Supplementary material: Selective plane illumination microscopy on a

chip

Petra Paiè,a,†

Francesca Bragheri,b,†

Andrea Bassi, a,*

Roberto Osellameb

aDipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy

bIstituto di Fotonica e Nanotecnologie, CNR, Piazza Leonardo da Vinci 32, 20133 Milano, Italy

*Corresponding author: andreabassi@polimi.it

TEXT

Chip fabrication by femtosecond laser micromachining

The chip was realized by using femtosecond laser micromachining 1-3. The

irradiation was performed by focusing through a 50x, 0.6 NA microscope

objective the second harmonic of a commercial femtosecond laser

(femtoREGEN, HIGH-Q Laser) emitting pulses of 400 fs, 1040 nm

wavelength and energy up to 23 μJ at 960 kHz repetition rate. Scan

velocities and pulse energies were varied in relation to the depth of the

irradiated structure with respect to the glass surface to compensate the

spherical aberrations. The geometry of the device was obtained by properly

translating the sample relative to the laser beam with a system of high

precision air bearing translation stage (Fiberglide 3D, Aereotech).

The different components, i.e. the lens and the microchannel network along

with their respective access holes for external tubes connection, were

irradiated in the same fabrication step with pulse energies of 270 nJ for the

lens, 350 nJ for the access holes and 500 nJ for the microchannel. The

translation speed was 1 mm/s for the lens and 2 mm/s for all the other

structures.

Two different irradiation geometries are possible, longitudinal and

transverse, in which the sample is translated, respectively, along and

perpendicularly to the beam propagation direction. In the first case,

exploited for the lens irradiation, the channel develops parallel to the writing

beam allowing a reduction of the wall roughness to few nanometers 4,5. The

desired acylindrical shape was obtained by irradiating from the bottom to

the top of the substrate 50 section of the lens (with 6 μm separation in

depth), each having the optimized profile designed for spherical aberrations’

reduction.

The transverse irradiation geometry was used to fabricate the H-

microchannel with square cross-section. In particular a multi-scan

irradiation approach was exploited: contiguous straight lines (with a

separation of 2 m) were scanned, forming the lateral surface of a

rectangular cross-section cylinder. For each channel branch six coaxial

cylinders were irradiated (with dimensions of 40x30, 90x70,180x150,

280x240, 380x330, 480x400 m2), so as to obtain the desired channel with

a cross-section of 500 m after the etching step. Similarly, access holes

were obtained by irradiating seven coaxial circular helices with diameters

equally spaced from 80 to 560 m. The total irradiation time was

approximately 2 hours.

Light sheet profile characterization

The lens was designed to have a long focal length (0.7 mm) as well as a

long depth of focus (0.4 mm) so as to illuminate the whole microchannel

section in a uniform manner. Being the minimum achievable waist limited

by the long depth of focus, we designed a waist of 5.5 μm, by considering

the lens filled with a high refractive index oil, n = 1.56 (Fig. S2).

In order to evaluate the light sheet profile we fabricated a separate device

with the lens facing a 800 μm wide and 35 μm height reservoir and we

filled it with a Rhodamine solution (Fig. S3a). The reservoir was centered at

the expected focusing position.

The light sheet profile was observed by capturing the Rhodamine

fluorescence signal with a CCD mounted on an standard inverted

microscope (DMI 3000M, Leica), using a 10x, 0.25 NA microscope

objective. The acquired fluorescence images were subsequently analyzed

(Matlab) to retrieve the beam radius along the entire channel and to

determine the waist of the beam and its position.

The routine automatically analyze the acquired fluorescence images first by

sectioning them along the beam propagation direction and then by fitting

the fluorescence signal of each section with a gaussian intensity profile to

find out the correspondent waist (Fig. S3b). A correction in the Gaussian

model has been inserted so as to take into account the effect of the depth of

field of the microscope objective used to acquire the fluorescence image.

The optimized lens shows a minimum waist close to the theoretical one,

equal to 5.8 μm. This result confirms that designed acylindrical profile

allows reducing the impact of spherical aberration so as to obtain a beam

waist size close to the theoretical one.

Pumping scheme

Pressure driven pumps (Fluigent, MCFS Flex) were used to inject and

control the sample and the buffer flow in the device. The H-shaped (Fig.

S1) geometry of the channel was chosen so as to exploit the laminarity of

fluids in microfluidic channels. Indeed thanks to this property the streams of

the two channels, sample and buffer, won’t mix when merging into the

central common branch and the fluids entering at sample or buffer input will

exit at sample and buffer output respectively. The velocity of the flow is

controlled by unbalancing the pressure at the input and output inlets

separately for each stream. The buffer stream was exploited to prevent

spheroids touching the channel wall. Indeed the interface between the two

streams can be moved by accordingly balancing the sample and the channel

driving pressures. This allows one to make the spheroids flowing

orthogonally with respect to the light sheet without touching the channel

wall that might induce a rotation of the sample

Spheroids

H2B-mCherry expressing tumor spheroids were prepared as described in

the references 6,7. Fixed samples were passed in dilutions of 2,2’-

thiodiethanol (TDE, Sigma) and PBS in a stepwise manner (25%, 50%,

68%, for 10 min), to finally match the index of refraction of fused silica

(n=1.46).The liquid used in the fluidic channels was a solution of water and

TDE, to which we added 0.1% (final concentration) low melting point

agarose (LMA Sigma) in order to slightly increase the liquid viscosity and

Electronic Supplementary Material (ESI) for Lab on a Chip.This journal is © The Royal Society of Chemistry 2016

consequently to better control the sample movement: this being particularly

important when the sample was slowly scanned through the light sheet.

Correction for light sheet in-homogeneities

During imaging we observed that the sample was illuminated by an in-

homogeneous excitation intensity. This was primarily due to the non-perfect

alignment of the optical fiber which was manually glued to the chip. In

order to correct non-homogeneous illumination, before or after the

measurement session, the channel was filled with a nanobeads solution,

which was circulated at high speed through the light sheet. This creates an

image on the sensor that mapped the intensity distribution of the light sheet

on the entire field of view (reference image).

All the acquired images were then divided by the reference image in order

to correct in-homogeneous illumination (Fig. S4). The correction was less

effective in high-throughput measurements, where the lower signal to noise

ratio of the data resulted in an amplification of the noise in the upper part of

the image (visible in Fig. 3).

Nuclei Segmentation and visualization

Nuclei segmentation was obtained using grayscale morphological image

processing operations 8, implemented in Matlab.

Sample sections, Maximum Intensity Projections and 3D volumes were

visualized using Fiji 9.

Nuclei segmentation

Nuclei segmentation was obtained using well known grayscale

morphological image processing operations8, implemented in Matlab. In

details we performed:

- volume erosion by a sphere with diameter of approximately half the size

of the nuclei (5 µm), followed by filtering by reconstruction (opening by

reconstruction);

- volume dilation by a sphere with 5 µm diameter, followed by filtering by

reconstruction (closing by reconstruction);

- volume opening by a 2 µm diameter sphere (consisting in image erosion

followed by dilation);

- calculation of the regional maxima of the volume;

- detection of the connected components of the final binary volume, which

correspond to the segmented nuclei.

The procedure was applied to the entire volume (c.a. 1000x1000x200

voxels), requiring c.a. 5 min/volume on an Intel I7-4770K with 32GB of

RAM memory. The procedure was also performed plane by plane,

requiring c.a. 300 ms/plane. For high-throughput acquisitions the

segmentation was applied only to a single plane of the acquired stack

corresponding to a depth of c.a. 70 µm within the sample (Fig. 3 and Fig.

S8). The Matlab code for the segmentation of the nuclei is available on

request.

Spheroid volume quantification

Segmentation of the spheroids and measurement of their volume was

performed by applying a series of binary morphological operations to the

acquired SPIM stacks, using Matlab. The acquired grayscale images were

first converted to a binary image by setting a proper threshold level. Then

morphological opening (image dilation followed by erosion) and filling

(consisting in the removal of regional minima that are not connected to the

image border) were performed, in order to segment the area of the spheroid

(green in Fig. S9) within the image. The batch of samples acquired in high-

throughput mode presented a large central empty region. This region was

obtained as the difference between the image processed by opening and

filling, and the same image processed only with the opening operation (red

in Fig. S9).

The time required for segmentation of a single plane was c.a. 30ms,

compatible with real time processing during high-throughput acquisition.

The procedure was repeated in all planes to measure the entire volume of

the spheroid and the volume of the empty region. The Matlab code for

segmentation and quantification is available on request.

FIGURES

Fig. S1 Schematic of the fluidic channel. (a) Schematic of the H-shaped microfluidic channel, with the microscope objective above the chip. The sample is moved by the flux through the fluidic network. (b) Two-dimensional scheme of the H- shaped fluidic network. The movement of the sample is carefully controlled by four tubes each of which provides a selected pressure to the liquid. The geometry has been chosen to avoid the spheroid hitting a wall due to its inertia after the 90° turn. The sample is observed from above with a microscope objective; due to the symmetry of the chip, observation from below (not shown) or double sided detection are also possible. Figures are not in scale.

Fig. S2 Schematic diagram of the procedure used to optimize the lens shape. The first interface of the lens is designed to be perpendicular to each ray composing the beam so that the beam propagation is not modified. The second interface is designed to focus all the beam radii in the same position. Each coordinate of the aspherical profile is retrieved by geometrical considerations on the known angles. The design parameters are summarized in the table on the right hand side. The desired focal length of the lens is indicated with f, the waist is w0 the and the confocal parameter is 2z0

a b

Fig. S3 Lens characterization. (a) Schematic of the chip used to characterize the focusing properties of the lens. The light emitted by the fiber creates a light sheet in a reservoir filled with Rhodamine. The chip is imaged under an inverted microscope to capture the light sheet

profile, in direction perpendicular to it. (b) Image of the light sheet profile obtained with the acylindrical lens in the Rhodamine-filled reservoir. (c) Results of the waist retrieval. The fluorescence image is sectioned along the beam propagation direction (x axis) and the intensity of each section is fitted with a gaussian profile (red) that allows determining the correspondent waist w0. A minimum value of 5.8 µm is obtained.

a

b

c

Fig. S4. Correction for light sheet in-homogeneities. Results of the correction for non-uniform illumination (see also Online Methods for details). Section of a H2B-mCherry expressing spheroid, acquired at z=100 µm in depth, before (a) and after correction (b). Maximum Intensity Projection (MIP) of the spheroid, before (c) and after correction (d). Scale bar, 100µm.

a b

c d

Original Corrected

xy xy

MIP MIP

Original Corrected

Fig. S5. H2B-mCherry expressing spheroid, acquired at various depths, shown with 14 µm step. Arrows indicate three nuclei undergoing mitosis. Scale bar, 100µm.

z=30 µm z=44 µm z=58 µm z=72 µm

z=86 µm z=100µm z=114µm z=128 µm

z=142 µm z=156µm z=170µm z=184 µm

z=198 µm z=212µm z=226 µm z=240µm

xy

Fig. S6. Segmented nuclei of a 250µm diameter H2B-mCherry expressing spheroid; (a) xy sections of the spheroid, acquired at various z-depths (left to right), shown with 45 µm steps; (b) xy sections with superimposed colored segmented nuclei; (c) xz section of the spheroids at various height (left to right), shown with 45 µm step; (d) xz sections with superimposed colored segmented nuclei. Each color corresponds to a different structure. Scale bar, 100µm.

a

b

c

d

xy

xz

xy

xz

Fig. S7. Details of nuclei undergoing mitosis. (a) Nuclei at the depths z=30um (b) z=128 µm. (c) z=212 µm. Nuclei can be observed in detail along different orientations. Yellow lines indicate the position of the transverse (xy) sagittal (zy) and coronal (xz) sections. Scale bar, 10µm.

xy xy xy zy zy zy

xz xz xz

a b c

Fig. S8. Nuclei count in high-throughput acquisitions. (a) Single section of a H2B-mCherry expressing spheroid (sample #1 in Fig.3) at z=70 µm in depth, acquired in high-throughput mode. Scale bar, 100µm. (b) Image in a with overlapped segmented nuclei (c) Statistical analysis of the fluorescence intensity in n=25 spheroids: histogram of the fluorescence intensity showing different intensity ranges (intensity 1 corresponds to the maximum signal detected in all samples) and the number of nuclei whose fluorescence intensity fits in each range. The nuclei count in each range is the sum over the n=25 samples. Standard deviation, calculated as the deviation in nuclei count within the n=25 samples, is shown for each range of the histogram.

Intensity (a.u.)

Nu

clei

#

a b

c

xy xy

High-throughput High-throughput

Fig. S9. Quantification of the volume of spheroids in high-throughput measurements. The acquired samples presented a central empty region whose volume was also quantified. (a) Single section of a 300µm diameter H2B-mCherry expressing spheroid acquired, at z=150 µm in depth, in high throughput mode. The green and red curves indicate the borders of the spheroid and its central empty region respectively. (b) Maximum Intensity Projection (MIP) of a stack of 200 planes, highlighting the entire volume of the spheroid (green) and empty region (red). Scale bar, 100µm. (c) Statistical analysis of n=25 spheroids: histogram of the spheroids’ volume (green) and histogram of the volume of the internal empty regions (red) showing different volume ranges and the percentage of samples whose volume fit in each range.

xy MIP

a b

Volume (x10-3

mm3)

Per

cen

tage

of

sam

ple

s (%

)

c

Spheroid volume Empty volume

High-throughput High-throughput

MEDIA CAPTION

Movie 1 legend

Side view of sample flow in microchannel, to observe the effect of the 90°

curvature in two microchannel with different shape. Sample rotation

induced in the ‘C’ shaped channel is avoided with the introduction of

auxiliary channels in an ‘H’ shaped configuration.

Movie 2 legend.

Optical sectioning of a cellular spheroid.

Real time acquisition of a H2B-mCherry expressing spheroid, slowly

moving through the light sheet (20µm/s). The images are captured at 12 Hz

and corrected for illumination in-homogeneities as described in the

Methods. Scale bar, 100µm

Movie 3 legend.

3D reconstruction of segmented nuclei.

Three dimensional reconstruction of a H2B-mCherry expressing spheroid.

The sample is virtually rotating around itself and after one rotation the

image of the 3D reconstruction of the segmented nuclei (red) is overlapped

to the original sample.

Movie 4 legend

High-throughput acquisition.

Real time acquisition of a H2B-mCherry expressing spheroid, quickly

moving through the light sheet (c.a. 150µm/s). Acquisition starts when the

mean fluorescence signal calculated over the entire image overcome a

certain threshold and continues until the signal decreases below the same

threshold. The acquisition continues for c.a. 2s depending on the sample

size. For visualization, a red circle appears at the frames corresponding to

the acquisition start. Black frames (90 frames) are concatenated to the

acquired stack after each acquisition to visualize the samples separately. No

image correction is applied to the data for illumination in-homogeneities.

Scale bar, 100µm.

† These Authors had equal contribution to this paper.

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