Microfluidics for microscopy imaging and treatment of Arabidopsis thaliana primary roots in vivo 

Microfluidics for microscopy imaging in plant biology allows to observe, in vivo, the biological response of plant roots to various stimuli at high temporal and spatial resolutions.

Plants are sessile organisms constantly balancing their internal state with their direct environment to optimize their growth. This application note describes a method to observe Arabidopsis thaliana primary roots in response to laboratory controlled stress conditions. This method is based on the combination of an Elveflow microfluidic setup and manually closable and reusable microfluidic chips. Observations can be performed using inverted vertical or horizontal stage microscopes. We carry our experiment with inverted vertical stage microscopes to keep the roots in their natural orientation. We use a combination of the Elveflow OB1 pressure controller and the Elveflow MFS2 digital flow sensors to reach high stability at low flow rates.

Introduction | Microfluidics and microscopy to study root responses

In general, microfluidics for microscopy imaging and study of plant root responses allows to:

• Study of the response to any treatments that can be added in solution (biotic/abiotic stresses)
• Observations in control then treatment granting internal controls for every plant
• Imaging of the very first instants of a treatment (e.g. phytohormone auxin [1]) and/or longer (e.g. NaCl [2])

The Elveflow setup allows precise flow stability, a key element to mitigate pressure shifts resulting in tissue mechanical stress and unspecific responses. Moreover, automation through macros in the ESI software improves experimental control and reproducibility.

Since the publication of the RootArray [3] and RootChip [4], a few microfluidic devices have been developed to observe Arabidopsis thaliana root responses to various stimuli (e.g [5,6], for reviews [7,8]). However, these devices are conventional glass-PDMS bonded closed microfluidic chips. This put a constraint on the user which has to grow the seedlings in specific conditions and then grow them in the chip. 

Recently, we developed a new microfluidic chip which is manually closable and reusable. This system allows the user to directly transfer mature seedlings grown in regular conditions into the device and close it with a microscopy coverglass [1].

Applications

• Imaging of Arabidopsis thaliana primary root responses to biotic and/or abiotic stresses
• Observations of very fast effects (order of seconds)
• Observation of the control and treated behaviors of the same plant

Experiment setup | Avoiding backflow for accurate minimal flow

Flow controller OB1

2x Flow sensors

Mux Wire valve controller

2 x 3/2 valves

And a custom made manually closable / reusable microfluidic chip

Working principle of the setup

The OB1 and flow sensors allow high stability of low flow rates (here we use 3 µL/min) while the 3/2 valves installed on each medium lines (pressure outputs) allows to control the direction of the flow. Either toward the chip or toward a collection tube. We implemented these valves to avoid back-flow and to constantly have minimal flow in both lines. Avoiding non-moving liquids limits precipitations, media to media contamination and dyes/solvents/drugs sticking to the tubing, elements which could impair experiments.

Figure 1: Setup schematic

Materials

Hardware

• 1 x OB1 mk3+ with two 0-200mbars pressure channels
• 2 x MFS2 0-7 µL/min digital flow sensor
• 1 x MUX WIRE valve controller
• 2 x 3/2 valves 
• 1 x Microscope (we use inverted vertical stage microscope, e.g. Zeiss Axioimager coupled to a Visitron spinning disk or a Zeiss LSM 980)

Optional

• 1 x Zeiss TTL trigger box
• 1 x BNC mal x male cable
• 1 x Perilstaltic pump (to retrieve the medium accumulating on top of the microfluidic chip)

Microfluidic accessories and other parts

• PTFE tubing (0.8mm ID x 1.6mm OD)
• PTFE tubing (0.3 mm ID x 1.6 mm OD)
• Tygon tubing (0.5 mm ID x 1.6 mm OD)
• 4 x Microfluidic Fittings 1/4″-28 Unions Kit
• 16 x 1/4-28 fittings + ferrules for 1/16″ OD stiff tubing  
• 2 x Microfluidic reservoir adapters for 15mL Falcon
• Biopsy puncher (Diameter 0.5 mm)
• Stainless steel pins (0.330 mm ID x 0.64 mm OD/23G)
• Microfluidic chip (non-commercial, contact us for designs)
• Acrylic microflluidic chip holder/clamp and microscope stage adapter (non-commercial, contact us for designs)
• 4 x M5, 2 cm screws
• Microscopy coverslips (22 x 40 mm, 1.5 mm thickness)
• Pecon microscope universal mounting frame KM

Optional

PEEK resistance capillary (1.6 mm OD). Depending on the length of your tubing you might need to adjust the resistance of the system to obtain optimal flow control.

Figure 2: Clamping contraption

From left to write: 1) Microfluidic photomask example. 2) Clamping contraption with a) microfluidic chip, b) chip mounting plateform, c) M5 2 cm screw, d) Microscope mounting counterpart and e) Microscope stage adapter. 3) Fully mounted contraption.

Software

• ESI software
• Microscope imaging software (e.g. Visitron Visiview or Zen Zeiss)

Reagents

• Filtered liquid plant media (e.g. ½ MS, 0.5mM MES, pH 5.8, 1% sucrose )
• Potential dye
• Potential treatment
• Isopropanol 50% or ethanol 70%
• mQ water
• Fluorescein Dextran >=10kDa (fluorescent tracer to follow treatment arrival/removal)

Biological material

3-5 day old Arabidopsis thaliana seedlings grown on solid media (Wild type, mutant, fluorescent reporter line…)

Quick start guide

1) Connect everything according to schematic and Elveflow mounting instructions

2) Optional: connect BNC cable from the OB1 TTL input to one of the trigger outputs on the Zeiss trigger box. To avoid instrument damages, always check the min/max voltage ranges in/out of the connected devices either in documentation or with a voltmeter.

3) Flood your system at max pressure with your treatment media then your control media. Times will vary from one system to the other (tubing length, elevation…)

Figure 3: Example of Elveflow Software Interface MACRO1: Media flush in the system

4) Set both pressure channel to 0µL/min

5) Prepare the chip:

• Rinse with isopropanol 50% or ethanol 70%
• Dry using paper towel
• Rinse with mQ or distilled water
• Dry using paper towel
• Apply tape to remove dust and other particles
• Set Control channel to 200 mbars to obtain a drop of medium on the chip
• Spread this medium in the microfluidics channels using, for example, the tip of a pipette
• Remove the medium excess so that is +/- flat with the chip 
  • If the medium forms a convex meniscus, the root might go on the side of the channels once you close the chip
  • If the medium forms a concave meniscus, a lot of air bubbles may appear upon closure of the chip.
• Slide the seedlings in the microfluidics channels with the cotyledons sticking out
• Place a microscopy coverslip in one quick motion from bottom to top (we also used a handi-vac with success
• Set the control pressure channel to 3 µL/min
• Gently tap on the air bubbles to make them exit the system (we use the back of a pair of tweezers)

Figure 4: Chip preparation

6) Enclose the closed microfluidic chip into the acrylic/screws clamping device

7) Mount the contraption on the microscope stage

Figure 5: Root in channel

Arabidopsis thaliana primary root observed in a microfluidic channel. This particular chip was obtained from a micro-milled acrylic mold (the drill head pattern is visible at the bottom of the channel).

8) Let the seedlings recover from the transfer mechanical stress for a least 20 minutes

9) Start the experiment with a macro to automate treatment

Figure 6: Example of Elveflow Software Interface MACRO2: Automated treatment

10) Optional: Start of the ESI macro triggered by the “Start experiment button” in the Zeiss Zen software

Figure 7: Example of Elveflow Software Interface MACRO3: Automated treatment, macro triggered by Zeiss Zen imaging

Figure 8: Zeiss Zen software Time Series settings to start ESI macro once the « Start Experiment » button is pressed.

Following media switch by fluorescence

To follow the media switch we add 3 µL of Fluorescein dextran 10 kDa (10 mg/mL) in 5 mL of control medium and 1.5 µL for 5 mL of treatment media. We observe the tracer in the channel background with a 488 nm laser excitation.

Microfluidic chip production

Our chip molds are produced either by CNC micro-milling (negative features) and polyurethane casting (positive features) or by UV-photolithography on silicon wafers (positive features). Chips were molded in PDMS (Sylgard) in a 15:1 ratio to increase stickiness to the closing microscopy coverslip [1]. PDMS-PEG co-polymer can be added up to 0.5% [9] in the un-polymerized PDMS mix to increase hydrophilic properties and to easily spread the medium in the open chip. This could be especially important in perfectly smooth chips molded from silicon wafers (compare to the ones obtained by micro-machining).

Connect the chip to the outside world

Directly connecting 1.6 mm OD PTFE tubing to a punched microfluidic chip is producing small depression in the PDMS and is not adapted to our open chips.

We used 23G stainless steel pins in combination with either:

• 0.5 mm ID x 1.6 mm OD PTFE tubing
• 0.5 mm ID x 1.6 mm OD Tygon tubing

Both tubing work well but Tygon flexibility is well adapted to the manipulations of the chip in the clamping device. You can also directly use pins compatible with the tubing used in your setup (important note: 25G is not fitting 0.3 mm ID PTFE tubing, too small and rigid).

Figure 9: Microfluidic connections

Key findings | Microfluidics for microscopy imaging and novel genetic reporter

This method allowed us to study the very fast response of seedlings to the major phytohormone auxin1 and discover the hormone receptor essential to trigger the root tissues depolarization. 

But also to characterize a novel genetic fluorescent potassium reporter using the system to study root response to NaCl know to induce a potassium leak from the tissues [2].

Movies illustrating the medium switches and the rapid root responses can be find in the corresponding scientific publication supplementary data [1,2].

Conclusions | Microfluidics for microscopy imaging

The pressure driven flow and the flow/pressure feedback with flow sensors in this Elveflow setup allowed us to characterize quick responses of Arabidopsis thaliana primary roots to various treatments with high flow stability and high spatial and temporal resolution.

The system can be adapted up to four treatments for sequential injections using four Elveflow pressure channels. Furthermore, the system can be used with conventional microfluidic chips [4,6,8] but was, in our hands, only tested with closable and reusable microfluidic chips [1].

Application note written by Nelson BC Serre, Yvon Jaillais and Matyáš Fendrych.

Acknowledgements

The research associated with this application note was supported by the European Research Council. ERC grant n°803048 (granted to M. Fendrych) and n°101001097 (granted to Y. Jaillais).

If you use this system in scientific publications please cite Elveflow components in your Material and Methods and our orignal paper describing the use of the closable and reusable microfluidic chip [1].

References

1. Serre NBC, Kralík D, Yun P, Slouka Z, Shabala S, Fendrych M. AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root. Nat Plants. 2021;7(9):1229-1238. doi:10.1038/s41477-021-00969-z
2. Wu SY, Wen Y, Serre NBC, et al. A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life. Dutzler R, ed. PLOS Biol. 2022;20(9):e3001772. doi:10.1371/journal.pbio.3001772
3. Busch W, Moore BT, Martsberger B, et al. A microfluidic device and computational platform for high-throughput live imaging of gene expression. Nat Methods. 2012;9(11):1101-1106. doi:10.1038/nmeth.2185
4. Grossmann G, Guo WJ, Ehrhardt DW, et al. The RootChip: An Integrated Microfluidic Chip for Plant Science. Plant Cell. 2011;23(12):4234-4240. doi:10.1105/tpc.111.092577
5. Massalha H, Korenblum E, Shapiro O, Asaph A. Tracking Root Interactions System (TRIS) Experiment and Quality Control. BIO-Protoc. 2019;9(8). doi:10.21769/BioProtoc.3211
6. Fendrych M, Akhmanova M, Merrin J, et al. Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nat Plants. 2018;4(7):453-459. doi:10.1038/s41477-018-0190-1
7. Sanati Nezhad A. Microfluidic platforms for plant cells studies. Lab Chip. 2014;14(17):3262-3274. doi:10.1039/C4LC00495G
8. Yanagisawa N, Kozgunova E, Grossmann G, Geitmann A, Higashiyama T. Microfluidics-Based Bioassays and Imaging of Plant Cells. Plant Cell Physiol. 2021;62(8):1239-1250. doi:10.1093/pcp/pcab067
9. Gökaltun A, Kang YB, Yarmush ML, Usta OB, Asatekin A. Simple Surface Modification of Poly(dimethylsiloxane) via Surface Segregating Smart Polymers for Biomicrofluidics. Sci Rep. 2019;9(1):7377. doi:10.1038/s41598-019-43625-5