D optofluidic microchip is
biocompatible with biological samples.
Supplementary Movie 2. The filtering functions of W-shape with high stability. The hole
size formed in the filter is designed as 7 m. The optofluidic microfluidic system consists of
two Y-shape glass microchannels, two MLAs and a W-shape filter.
At the time of 0, 4s in the video: Two small cells with 50-m length and 6-m width passed
the filter. However, at the time of 5 s in the video, a big cell with 72-m length and 8-m
width is hindered by the filter. At the time of 9 s in the video, a small cell with 45-m length
and 5.5-m width also passed the filter. To clearly observe the filtering process, the flow
speed of water is adjusted to be as low as 50-100 m s-1, since the swimming speed of cell is
about 50-80 m s-1.
15