Microfluidics to follow spatio-temporal dynamics at the nucleo-cytoplasmic

interface during plant root growth

Gilles Dupouy1, Gaurav Singh1,2, Leona Marlene Schmidt-Speicher3, Elise Hoffmann1,

Stéphanie Baudrey4, Ralf Ahrens3, Andreas E. Guber3, Michael Ryckelynck4, Etienne
Herzog1, Marie-Edith Chabouté1*, Alexandre Berr1*

1
Institut de Biologie Moléculaire des Plantes (IBMP), CNRS, Université de Strasbourg,
Strasbourg, France
2
Université Aix Marseille, CEA, CNRS, BIAM, UMR7265, 13108, Saint-Paul-lez-
Durance, France
3
Institute of Microstructure Technology (IMT), Karlsruhe Institute of Technology (KIT),
Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen, Germany
4
Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002,
Strasbourg, 67000 France

*: Corresponding authors:
marie-edith.chaboute@ibmp-cnrs.unistra.fr
alexandre.berr@ibmp-cnrs.unistra.fr

Running head: Arabidopsis root microfluidic device

Key Words: microfluidic, confocal microscopy, live cell imaging, nucleus,

cytoskeleton, plant root, Arabidopsis thaliana.

1
Abstract

Nuclear dynamics refers to global/local changes in the molecular and spatial

organization of genomic DNA that can occur during development or in response to

environmental stress signals and eventually impact genomic functions. In plants,

nuclear dynamics relies notably on the connection of the nucleus with the cytoskeleton

during development. It orchestrates genomic functions in response to developmental

and environmental cues. This is particularly true in the plant root system, which is

constantly exposed to a wide range of internal and external stimuli. Currently, studying

nuclear dynamics in a growing root is challenging due to limitations regarding real-time

imaging for quantitative analyses under controlled conditions. Microfluidic systems for

plant cell studies are valuable analytical tools that provide a precise control of culture

conditions together with live-imaging capabilities at high temporal and spatial

resolutions. Herein, we describe a microfluidic platform to unravel dynamically and

non-invasively nuclear organization in the seedling root system exposed to various

treatments. As exemplified here, our microfluidic platform can be conveniently used for

real-time microscopy imaging and quantitative analysis of fine nuclear morphological

changes upon modifying cytoskeleton dynamics. Importantly, our system can be

applied to a wide variety of microscopic means including high-resolution microscopy

to investigate diverse sub-cellular compartment or nuclear domains in Arabidopsis

thaliana roots.

2
1. Introduction

The nucleus is a complex and sophisticated organelle where major cellular processes,

including DNA protection, compaction, repair, replication and transcription, take place.

The nuclear content is isolated from other cell compartments by the nuclear envelope,

which acts as a key nexus between the cytoskeleton and the nuclear interior. As a

central hub, the nuclear envelope serves for signal sensing, transduction, molecule

trafficking through nuclear pores and intra-nuclear attachments of particular chromatin

domains (for review see [1][2]). Underneath the nuclear envelope, the nucleoskeleton

appears as a dynamic nuclear network, bridged to the cytoskeleton via the linker of

nucleoskeleton and cytoskeleton (LINC) complex, that ensures the overall structural

integrity of the nucleus and provides support for nuclear functions. The nucleus is

organized in functionally-defined subregions such as the nucleolus, where rRNAs are

transcribed and processed, chromosome territories corresponding to regions

preferentially occupied by particular chromosomes, heterochromatin corresponding to

highly condensed chromatin regions (e.g., chromocenters in Arabidopsis thaliana)

versus euchromatin corresponding to less-dense ones enriched in active genes, as

well as higher-order chromatin foldings [3]. In addition, the nucleus is also highly

dynamic. This is illustrated during each cell cycle with the assembly/disassembly of the

nuclear envelope, nuclear movements during the day/night cycles, changes in nuclear

shape and its mechanical properties, chromosome folding and positioning in the

nuclear space, as well as dynamics in the chromatin organization during development

[4] or in response to environmental stimuli [5][6].

Arabidopsis thaliana was widely used as a model species to track nuclear and

chromatin dynamics. In normal conditions, Arabidopsis interphase nuclei harbor

conspicuous heterochromatic regions grouped at chromocenters next to the nuclear

3
envelope, and telomeres often associated with the nucleolus [7][8]. Interestingly, the

nuclear organization can dynamically undergo drastic/subtle macro/micro-changes in

response to a wide range of external and internal signals, which are used to regulate

various developmental and stress responses. Indeed, dynamic changes in the higher-

order nuclear organization were for example reported depending on light availability

[9], mechanical stress [10], heat stress [11], or even osmotic and salt stresses [12][13].

From these few examples, the nucleus was proposed to act as a rheostat to integrate

stress signals from outside to inside and modulate transcriptional control and

chromatin biology, as well as epigenetic processes [14][15].

Yet, these data were mainly gained from fixed material at single time-point and should

therefore not depict a complete and comprehensive view of the nuclear dynamics from

its early to late response towards external stimuli, as well as its recovery upon return

to normal conditions. In this context, one of the challenges was to establish systems

for live-cell imaging allowing rapid and reversible treatments while ensuring the long-

term integrity of the living material. Towards this end, microfluidic devices were

developed [16-23] by gathering around a common interest complementary expertise

of biologists, physicists, engineers and mathematicians in order to acquire and

quantitatively analyze images and finally establish biological principles [24]. Here we

report a method to setup a microfluidic system that enables dynamic live-cell imaging

of fluorescent markers at the nucleo-cytoplasmic interface during Arabidopsis thaliana

primary root growth at a level previously inaccessible. Interestingly, the proposed

system allows to easily and rapidly apply any treatments that can be added in solution

(biotic/abiotic stresses) via passive pumping and proper washout conducted in parallel

with long-term (i.e., up to 36h) high-resolution in vivo imaging. For example, drugs

affecting the integrity of key cytoskeleton components (for an exhaustive list see [25])

4
can be easily applied as exemplified in this protocol. Drugs known as “epidrugs” able

to target chromatin-related enzymes (for a review see [26]) can also be used to

address the impact of chromatin changes on nuclear dynamics or on the cytoskeleton

network. Finally, the microfluidic system is also suitable to apply chemical agents such

as polyethylene glycol (PEG), mannitol, and salt (NaCl) to simulate osmotic stress. An

interesting outcome from this system is that the very first instants of a treatment can

be imaged. Another advantage of the system is that it is suitable to grow and image

seedlings at high temporal and spatial resolutions for up to 36-48 h. Moreover, the

system can be later recovered by simply replacing the feeding medium again with the

original one. Finally, as a demonstration, we show how our microfluidic system can be

applied to follow morphological changes of Arabidopsis nuclei in the root meristem

when altering microtubule dynamics.

2. Materials

2.1 Master mold preparation

1. Computer equipped with a Computer-Aided Design (CAD) software

2. Mold material for micro-milling

3. CAM software for the generation of the milling machine G-code

4. Micro-milling machine equipped with milling tools (e.g., circular shaft mills with

typically several blades evenly distributed around the circumference)

5. Cutting fluid (e.g., isopropyl alcohol, water)

6. Binocular microscope

7. Photomask

8. Silicon wafer

9. Acetone

5
 10. Isopropyl alcohol

11. Deionized water

12. Compressed N2

13. Plasma cleaner

14. Hot plate

15. SU-8 photoresist

16. Spin coater

17. Mask aligner equipped with a UV light source

18. SU-8 developer

2.2 Polydimethylsiloxane (PDMS) molding and microfluidic device primary

assembly

1. Sterile polystyrene petri dishes (93 diameter x 21mm height)

2. Disposable fork and beaker

3. Vacuum pump

4. Beaker with three dripless pouring spouts (e.g., Tripour Beaker, Haslab, or

similar)

5. Sterile carbon steel surgical blades

6. Protective glasses

7. Adhesive tape

8. Nitrile gloves

9. Drill equipped with a 1 mm diameter drill bit

10. Cleaning wipes

11. Cover glass 22 x 32 mm

12. Compressed N2

6
 13. Plasma etcher

14. 100 W oxygen-plasma

15. Cylindrical tool used as a rolling pin

16. Oven

17. Syringe electric pump

18. Polytetrafluoroethylene (PTFE) microtubing (0.56 inner x 1.07 mm outer

diameter)

19. Electric syringe pump

20. Isopropanol

21. Acetone

22. Deionized water

23. Polydimethylsiloxane (PDMS) elastomer base and curing agent

2.3 Seedling preliminary growth inside tips

1. Laminar flow hood

2. Electric syringe pump system that can receive up to two syringes

3. Sterile polystyrene petri dishes (93 diameter x 21mm height)

4. Beakers

5. Multichannel micropipette 8-ch 1-10 µL

6. Pipette tips 0.1 – 20 µL

7. Sterile carbon steel surgical blades

8. Extra thin tips straight tweezer

9. Fine straight tweezer

10. Sterilized toothpicks

11. Micropore surgical tape

7
 12. Cleaning wipes

13. Nitrile gloves

14. Oven (65°C)

15. pH meter

16. Safety laboratory gas burner with propane gas connection

17. In vitro growth chamber

18. Ultra-pure sterile water

19. Liquid ½ MS medium: 2.2 g/l Murashige and Skoog basal medium, 0.5%

Sucrose, pH 5.7

20. Solid ½ MS medium: 2.2 g/l Murashige and Skoog basal medium 0.5%

Sucrose, 1% Agar, pH 5.7

21. 70% Ethanol

2.4 Assembly of the microfluidic system

1. Laminar flow hood

2. Sterile polystyrene petri dishes (93 diameter x 21mm height)

3. Vacuum chamber

4. Lab marker

5. Hypodermic needles 0.7 mm diameter x 38 mm length

6. Reusable biopsy punch 1 and 7 mm

7. Sterile carbon steel surgical blades

8. Polytetrafluoroethylene (PTFE) microtubing (0.56 inner x 1.07 mm outer

diameter)

9. Fine straight tweezer

10. Microcentrifuge tube 0.65 mL with snap cap

8
 11. Paper towel

12. Sterile water

13. Syringes 50 mL

14. Electric syringe pump system that can receive up to two syringes

15. Liquid ½ MS medium: 2.2 g/l Murashige and Skoog basal medium, 0.5%

Sucrose, pH 5.7

16. Extra thin tips straight tweezer

17. Fine straight tweezer

18. Micropore surgical tape

19. Nitrile gloves

20. Oven (65°C)

21. Safety laboratory gas burner with propane gas connection

22. Binocular microscope

23. pH meter

24. Cleaning wipes

25. 70% ethanol

2.5 Loading the microfluidic device with seedlings grown inside tips

1. Laminar flow hood

2. Binocular microscope or a magnifying glass

3. Fine straight tweezer

4. Micropore surgical tape

5. In vitro growth chamber

6. Electric syringe pump system that can receive up to two syringes

7. Cleaning wipes

9
 8. Nitrile gloves

9. Oven (65°C)

2.6 Microfluidic real-time imaging

1. Confocal laser scanning microscope

2. Liquid ½ MS medium: 2.2 g/l Murashige and Skoog basal medium, 0.5%

Sucrose, pH 5.7

3. Electric syringe pump system that can receive up to two syringes

3 Methods

3.1 Master mold preparation

This section describes two distinct procedures classically used to prepare molds that
will precisely mirror the microfluidic device. Preparing molds by micro-milling involves
the use of a high-speed micrometer milling tool to precisely cut patterns into a mold
material, in our case a synthetic polymer named Polymethylmethacrylate (PMMA) or
acrylic glass (see Note 1). Preparing molds by photolithography is based on the use
of a photoresist material, in our case SU-8 (see Note 2) that is patterned by exposure
to UV light through a mask. Both methods can produce high-quality molds with
extremely high resolution, high repeatability and relatively easy customization of molds
to fit specific design requirements. Unfortunately, both methods are complex and
require specialized/expensive equipment such as a clean room, as well as skilled
operators to perform effectively. Regarding technical aspects, photolithography can
produce molds with higher resolution than micro-milling with feature sizes as small as
a few hundred nanometers (compared to a few micrometers for micro-milling). Finally,
micro-milling is a highly versatile method since it can be applied to a wide range of
materials, including metals, plastics, and ceramics, while photolithography is limited to
photoresist materials. In the example provided here, the mold produced by micro-
milling contains three cultivation channels, each with a quadratic cross-section of 200

10
µm x 200 µm (Fig. 1a and c). In addition, two 0.8 mm diameter pillars are added per
channel at the bottom of the mold for both nutritional fluid in- and outlets. The seedling
inlet is milled in the chip after PDMS solidification. A single mold can be reused multiple
times and multiple different molds can be designed, for instance with a zig-zag
structure designed directly ahead of the cultivation channels to ensure the proper
mixing of several liquid media injected simultaneously (Fig. 1b). Exact parameters and
conditions for both micro-milling and photolithography may vary depending on the
specific materials and equipment used, as well as the desired pattern and resolution.
The entire procedure for both methods require from one to several hours depending
on the complexity of the mold design.

1. Design the 3D mold pattern using a Computer-Aided Design (CAD) software. The
pattern should include all features required for the microfluidic device, such as
channels or reservoirs at the microscale level. This step is common to both methods.

2. Mold preparation

2a. For micro-milling, generate a list of G-code commands (i.e., the programming
language specific for the milling machine) corresponding to the design using a
Computer-Aided Manufacturing (CAM) software compatible with the CAD software.
The G-code should specify the tool path, cutting speed, and cutting depth.

3a. Mount the mold material, in our case PMMA, onto the work table of the milling
machine using clamps. Load the milling tool with a diameter of 50 µm into the spindle
of the milling machine and set cutting parameters such as cutting speed, depth and
feed rate (see Note 3).

4a. Apply cutting fluid to the cutting area to lubricate and cool both the mold and the
tool during the milling process. Then, start the milling process and monitor the progress
to ensure that the mold features are being accurately machined.

11
5a. Once the milling process is complete, remove the mold from the machine and clean
it with a soft brush and compressed air to remove any debris or residue. Inspect the
mold under a binocular microscope to ensure that all features are properly machined
and that there are no defects or inaccuracies.

2b. The procedure described below provides a general guide for performing
photolithography using a single photomask (see Note 4). Prior photolithography,
photomasks printing should be performed (e.g., in our case one mask for the three
parallel main channels was sufficient) at high resolution (50,800 dpi) in negative mirror
and usually by a specialized company (e.g., Selba S.A.).

3b. Clean a 3 inches silicon wafer by immersing it successively in acetone, isopropyl

alcohol and deionized water for 5 minutes each. After removal from water, dry the
wafer using compressed N2.

4b. Activate the wafer in a plasma cleaner (40 kHz, 100 Watts) for 30 seconds with 0.5
mbar of oxygen at 25% power and pre-bake it for 3 minutes at 95°C on a hot plate to
evaporate any remaining solvent.

5b. Dispense SU-8 2000.5 photoresist on the center of the activated wafer for a 0.5
µm layer (i.e., measure circa. 10g, see Note 5) and evenly distribute the photoresist
on the wafer surface using a spin coater following manufacturer instructions, first at
500 rpm for 5 seconds, then at 3,000 rpm for 30 seconds (see Note 6).

6b. Expose the wafer to UV light for 20 seconds (i.e., corresponding to an energy
exposure of 80 mJ/cm2) using a mask aligner and post-bake it for 3 min at 95°C to
further harden the SU-8 photoresist.

7b. Generate a second layer of 100 µm on the first 0.5 µm one by adding two 50 µm
layers of SU-8 2025. Pre-bake the wafer for 15 minutes at 95°C on a hot plate. Spread
the first 50 µm layer of the photoresist using a spin coater, first at 500 rpm for 5

12
seconds, then at 1,700 rpm for 30 seconds and expose it to UV light for 95 seconds
(i.e., corresponding to an energy exposure of 380 mJ/cm2) using the mask aligner with
the photomask on the top of the resist. Finally, post-bake the wafer for 10 min at 95°C.

8b. Remove unbound photoresist by placing the wafer on the spin coater, covering it
for 3 min with SU-8 Developer and spinning it 25 seconds at 2,500 rpm. Repeat the
development step 3 times and finally wash the wafer with isopropyl alcohol followed
by 25 seconds of spinning at 2,500 rpm still in the spin coater.

9b. Repeat the same procedure to add the second 50 µm layer with a pre-bake of 10

min and finally remove cracks of the photoresist by a hard bake for 10 minutes at

200°C. At this step, the design of the channels should be clearly visible (see Note 7).

3.2 Polydimethylsiloxane (PDMS) molding and microfluidic device primary

assembly

This section describes the PDMS-casting process used to manufacture microfluidic

devices (Fig. 1d and e). A mold precisely mirroring the microfluidic device design is

preliminary produced by either micro-milling or photolithography as described above.

The PDMS casting process requires about two and a half hours, including the PDMS

polymerization time.

1. Thoroughly mix the PDMS elastomer base with the curing agent (i.e., the

catalytic agent which when added to resin causes polymerization) in a 1/10 ratio

until obtaining a homogeneous mixture. Because of the very strong adhesive

properties of PDMS, the use of disposable fork and beaker for mixing is

preferable.

2. Pour the PDMS mixture into a mold beforehand placed into a Petri dish until

covering up the above-mentioned pillars with a 5 mm PDMS layer. In addition,

13
 pour a 5 mm layer of PDMS in a second Petri dish that will serve to later

assemble the bubble trap system (see step 3 in section 3.4).

3. Degas the PDMS mixture by placing the mold under vacuum for 20 minutes.

The removal of air bubbles trapped in the PDMS mixture will prevent the

formation of unwanted cavities upon casting in the final microfluidic device.

4. Cure the PDMS mixture in the mold in an oven at 65 °C for one hour.

5. Let the cast PDMS cool down to room temperature until solidification. Then,

very carefully demold PDMS microfluidic device using a clean scalpel blade

while avoiding to damage both the mold and the microfluidic device. Small

pieces of adhesive tape can be used to remove PDMS debris and dust. Wear

protective glasses during the demolding procedure to prevent any injuries due

to eventual flying sparkles and wear nitrile gloves to avoid leaving fingerprints

on the PDMS surface.

6. Drill straight through the PDMS microfluidic device at locations coincident with

the above-mentioned pillars using a drill equipped with a 1 mm diameter drill bit

to open the nutritional fluid in- and outlets. Do the same for the seedling inlets

by applying a 45° angle. Such angle will later facilitate the root seedling entering

into the cultivation/observation channel (Fig. 1d and e).

7. Before final assembling, clean the PDMS microfluidic device from any drilling-

residue by consecutively dipping it in an isopropanol followed by a deionized

water bath for 5 minutes each. Do not use solvents such as acetone as PDMS

swells in it.

8. Meanwhile, clean a 22 x 32 mm glass cover that will serve as microfluidic device

background in acetone, followed by isopropanol and deionized water for 5

minutes each.

14
 9. Carefully dry all parts using compressed N2.

10. Place the PDMS microfluidic device and the glass cover with their bonding

surfaces facing up inside a plasma etcher and activate bonding surfaces with a

100 W oxygen-plasma for 7 seconds (see Note 8).

11. Open the etching chamber and immediately assemble the glass cover on the

PDMS microfluidic device by carefully applying pressure on the glass cover

surface using a cylindrical tool as a rolling pin.

12. Place the microfluidic device in an oven at 65 °C for 15 minutes to finalize the

assembly.

13. Test the microfluidic device for blockages and leakages by connecting it to a

syringe electric pump and pumping isopropanol at a flow rate of 500 µL / min

through the different channels (see Note 9).

14. Finally, dry the functional microfluidic devices with compressed N2 and package

them. Be aware that PDMS-based microfluidic devices cannot be sterilized via

ultraviolet-radiation or in an autoclave (see Note 10). Therefore, use 70%

ethanol for sterilization. The durability of the bond between the PDMS and the

glass cover is stable for several months but not indefinitely.

3.3 Seedling preliminary growth inside tips

This section describes the procedure for germination and early cultivation of plantlets

inside ½ MS agar medium filled pipette tips, a preliminary step necessary before

loading the microfluidic device (Fig. 2a-d). The entire procedure requires about an

hour and should be carried out under sterile conditions in a laminar flow hood. Be

aware that the microfluidic device described here was configured to allow the growth

of a total of three plantlets in parallel per device. The model of electric syringe pump

15
listed in the “Material” section can receive up to two syringes, for example to supply

two distinct microfluidic devices.

1. For each fluorescent Arabidopsis tagged line (see Note 11), sterilize

approximately 30 seeds using your standard seed sterilization protocol. While

the seeds are air-drying, prepare tips as follows.

2. Pour approximately 25 mL of melted ½ MS agar medium into a sterile tall Petri

dish (i.e., one Petri dish per fluorescent Arabidopsis tagged line).

3. Prior solidification, draw 4 µL of the melted medium inside 10 µL pipette tips

using a pipette (i.e., this procedure can be speeded up using a multichannel

pipette as illustrated in Fig. 2a).

4. Wait 20 seconds for the ½ MS agar medium to solidify inside tips and finally

eject filled pipette tips aside in a second sterile and empty Petri dish. Prepare a

total of approximately 30 pipette tips for each fluorescent Arabidopsis tagged

line analyzed.

5. When the required number of pipette tips is prepared, cut each tip just below

the top level of the solidified ½ MS agar medium using a sterile scalpel to obtain

a flat surface (Fig. 2b; see Note 12).

6. Plant cut tips inside the ½ MS agar medium filled Petri dish (see step 3 in section

3.3) using a fine straight tweezer in order to maintain them in a vertical position.

The use of a tall Petri dish is recommended here to avoid any contact between

cut tips and the Petri dish lid (Fig. 2c).

7. At this stage, sterilized seeds must be dried. Drop one seed per cut tip on the

top of the agar surface using a tweezer or a sterilized toothpick. When all cut

tips are loaded, close the Petri dish and seal it with micropore surgical tape.

16
 8. For stratification, place Petri dishes containing cut tips loaded with seeds at 4°C

for at least two days in the dark.

9. After stratification, transfer Petri dishes to a long day (16 h light, 8 h night, 21°C)

in vitro growth chamber for 4-5 days until root tips reach the edge of cut tips

(Fig. 2d).

3.4 Assembly of the microfluidic system

This section describes the assembly procedure allowing to connect the microfluidic

device to the electric syringe pump (Fig. 2e-j). The entire procedure should be carried

out under sterile conditions in a laminar flow hood and requires about one and a half

hours.

1. Sterilize the microfluidic device by immersing it in 70% ethanol in a Petri dish

and by placing the device in a vacuum chamber for at least 5 minutes. Besides

sterilizing the device, this 70% ethanol bath will also later prevent the formation

of air bubbles in microchannels.

2. While the sterilized microfluidic device dries, continue by preparing the Petri

dish that will serve as a container for the microfluidic device to keep sterile and

humid conditions during the entire experimental process. Label with a marker

on the lid of a Petri dish the positions of the in- and outlet tubing of the

microfluidic device and bore openings at these positions using a red-hot

hypodermic needle while ensuring that the diameter of the holes is about the

outer diameter of the PTFE microtubing (i.e., slightly higher than 1.07 mm; Fig.

2e).

3. Continue by preparing the bubble trap system. Slice a PDMS cylinder from the

Petri dish prepared at step 3.1.2 using a 7 mm biopsy punch (i.e., corresponding

17
 to the internal diameter of a 0.65 mL microcentrifuge tube) and drill 4 holes in it

using a 1 mm biopsy punch (Fig. 2f).

4. Using a sterile surgical blade, cut an adequate number of micro-tubing pieces

(i.e., in the microfluidic device presented here, 3 x 20 cm long for inlet tubing

and 3 x 10 cm long for outlet tubing).

5. Using the same sterile surgical blade, cut an additional microtubing that will

serve as a primary tubing connected at one of its ends to the syringe. Be aware

that the length of this tube may vary depending on the space available at your

acquisition tool (e.g., a 20 cm length primary tube for a LSM700 Laser Scanning

Zeiss Microscope or a 40 cm length for a Zeiss Axio Observer Z1 Spinning Disc

Confocal Microscope).

6. Using a tweezer, insert the three 20 cm inlet tubing and the primary tubing into

the holes of the PDMS cylinder prepared at step 3 section in 3.4 and let tubing

protruding from the PDMS at one side of the cylinder for about 0.5 cm long (Fig.

2f).

7. Insert the resulting PDMS cylinder in a 0.65 mL microcentrifuge tube like a cork

with the 0.5 cm long excess tubing at the inside of the microcentrifuge tube (Fig.

2f).

8. Using a tweezer, pass the other end of the in- and outlet tubing through the

holes drilled in the Petri dish lid prepared at step 2 in section 3.4 in order to

leave around 1 cm of microtubing length on the interior side of the Petri dish.

Still from the interior side of the Petri dish, tubing can be cut with a 45° angle to

help inserting them inside the PDMS-based microfluidic device (Fig. 2g).

18
 9. Assemble all parts by carefully pushing the in- and outlets tubing from the Petri

dish lid inside their respective in- and outlets holes on the PDMS microfluidic

device using a tweezer (Fig. 2h).

10. Dispose a piece of autoclaved paper towel at the periphery of the Petri dish

while leaving enough space at its center to later dispose of the microfluidic

device.

11. Add sterile water to the paper towel contained in the Petri dish to keep humidity

in the system and place the PDMS microfluidic device inside (Fig. 2i).

12. Fill a 50 mL syringe with ½ MS liquid medium, place a needle at its tip and

mount the whole on the electric syringe pump.

13. Using a tweezer, carefully push the other end of the primary tubing onto the

needle. Be careful not to damage the tubing with the needle tip.

14. Setup the liquid flow rate at 2,000 µL / minute and fill the entire microfluidic

system until liquid reaches outlet tubing ends.

15. Carefully control whether all tubing and channels are filled completely and all

air bubbles are removed. If necessary close the Petri dish with micropore

surgical tape and inspect the system under a binocular microscope.

16. Stop the electric syringe pump and continue with the final assembling step of

the microfluidic device still under sterile conditions.

3.5 Loading the microfluidic device with seedlings grown inside tips

This section describes the assembly of seedlings grown inside tips on the microfluidics

system. This final assembly requires 5-10 minutes and should be carried out under

sterile conditions in a laminar flow hood. Be careful not to let root tips grow beyond the

tip cone edge to prevent any damage or additional stress during this final assembly. A

19
regular control of seedling growth using a binocular microscope or a magnifying glass

is highly recommended. Usually, around 20% of the prepared cut tips loaded with a

seed can be used for following steps due to proper seedling growth.

1. Gently remove a single cut tip holding a properly grown seedling from the agar

plate (i.e., for better grip, avoid extra thin tweezers) and carefully insert it,

exerting a gentle pressure along a spiral path, inside one of the seedling inlet

holes. Be careful not to insert it too deep as this could break the underneath

cover glass and result in fluid leakage and contaminations.

2. Repeat this step as many times as necessary to fill each seedling inlet hole

available on the microfluidic device (i.e., three in the example shown in this

protocol).

3. Close the Petri dish containing the fully assembled microfluidic device and wrap

it with micropore surgical tape.

4. Transfer the system in an in vitro growth chamber and set the flow rate to 350

µL / min for 30 secondes while flicking the outlet tubing to remove any air bubble

inside the microchannels.

5. Position the syringe electric pump at the same height compared to the Petri dish

containing the fully assembled microfluidic in order to maintain the inlet tubing

as vertically as possible and tilt the Petri dish at an angle of approximately 45°

to facilitate the entrance of the root inside the observation channel (fig. 2j).

6. Finally, reduce the flow rate to 8 µL / minute and let seedlings grow (see Note

13).

3.6 Microfluidic real-time imaging

20
This section describes the real-time imaging procedure of root-grown inside the

microfluidic device. Be aware that the microfluidic system presented here was

optimized for an inverted microscopy platform and that the settings have to be adjusted

for other imaging acquisition systems.

1. When roots have reached at least 0.5 cm inside the cultivation/observation

channels (i.e., usually after 3-4 days of growth), the system can be used for

imaging.

2. Be careful to keep the microfluidic system plugged to its fluid supply during the

entire real-time imaging procedure.

3. Unwrap the Petri dish and open it in order to place the microfluidic device on

the microscope platter with the glass cover facing downward (see Note 14).

4. Start the image acquisition process.

5. To continuously follow the effect of drugs or chemicals (e.g., cytoskeletal or

epidrugs) on the nucleo-cytoplasmic interface dynamic, place an additional

syringe containing ½ MS medium with the drug (at the concentration of interest)

on the second slot of the electric syringe pump (see Note 15).

6. Move the needle connected to the primary tubing from the syringe containing

the control ½ MS medium to the other one containing the drug and remove the

old syringe.

7. Before initializing image scanning, temporally increase the flow to 350 µL /

minute for a 1 min period in order to entirely replace the control medium by

medium containing the drug. Afterwards, reset the flowrate back to 8 µL /

minute.

8. Restart the image acquisition process.

9. Real-time imaging settings

21
9a. using a confocal microscope LSM 700. Perform a Z-stack every minute at high

speed (e.g., circa 20 slices with a step size of 1 µm every minute for 20 minutes

(10 minutes growth in controlled conditions, 10 minutes in presence of the

medium-of-interest for the study) followed by 90 minutes with one Z-stack every

three minutes (recovery from the treatment). Set the excitation parameters and

signal amplification to obtain a good signal to noise ratio, using a pinhole of 1

Airy unit.

9b. using a confocal spinning disk. The scan speed can be much faster in

comparison to the above microscope while still keeping a high-resolution image.

This allows higher temporal resolution with images (circa 20 slices, 1 µm z-step)

every 2 seconds using a 40x magnification objective. Laser intensity has to be

adjusted depending on each system. Under optimal conditions, the signal is

stable over 5-10 minutes imaging time, for GFP and RFP reporter proteins. This

fast-scanning microscope is especially useful for live visualization of highly

dynamic structures such as organelles or cytoskeleton. However, note that

increasing temporal resolution increases the size of the image file. This is

especially important to consider for downstream image analysis.

10. Proceed to image analysis using ImageJ or Fiji and quantify parameters of

nuclear morphology and chromatin organization using dedicated macro tools

such as NucSeg (https://github.com/mutterer/NucSeg; [12]) or NucleusJ2.0

(https://gitlab.com/DesTristus/NucleusJ2.0; [27]). Further analyze chromatin

using ImageJ plugins such as Nuclear Object DetectionJ (NODeJ) to identify

and analyze high intensity domains such as heterochromatin domains from 3D

images (https://gitlab.com/axpoulet/image2danalysis; [28]). Image analysis

workflows were recently proposed to quantify different structures of plant nuclei

22
 imaged in 3D [29][30]. Note that quantitative analyses require careful

normalization of signal intensities and correction for signal loss due to

photobleaching, signal increase due to stress-induced autofluorescence, or

both. Further practical considerations for carrying out quantitative analyses of

chromatin organization in plant cells are reviewed by [29]. Similarly, quantitative

evaluation of cytoskeletal organizations (i.e., orientation, parallelism, bundling

and density) can be performed directly using ImageJ [31] or through plugins

such as FibrilTool (https://github.com/marionlouveaux/FibrilTool_Batch; [32]).

3.7 Representative experiment

The system described here enables us to dynamically follow the impact of any

treatment that can be added in solution on nucleo-cytoplasmic structures in

Arabidopsis roots. As an example, the behavior of fluorescent chromatin and nuclear

envelope markers was followed during treatment of Arabidopsis roots with low

concentration of oryzalin (200 nM) to alter microtubules dynamics as previously

described [33] (Fig. 3). As part of the cytoskeletal-nucleoskeletal bridging complexes,

SUN2-RFP localized to the nuclear envelope, while the tagged centromeric specific

histone 3 variant EYFP-CENH3 protein is seen at the heterochromatic chromocenters.

Using these markers, we can follow changes in the nuclear shape as well as changes

in CENH3 positioning. A similar approach can be used to dynamically investigate the

influences of different treatments and/or stimuli on nuclear parameters at other root

parts, including the root cap, elongation zone, mature zone and also root hairs. Finally,

the system can also be used to simply follow root growth.

4. Notes

23
1. As a versatile material, PMMA has a number of properties that make it an attractive
material. It has an excellent optical clarity as well as a high resistance to impact, UV
radiation and many chemicals including acids and alkalis.
2. SU-8 is a commonly used negative-tone epoxy-based photoresist material used in
microfabrication processes that has a thickness range of 5-200 μm and a high chemical
resistance and mechanical strength. Exposure to ambient light should be minimized
since SU-8 is sensitive to the UV contained in it, which can cause unintended exposure
and reduce the contrast of the pattern. Rather use a yellow or red safe light in the
lithography area and cover the photoresist-coated substrate when transferring it
between equipment.
3. Cutting parameters should be carefully selected to achieve the desired results since
they can affect the quality of the cut, the accuracy of the mold features, and the lifespan
of the cutting tool.
4. Typically, a photomask can be used in the range of 50 to 100 cycles. It is important
to verify the quality of the photomask by inspection or metrology before re-using it.
After photolithography and prior reuse, rinse the photomask with isopropyl alcohol to
ensure complete removal of any residual photoresist or contaminants. Finally, dry the
photomask with nitrogen gas.
5. The SU-8 resin being highly viscous, the easiest way to pour it onto the wafer is
directly from the bottle. Dispense a volume equivalent to 10g for uniform coating. A too
small volume can result to a highly uneven surface after spinning.
6. There are typically two spin coating steps during the photolithography process to
ensure a uniform and controlled thickness of the photoresist layer on the wafer surface
and minimize defects such as bubbles and edge bead. The first spin coating step is
referred to as the "prime" or "adhesion" layer while the second is referred to as the
"main" or "photoresist" layer. Spin coating steps are following manufacturer
instructions.
7. To facilitate the subsequent removal of cast PDMS, few droplets of
tridecafluoro1,1,2,2-tetrahydrooctyl-1-trichlorosilane can be added on the surface of
the mold.

24
8. Plasma etching is a dry-etching technique that combines the principles of physical
etching due to the impact of non-reactive ions on the material’s surface and the
principles of chemical etching due to chemical reactions between the etching gas and
the material surface. If the etch time is kept short, it can be used for activating surfaces
as well. Such a surface activation is done for bonding PDMS to glass (i.e., both having
similar surface-chemistry). Through activation by an O2-plasma etching process PDMS
forms Silanol-groups (Si-OH) to which glass often binds itself. When the activated
glass and PMDS are brought together e.g., by pressing them together, the Si-OH-
groups of glass and PDMS condense and form strong intermolecular bonds through
newly formed Si-O-Si-groups.
9. PDMS not sticking to the glass cover could result from glass cover and/or PDMS

surfaces are not clean enough. To avoid this, make sure that the coverslip has been

thoroughly cleaned with water and that both glass cover and PDMS surfaces have

been totally dried out using compressed air.

10. Be aware that PDMS-based microfluidic devices cannot be sterilized by

autoclaving as it can provoke swelling that may cause leakage rendering the chip

unusable.

11. Practically, all transgenic plants with translational fusion of a protein-of-interest with

a fluorescent tag can be used, provided that the fusion protein is expressed at least in

root tissues. Below is a list of possible Arabidopsis transgenic plants expressing fusion

proteins in different compartments.

1. Nuclear envelope markers: such as pSUN1::SUN1-GFP [34];

pSUN2::SUN2-RFP [35]; p35S::GFP-SINE1-5 [36]; or GFP-Tagged

Nucleoporins [37].

2. Nuclear periphery markers: such as the nucleoskeleton pKAKU4::KAKU4-

tRFP or -EYFP [38]; pCRWN1::CRWN1-EYFP, pCRWN2::CRWN2-EYFP and

pCRWN3::CRWN3-sGFP [39] and pGIP1:AtGIP1-GFP [40].

25
 3. Nucleoplasm makers: such as tagged histones for the H3 isoforms [41]; the

linker H1 variants [42]; or the heterochromatin associated p35S::EYFP-

AtCENH3 [43] and pH2A.W.6::H2A.W.6-RFP [44].

4. Cytoskeleton markers: such as p35S::GFP–FABD2 [45] and p35S::GFP-

Lifeact [46] for actin filaments or p35S::mCherry-MBD [47] and p35S::YFP-

TUA5 [48] for microtubules.

12. Agar can dry up too quickly in the tip after sowing if the agar surface in the tip is

not flat when cutting the tip after filling with ½ MS medium. Therefore, make sure to

cut underneath the meniscus to leave a flat surface.

13. Contamination may occur during microfluidic device plugging or syringe exchange.

To avoid high contamination rate, make sure to cut tubing with a scalpel beforehand

soaked in 70% ethanol and flame sterilized. When changing the syringe make sure to

keep the pump surroundings sterile. Finally, make sure that the coverslip is intact

before plugging the system as contaminations may come from microleakage.

14. Images can appear not steady during acquisition under a microscope. PDMS being

a squishy material the positioning of the microfluidic device on the microscopic plate

can be tricky to keep it steady. Make sure that the PDMS block is shorter than the

coverslip and well centered on it during the assembly procedure.

15. When applying precious drugs or purified bioactive compounds with limited

availability, prefer using smaller syringes (< 50 mL) with an adapted pump system.

Acknowledgments

The work needed to establish this protocol was funded by the Centre National de la

Recherche Scientifique (CNRS), the Human Frontier Science Program (HFSP, grant

RGP 2018, 009), the Agence Nationale de la Recherche (ANR-18-CE20-0011-01

26
project REWIRE and ANR-20-CE13-0025 project MechaNUC). In addition, this work

was also partly funded by the European Fund for Regional Development (Interreg

Upper Rhine, project DialogProTec, coordinator: Prof. Peter Nick, Botanical Institute,

Karlsruhe Institute of Technology, Karlsruhe, Germany). The authors gratefully

acknowledge Prof. Anne-Catherine Schmit for her contribution to the establishment of

this protocol, as well as Louis-Thibault Corbin for technical assistance.

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30
Figure legends

Figure 1: Molding of the PDMS microfluidic device. (a) Mold used for casting a replica

of the microfluidic device adapted to the simultaneous growth of three seedlings. The

zoom-ring shows one of the 0.8 mm pillars that will serve as a nutritional fluid outlet.

(b) Example of an alternative mold design in which three inlet-channels for three

different substances converging into a single mixing channel with a “zig-zag” structure

designed for an optimal substance mixing. The zoom-ring shows the 0.8 mm pillars

that will serve as a seedling root inlet upstream of the cultivation/observation channel.

(c) Scheme of the PDMS microfluidic device obtained from the mold presented in (a).

This device contains three cultivation/observation channels in which seedling roots are

grown. Each cultivation/observation channel has a nutritional fluid inlet on one side

and a nutritional fluid outlet on the other side for connecting nutritional fluid inlet and

outlet tubing, respectively. In addition, each cultivation/observation channel has an

opening for inserting a tip in which a seedling is grown. Each opening forms a 45°

angle with its cultivation/observation channel. (d) Top (left) and side (right) views of the

PDMS-based microfluidic device bonded via plasma bonding to a cover glass on the

observation side.

Figure 2: Assembly of a microfluidic device for root imaging. 10 µL tips are filled with

melt ½ MS agar medium (a), cut just above the 2 µL mark (b) and plant vertically on a

solidified ½ MS agar plate before loading sterilized seeds on it. (d) Tips containing 4-

5-day-old seedlings just before transfer on the microfluidic device. (e) Preparation of

the Petri dish that will serve as a container for the microfluidic device. (f) Assembly of

the bubble trap system from a PDMS cylinder (top left), that will be inserted in a 0.65

31
mL microcentrifuge tube (middle left) after inserting the three inlet tubings and the

primary tubing (bottom left). (g) Follow-up of the tubing mounting, including the three

outlet tubings, through the Petri dish lid prepared in (e). (h) Connection of the PDMS

microfluidic device to the different in- and outlet tubing. (i) Microfluidic device

assembling completion by placing a waterlogged autoclaved paper towel at the

periphery of the Petri dish to keep the system humid. (j) Microfluidic device loaded with

a seedling grown inside a tip, connected to an electric syringe pump and placed in an

in vitro growth chamber before imaging. (k) Microfluidic real-time imaging on a confocal

LSM 700 (Zeiss) microscope. (l) Scheme of the microfluidic system in its entirety.

Figure 3: Example of long-term high-resolution visualization of chromatin/nuclear

envelope markers in meristematic cells during main root growth inside a PDMS

microfluidic device. The scans were performed at regular intervals after oryzalin

treatment using low concentration (200 nM) for 10 hours. The nuclear envelope

appears highlighted in red (pSUN2::SUN2-RFP) and centromeric regions in green

(p35S::EYFP-CENH3). Propidium Iodide at 2 µg/mL allows the visualization of cell

walls (also in red). Numbers highlight connected cells which can be followed

throughout the scanning, showing progressive nuclear deformation and changes in the

position of the centromeric signals. Bar = 10 µm.

32