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Feb 2018

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Fabrication and Use of the Dual-Flow-RootChip for the Imaging of Arabidopsis Roots in Asymmetric Microenvironments
制造和使用dual-flow-RootChip在不对称微环境下进行拟南芥根组织成像   

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Abstract

This protocol provides a detailed description of how to fabricate and use the dual-flow-RootChip (dfRootChip), a novel microfluidic platform for investigating root nutrition, root-microbe interactions and signaling and development in controlled asymmetric conditions. The dfRootChip was developed primarily to investigate how plants roots interact with their environment by simulating environmental heterogeneity. The goal of this protocol is to provide a detailed resource for researchers in the biological sciences wishing to employ the dfRootChip in particular, or microfluidic devices in general, in their laboratory.

Keywords: Dual-flow-RootChip (Dual-flow-RootChip), Lab-on-a-chip (Lab-on-a-chip), Environmental sensing (环境感知), Calcium signaling (钙信号), Root development (根组织发育), Root hairs (根毛), Cell-cell communication (细胞间通讯), Microfluidics (微流体), Plant-microbe interactions (植物-微生物互作)

Background

Conditions belowground are highly heterogeneous and dynamic, hence plant roots are exposed to various stimuli and consequently have to adapt to this complex environment. Despite the importance of these developmental adaptations, the underlying mechanisms still remain to be elucidated. Microfluidic devices have proven useful to cultivate specimens in controlled microenvironments and facilitate access for live imaging of dynamic processes from the subcellular to the organismic level (Crane et al., 2010). Thanks to the possibilities of microfluidics to manipulate small fluid volumes in a controlled manner, conduct experiments in high-throughput, extract quantitative information and perform time-lapse measurements, microfluidic devices have found their way into organismal studies. For the model plant Arabidopsis thaliana, a series of microfluidic devices have been developed that enable the monitoring of gene expression during root development (Busch et al., 2012), signaling events (Keinath et al., 2015) and sensor-based imaging of nutrient uptake (Grossmann et al., 2011; Lanquar et al., 2014). Additionally, more recent advances using microfluidic platforms have included high-resolution phenotyping (Jiang et al., 2014; Xing et al., 2017) and the investigation of root-microbe interactions (Parashar and Pandey, 2011; Massalha et al., 2017). While the root microenvironment can be precisely controlled in these perfusion devices, environmental complexity, a hallmark of natural root environments, was challenging to simulate (Stanley et al., 2016; Stanley and van der Heijden, 2017). The dfRootChip was therefore developed to enable the study of single Arabidopsis roots in asymmetric microenvironments at the cellular level to investigate gene expression, signaling and development (Stanley et al., 2018). Importantly, the dfRootChip can be implemented in a range of applications, which include performing localized treatments with drugs, differential nutrient or stress conditions, probing host-microbe interactions (e.g., pathogenic and beneficial interactions, potential biocontrol agents), and investigating root physiology and root hair development.

The current protocol was developed to provide fundamental know-how to researchers wishing to implement this platform. This protocol therefore provides a detailed explanation of how to fabricate the dfRootChip, using photo- and soft lithography, and how to cultivate Arabidopsis seedlings within the dfRootChip. Due to the wide applicability of microfluidics in biology, a number of steps in this protocol will also aid the fabrication and handling of other device designs. Furthermore, this protocol illustrates how the dfRootChip can be utilized in three different experimental settings. Specifically, we highlight how to perform (i) symmetric and asymmetric root treatments over longer time-periods (hours to days), (ii) localized inoculation of plant roots with bacteria and (iii) rapid asymmetric treatments with the dfRootChip. We exemplify these applications by utilizing different phosphate treatments, the bacterium Pseudomonas fluorescens and a calcium elicitor treatment respectively.

Materials and Reagents

Note: Catalog numbers are provided for commercial, non-custom-made products (see Note 1).

  1. Polyester film photolithography mask (Micro Lithography Services Ltd. UK, custom-made)
  2. 100 mm silicon wafers (Silicon Materials)
  3. SU8 3050 photoresist (MicroChem)
  4. Plastic cups (Semadeni, catalog number: 8323 )
  5. Plastic spatulas (Semadeni, catalog number: 3340 )
  6. Glass coverslips, 75 mm x 50 mm, No. 1 (Th. Geyer, catalog number: 11678524 )
  7. Cutting blades (Häberle Labortechnik, catalog number: 9156110 )
  8. Scotch® MagicTM Invisible tape (3M)
  9. Microcentrifuge tubes 1.5 ml (Eppendorf Safe-Lock, Eppendorf, catalog number: 0030120086 )
  10. Sterilised filter tips 100-1,000 μl (Pipetman Diamond Tips D1000ST, Gilson, catalog number: F171501 )
  11. 0.2 μm sterile syringe filters (Lab Logistic Group, catalog number: 9.055 511 )
  12. Sterilised filter tips 0.1-20 μl (Pipetman Diamond Tips DL10ST, Gilson, catalog number: F171201 )
  13. Sterilised filter tips 2-200 μl (Pipetman Diamond Tips D200ST, Gilson, catalog number: F171301 )
  14. 94 mm diameter sterile Petri dishes (HUBERLAB, catalog number: 7.663 161 )
  15. Parafilm® (Bemis, HUBERLAB, catalog number: 15.1550.01 )
  16. Silicone tubing (TYGON® 0.020” ID x 0.062” OD; type ND-100-80) (Th. Geyer, catalog number: AAD04103
  17. Gauge 23 dosage needles with Luer lock fitting (Gonano Dosiertechnik, catalog number: IP423050-EAR-BULK )
  18. Syringes 20 ml (VWR, BD PlastipakTM, catalog number: 613-3922 )
  19. Rotilabo-screw neck ND24 vials (Carl Roth, catalog number: LC88.1 )
  20. Screw caps with bore hole (Carl Roth, catalog number: LC97.1 )
  21. Septa Ø 22 mm, ND24, 1.6 mm, 55° (Carl Roth, catalog number: LC98.1 )
  22. Mini 3-way stopcock, 2 x Luer female, 1 x Luer male (NeoLab, catalog number: 270124190 )
  23. Male-male luer connectors (Vygon, catalog number: 893.00 )
  24. Polystyrene cuvettes (SARSTEDT, catalog number: 67.742 )
  25. 120 x 120 mm2 Petri dishes, sterile (Carl Roth, catalog number: PX67.1 )
  26. Aluminum foil (can be purchased from any supermarket)
  27. Arabidopsis thaliana seeds; lines are chosen based on individual needs
  28. Optional: Pseudomonas fluorescens WCS365-GFP strain (Haney et al., 2015)
  29. mrDev-600 developer solution (Micro Resist Technology)
  30. Isopropyl alcohol [(CH3)2CHOH] (Sigma-Aldrich, catalog number: W292907 )
  31. Chlorotrimethylsilane [(CH3)3SiCl] (Sigma-Aldrich, catalog number: 92361 )
  32. Sylgard 184 Kit [Poly(dimethylsiloxane), PDMS] (Biesterfeld Helvetia, catalog number: 5498840000 )
  33. Acetone (CH3COCH3) (Sigma-Aldrich, catalog number: 00560 )
  34. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
  35. Ethanol (EtOH) (Sigma-Aldrich, catalog number: 51976 )
  36. Deionised water, purified by reverse osmosis or ultrafiltration; referred to as "purified water" below
  37. Sodium hypochlorite 14% Cl2 in aqueous solution (NaClO) (VWR, catalog number: 90350.5000 )
  38. Micro agar (Duchefa Biochemie, catalog number: M1002.1000
  39. Hoagland’s No. 2 Basal Salt Mixture (Sigma-Aldrich, catalog number: H2395-10L )
  40. MES hydrate (C6H13NO4S•xH2O) (Sigma-Aldrich, catalog number: M8250 )
  41. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  42. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  43. Potassium dihydrogen phosphate (KH2PO4) (AppliChem, catalog number: A3620 )
  44. Magnesium sulfate heptahydrate (MgSO4•7H2O) (Merck, catalog number: 1.05886.1000 )
  45. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: 31263 )
  46. Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA•2H2O) (AppliChem, catalog number: A3553 )
  47. Calcium nitrate tetrahydrate (Ca(NO3)2•4H2O) (Sigma-Aldrich, Fluka, catalog number: 21197 )
  48. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  49. Copper(II) sulfate pentahydrate (CuSO4•5H2O) (Grüssing, catalog number: 12079 )
  50. Zinc sulfate (ZnSO4) (Sigma-Aldrich, catalog number: Z1001 )
  51. Sodium molybdate (Na2MoO4) (Sigma-Aldrich, catalog number: 243655 )
  52. Manganese chloride dihydrate (MnCl2•2H2O) (Grüssing, catalog number: 12097 )
  53. Cobalt(II) chloride hexahydrate (CoCl2•6H2O) (Carl Roth, catalog number: T889 )
  54. Potassium chloride (KCl) (AppliChem, catalog number: A3582 )
  55. Luria Bertani broth (Sigma-Aldrich, catalog number: L3022 )
  56. Kanamycin sulfate (Carl Roth, catalog number: T832.3 )
  57. ½x Hoagland’s medium (½x HM) (see Recipes)
  58. ½x Hoagland’s agar medium (see Recipes)
  59. Phosphate rich medium (see Recipes)
  60. Phosphate deficient medium (see Recipes)
  61. Lysogeny broth (LB) medium (see Recipes)
  62. 100 mM salt solution (see Recipes)

Equipment

  1. Duran® laboratory bottles 500 ml (DWK Life Sciences, DURAN, catalog number: 21 801 44 5 )
  2. Duran® laboratory bottles 1,000 ml (DWK Life Sciences, DURAN, catalog number: 21 801 54 5 )
  3. Metal pins (New England Small Tube, catalog number: NE-1310-02 )
  4. Schott® culture tubes, 160 mm x 16 mm (DWK Life Sciences, DURAN, catalog number: 26 135 21 5 )
  5. Compressed air (Oil-free compressor, 9 L, 8 bar) (e.g., IMPLOTEX, catalog number: NEW-325 )
  6. Glass beakers (HUBERLAB, catalog number: 9.0112.43 )
  7. Forceps (VWR, RGS Solingen, catalog number: 232-0078 )
  8. Precision air regulator (Ashcroft, Ingersoll-Rand, catalog number: PR4021200 )
  9. 250 ml Erlenmeyer flask (Sigma-Aldrich, DWK Life Sciences, DURAN, catalog number: Z232815 )
  10. Spatula (HUBERLAB, catalog number: 13.1556.05 )
  11. Vortex (HUBERLAB, catalog number: 17.1378.01 )
  12. Pipettes (Pipetman classic P1000, Gilson, catalog number: F123602 )
  13. Pipettes (Pipetman classic P200, Gilson, catalog number: F123601 )
  14. Pipettes (Pipetman classic P10, Gilson, catalog number: F144802 )
  15. pH meter (Mettler-Toledo International, model: FiveEasyTM FE20 )
  16. Autoclave (Systec, model: VX-75 )
  17. Drying oven (SalvisLab, model: VC 20 )
  18. Standard refrigerator (4-6 °C)
  19. Biosafety cabinet equipped with UV (Kojair, model: Kojair® SilverLine BlueSeries 200 )
  20. Precision balance (Mettler-Toledo International, model: PB3001 )
  21. Analytical balance (Mettler-Toledo International, model: AB104-S/FACT )
  22. Spin coater (SAWATEC, model: SM-180-BM )
  23. Hot plate (SAWATEC, model: HP 160 III BM )
  24. 125 mm x 125 mm x 2 mm soda lime glass plate (Willi Möller)
  25. Mask aligner (Karl Süss, model: MA6 )
  26. Custom-made plastic chip holder (frame with an aperture for the RootChip and outer dimensions that fit the microscope stage)
  27. Incubator (Memmert, model: UM400 )
  28. Incubator with flask shaker (Eppendorf, New BrunswickTM, model: Innova® 44
  29. Wet bench with filtered laminar air flow (Goller Reinraum)
  30. Spin coater (Laurell Technologies, model: WS-650-23
  31. Utility knife
  32. Hole puncher (Syneo, model: Accu-Punch MP10 )
  33. Hole punch (cutting edge diameter, 0.71 mm) (Syneo, catalog number: CR0350255N20R4 )
  34. Hole punch (cutting edge diameter, 1.02 mm) (Syneo, catalog number: CR0500355N18R4 )
  35. Hole punch (cutting edge diameter, 4.75 mm) (Syneo, catalog number: HS1871730P1183S )
  36. Ultrasonic cleaner (BANDELIN electronic, catalog number: 301 )
  37. Plasma cleaner (Diener electronic, model: FEMTO40kHZ
  38. Vacuum pump (Pfeiffer Vacuum, catalog number: PK D56 712 )
  39. Vacuum desiccator (Thermo Fisher Scientific, catalog number: 5311-0250 )
  40. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM PicoTM 21 , catalog number: 75002553)
  41. Climate chamber (Panasonic, PHC, model: MLR-352H )
  42. Stratalinker® UV crosslinker (Stratagene, model: 1800 , catalog number: 400071)
  43. Syringe pumps (World Precision Instruments, model: AL-6000 )
  44. Stereo microscope (Nikon, model: SMZ1270 )
  45. Spectrophotometer (GE Healthcare, NovaspecTM Plus, catalog number: 80-2117-50 )
  46. Inverted microscope (any model, specific to user and application)
  47. Optional: source of pressurized, clean, dry air

Software

  1. AutoCAD Mechanical 2011 (AutoDesk, USA)
  2. Fiji (Schindelin et al., 2012)

Procedure

  1. Master mould fabrication (process outlined in Figure 1; see Note 1)
    1. Coat a 100 mm diameter silicon wafer with SU-8 3050 photoresist using a spin coater (SAWATEC). The film thickness of approximately 115 μm can be achieved by adapting the spin speed according to the photoresist manufacturer specifications (see Note 2).
    2. Place the photoresist-coated wafer onto a hot plate at 95 °C and bake for 20 min (soft bake, see Note 2). A larger silicon wafer or a sheet of aluminum foil can be used to protect the hotplate from contamination by the photoresist.
    3. Attach the photolithography film mask to a 125 mm soda-lime glass plate and, using a mask aligner with a built-in ultra-violet light source, pattern the dfRootChip design onto the photoresist by exposing the resist to an ultra-violet light source with an exposure energy of 500 mJ/cm2 (λ = 365 nm) (see Notes 2 and 3). The function of the soda-lime glass plate is to carry the mask and to hold it rigid to ensure good contact with the resist.
    4. Bake the photoresist on a hot plate at 95 °C for 20 min (post-exposure bake, see Note 2).
    5. To develop the structures (i.e., remove the unexposed photoresist), completely immerse the silicon wafer in an SU-8 developer for ca. 15 min with agitation (mr-Dev 600 developer solution). Then, rinse the structures with fresh developer solution for ca. 10 sec (see Note 2).
    6. Rinse the master mould with isopropyl alcohol for ca. 10 sec and air-dry thoroughly. To ensure that the structures are completely dry, use filtered, compressed air (see Note 2). If a white film is produced when rinsing, the master mould has been under developed. If this is the case, immerse the master mould in fresh developer solution until the film has been removed and rinse again with IPA.
    7. Place the master mould and an open glass vial containing 50 μl of chlorotrimethylsilane inside a vacuum desiccation chamber. Next, evacuate the chamber by applying a vacuum pressure of 50 mbar, then close the valve to seal the chamber and allow the master mould to incubate in the chlorotrimethylsilane vapor for at least 1 h.


    Figure 1. dfRootChip fabrication. A. The photo- and soft lithography processes involved in the fabrication of the dfRootChip are illustrated. First, a silicon wafer is coated with SU8 photoresist (1), after which a photolithography mask (2) is used to expose specific regions of the photoresist to collimated UV light (3). During exposure, the photoresist is polymerized (4) and then developed. Development removes regions of unpolymerised photoresist, resulting in a master mould with patterned microstructures (5). Poly(dimethylsiloxane) (PDMS) is then cast and cured (6), after which it can be removed from the master mould (7) and cut, punched and bonded to a glass coverslip coated with a thin layer of PDMS to form a PDMS device (8). B. To complement the “side-view” illustrations in A, “top-view” illustrations of the photolithography mask, master mould and PDMS device have been included for clarity. C-E. Photographs of an exemplar photolithography mask (mounted on a soda-lime glass plate), a master mould and PDMS device are displayed in C, D and E respectively. F. The specific dimensions and positions of the inlets and outlet are displayed for clarity.

  2. PDMS device fabrication (for detailed video instructions on PDMS device fabrication see Video 1; process outlined in Figures 1 and 2; see Notes 1 and 4)
    1. Prepare poly(dimethylsiloxane) (PDMS) using a 10:1 ratio of base to curing agent (Sylgard 184 Kit).
    2. Mix thoroughly using a spatula.
    3. Degas the mixture using a vacuum pressure of 50 mbar for 1 h.
    4. Pour the PDMS onto the master mould (see Note 5). 
    5. Coat five clean coverslips (rinsed with acetone, IPA and blow-dried) with a thin layer of PDMS (Laurell Technologies, spin coat at ~1,200 rpm for 25 sec). 
    6. Cure the master mould and coated coverslips overnight in an oven at 70 °C.
    7. Allow the master mould to cool to room temperature and remove the cured PDMS.
    8. Cut out the PDMS devices using a utility knife. In the cutting device shown in Figure 2E, the knife blade is fixed in a custom-made metal frame and pushed down to cut cured PDMS slabs.
    9. Punch holes to create the root inlets (Ø = 1.02 mm), solution in- and outlets (Ø = 0.71 mm) and reservoirs (Ø = 4.75 mm). The root inlets should be punched at ca. 30-45° angle. 
    10. Wash the devices and coverslips in each of the following solutions for 5 min using an ultrasonic bath: 0.5 mM NaOH, 70% EtOH and purified water. Rinse the devices in purified water between each washing step. 
    11. Dry the devices and coverslips with compressed air and place in the oven at 70 °C for 1 h.
    12. Bond the dried devices to the coverslips using a plasma cleaner (see Note 6). Scotch tape can be used to remove dust particles from the surface of the PDMS before bonding.


    Figure 2. PDMS device fabrication. The processes involved in preparing a PDMS device are illustrated (see Procedure B for detailed information). The PDMS is prepared by mixing the base and curing agent together (A) and degassing the mixture (B). The degassed PDMS is then poured on top of the master mould (C) and then removed after curing in an oven (D). After cutting out (E) and punching holes (F) into the PDMS, it is washed (G), dried (H) and bonded to a glass substrate (I-L). For detailed video instructions on PDMS device fabrication see Video 1.

    Video 1. PDMS device fabrication. The video shows the entire process of PDMS device fabrication outlined in Procedure B and Figure 2 of the protocol.

  3. Preparing dual-flow-RootChip for on-chip plant cultivation (see Note 1)
    1. After bonding, the devices should be filled immediately with half-strength Hoagland's medium (½x HM, see Recipe 1). To do this, manually pipette ½x HM into each microchannel via the root inlet. The microchannel is filled when the medium exits through the inlets and outlet. Check for air bubbles, and use more medium as necessary to remove air bubbles. Top up the inlets/outlets and reservoirs with ½x HM to ensure that they are completely filled.
    2. Sterilize the devices using UV light for 30 min with a UV crosslinking device.

  4. Surface seed sterilization (see Notes 1 and 7) 
    1. Place one small spatula full of Arabidopsis thaliana seeds (about 100) into a 2 ml microcentrifuge tube.
    2. Fill the microcentrifuge tube with 1 ml of sterile-filtered sodium hypochlorite solution (5%).
    3. Shake tube by hand to suspend seeds, then place on vortex for 3-5 min on a medium level.
    4. Place the tube with the seeds into a centrifuge at ca. 100 x g for a few seconds to spin down the seeds. Continue the procedure under sterile conditions (biosafety cabinet).
    5. Pipette off the supernatant. 
    6. Re-suspend the seeds in 1 ml of autoclaved purified water.
    7. Place on vortex for 20 sec.
    8. Repeat Steps D4-D7 until seeds have been washed with water three times.
    9. Store the seeds in the fridge (4 °C, in purified water) for three days for stratification.

  5. Seed farm preparation (see Figure 3; see Notes 1 and 7; a video of the seed farm preparation procedure has been published in Grossmann et al., 2012).
    Note: The steps in this section should be carried out under sterile conditions (biosafety cabinet).
    1. Prepare medium of choice containing 0.7% plant agar (e.g., see Recipe 2). The medium used in this step is dependent upon the experiment to be conducted. 
    2. Whilst still warm (ca. 50 °C), pour the agar-containing medium into sterile Petri dishes (e.g., 10) and fill autoclaved 0.1-20 μl pipette tips (Gilson DL10ST) with 5 μl of the same medium (ca. 48 tips per seed farm). Allow the medium to cool completely. Petri dishes that are not used in the preparation of seed farms can be sealed with Parafilm® and stored in the fridge for later use. 
    3. Cut the pipette tips to a final length of about 5 mm using a heat sterilized cutting blade.
    4. Set the pipette tips upright into agar plates (ca. 48 tips per plate).
    5. Take the previously surface-sterilized Arabidopsis seeds and, using a pipette, place one seed on top of each pipette tip.
    6. Seal the plates with Parafilm® and place them in a climate chamber. Grow the plants continuously under long day conditions (16 h light at 100 μE m-2 sec-1, 22 °C, 50-70% relative humidity).


    Figure 3. Arabidopsis seed farm. A. This photograph illustrates a “seed farm” that accommodates 5-day-old Arabidopsis seedlings germinated on agar-filled, cut pipette tips. The seed farm is created by placing Arabidopsis seeds on top of medium-filled pipette tips maintained in an agar plate. B. Prior to transfer onto the chip, seedlings with the proper root length are selected under a dissecting microscope (marked with blue dots on the bottom of the Petri dish in A).

  6. Plant selection (see Note 1; a video of the selection procedure has been published in Grossmann et al., 2012)
    1. Take a 5-day-old seed farm (see Note 8) and place under a stereo microscope.
    2. Adjust the light levels and direction of the Petri dish until the roots are visible. 
    3. Identify 8-10 plants per dfRootChip, where roots are at the same developmental stage. Preferably, the roots have just reached the end of the pipette tip, but have not grown past the opening. Slightly shorter roots can work if all roots in one device are at the same developmental stage. 
    4. Mark selected plants for later identification.

  7. Transferring plants onto the dual-flow-RootChip (see Notes 1 and 7; a video of the transfer procedure has been published in Grossmann et al., 2012).
    Note: These steps require sterile conditions (biosafety cabinet). 
    1. Sterilize two pairs of forceps and take a pre-bonded and filled dfRootChip (see Procedure C).
    2. Using forceps, gently insert the pre-identified pipette tips containing plants into the plant inlets (see Procedure F). To prevent the introduction of air bubbles into the microchannels, first put a small amount of medium over the plant inlets.
    3. Ensure that the pipette tips are inserted into the plant inlet until they almost touch the bottom of the channel. This will help to ensure consistent root growth into the microchannels.
    4. After all of the plant inlets are filled, place the dfRootChip into a sterile Petri dish.
    5. Fill the Petri dish with ca. 15 ml of ½x HM (see Recipe 1).
    6. Seal the Petri dish with Parafilm® and place in the climate chamber for ca. 3-4 days, or until several root tips are visible in the microchannels.

  8. Performing symmetric and asymmetric treatments with the dual-flow-RootChip: Exemplified with phosphate treatments (see Figure 4 and Note 1)
    1. Prepare tubing: for each plant on the dfRootChip prepare two identical lengths of tubing with a luer-lock dosage needle at one end and a metal connector pin at the other (to connect the syringes to the device via the inlets, “inlet” tubing), plus another length of tubing with metal connector pins at one end only (to connect the device outlets to a waste container, “outlet” tubing) (Figure 4C). When choosing the proper inlet tubing length, the required distance for full lateral movement of the chip mounted at the microscope stage needs to be considered.
    2. Autoclave all tubing, then place in a biosafety cabinet. 
    3. Take a dfRootChip containing Arabidopsis plants (see Procedure G) and place in a biosafety cabinet. Using a magnifying glass or dissection microscope, ensure that the roots have grown into the microchannels and have reached a sufficient length, preferably at the beginning of the channels. Also, make sure that the plant itself appears healthy, with green cotyledons.
    4. Connect the “outlet” tubings to the dfRootChip outlets via the metal pins. 
    5. Inside the biosafety cabinet, fill sterile syringes with the desired media (see Note 9). To perform experiments with 5 plants for example, 10 syringes will be required. To perform asymmetric treatments, fill half of the syringes with the first medium of interest (e.g., see Recipe 3) and half with a second medium of interest (e.g., see Recipe 4).
    6. Connect each “inlet” tubing to a syringe using dosage needles. Manually push the medium through each tubing, avoiding air bubbles. In case air-bubbles remain inside the chamber, these will be pushed out as soon as the perfusion is started.


    Figure 4. Experimental set-up for performing symmetric and asymmetric treatments with the dfRootChip. A chip holder (A) is used to house the dfRootChip (B) when performing experiments. The dfRootChip is connected to a syringe pump (C-E) to perfuse the roots with the treatment(s) of choice. A cover and wet tissues are used to maintain the humidity around the plants. The set-up can then be transferred to a microscope to image the roots (F, scale bar = 250 μm).

    1. Load the syringes onto the syringe pump, enter the syringe diameter and start perfusion at a high initial flow rate (e.g., 100 μl/min). Note that the syringe pumps can remain outside of the biosafety cabinet. 
    2. Start the pump and leave it to run until the medium is pushed out of the loose tubing ends. 
    3. Set the flow rate to 5 μl/min (Figure 4D).
    4. While the pump is still running, connect the “inlet” tubings to the inlets via the metal connector pins using sterilized forceps. This ensures that no air bubbles are introduced into the microchannels. 
    5. The dfRootChip is now ready to be imaged. Place the dfRootChip into the chip holder (Figure 4A) and transport the setup to an inverted microscope.

  9. Localized inoculation of Arabidopsis roots with bacteria trapped by the micropillar array: Exemplified with Pseudomonas fluorescens strain WCS365-GFP (Haney et al., 2015) as proof of concept (see Note 1)
    1. Culture Pseudomonas fluorescens at 28 °C with shaking (200 rpm) overnight in 100 ml LB medium supplemented with 50 μg/ml kanamycin.
    2. Check the OD (600 nm) of the bacterial culture. For bacteria to be in log phase, OD should be in the range of 0.1-0.2.
    3. Transfer the culture into a 20 ml Falcon tube and centrifuge at 12,000 x g for 5 min.
    4. Discard the supernatant and resuspend the pellet in 20 ml of ½x HM.
    5. Again centrifuge at 12,000 x g for 5 min.
    6. Repeat Steps I4 and I5 to remove any traces of LB and kanamycin.
    7. Gently resuspend the bacterial pellet in ½x HM and use this to fill the syringe(s).
    8. To inoculate Arabidopsis roots with P. fluorescens, connect the syringe(s) to the dual-flow-RootChip, as detailed in Procedure H.

  10. Performing rapid asymmetric treatments in the dual-flow-RootChip: Exemplified with a calcium elicitor treatment using 100 mM NaCl solution as proof of concept (see Figure 5 and Note 1)
    1. Prepare pressurizable vials (screw-neck vials; two per plant on the dfRootChip): Place a septum into the lid of the bottle and puncture it with a metal pin leaving the pin in the septum. Connect a piece of tubing long enough to reach the bottom of the bottle to the inside end of the pin and a length of tubing to the outside end of the pin. Autoclave the bottle and attached tubing. In addition, autoclave, per plant, two extra lengths of inlet tubing with metal pins on one end and one outlet tubing with one pin. 
    2. Place a dfRootChip containing Arabidopsis plants (see Procedure G) in a biosafety cabinet. Ensure that the roots have grown into the microchannels and have reached a sufficient length, preferably at the beginning of the channels (see Step H3).
    3. Fill one of the sterile vials with a control medium (e.g., see Recipe 1), and another with the treatment medium (e.g., 100 mM NaCl, see Recipe 6).
    4. Add a luer-lock stopcock valve set (ethanol-sterilized) in a “closed” configuration to the end of the tubing coming from each of the vials. Add the extra lengths of tubing to the stopcock outlets.
    5. Pressurise the vials by connecting the extra length of tubing to your source of pressurized, clean, dry air and puncture the septum with the pin. Open each of the stopcocks to fill the tubing completely with medium. Once the tubing is filled close the stopcock again.
    6. Connect the vial containing the control medium into one of the medium inlets and open the stopcock. In our setup, a pressure of 5 PSI results in a volumetric flow rate of 20 μl/min. Initially, the roots are perfused symmetrically, and the control medium will flow out of the medium outlet and the second medium inlet.
    7. Connect the vial containing the treatment solution into the second medium inlet. The active flow from the control medium will prevent the treatment solution from entering the microfluidic channel.
    8. When ready to apply a treatment, open the stopcock associated with the treatment vial to start rapid asymmetric perfusion.
    9. To stop the asymmetric treatment, close the stopcock associated with the treatment vial. Within seconds, the perfusion will return to symmetric control conditions.


    Figure 5. Experimental set-up for performing rapid asymmetric treatments in the dfRootChip. This figure illustrates how the air-pressure sources, pressurizable vials and stopcocks are connected and interfaced with the dfRootChip.

Data analysis

  1. With regard to experimental design, each dfRootChip enables several experiments (i.e., technical replicates) to be performed in parallel. In addition, a minimum of three biological replicates should be performed for each data set. Examples for analyzing data sets acquired using the dfRootChip can be found in the original article Stanley et al. (2018).
  2. With regard to data analysis, we recommend using the freehand line tool and kymograph function in Fiji to analyze, for example, primary root and root hair growth rates. To access a wealth of information regarding image analysis, we recommend visiting the following website: http://www.plant-image-analysis.org

Notes

  1. Please contact us for further information and advice.
  2. This protocol has been used in our previously published work (Stanley et al., 2018). Parts of the described procedures that are not specific to the use of the dfRootChip are similar to or adapted from previously published work (Grossmann et al., 2011 and 2012; Stanley et al., 2014) but are recapitulated to provide a comprehensive and complete protocol. 
  3. For optimal results, we recommend that the entire fabrication procedure is performed inside a Class 1000 clean room. It is obligatory that the master mould fabrication procedure is conducted under yellow light. We also recommend that all wet chemistry operations are performed in a wet bench with filtered laminar airflow. If clean room facilities are not available, this can be achieved in collaboration with research groups or other commercial services that enable fabrication to be done. Please contact us for further information and advice.
  4. The channel patterns for the dfRootChip were drawn using AutoCAD Mechanical 2011. The design file can then be printed as polyester film photolithography mask by a commercial provider. The mask layout was arranged such that several dfRootChip replicas could fit in a single 100 mm silicon wafer. Please contact us for further information and advice.
  5. The processes involved in PDMS device fabrication should be performed in a bench with filtered laminar airflow.
  6. The master mould can be placed in a 3D-printed plastic holder, as illustrated in Figure 2. Alternatively, aluminum foil can be shaped around a glass Petri dish and used to hold the master mould when pouring PDMS on top of the microstructures.
  7. When using the plasma cleaner the following conditions were employed: power, 50%; treatment time, 1 min. However, this can vary between products, and the ideal conditions must be determined for each plasma cleaner. 
  8. Work under sterile conditions (e.g., in a biosafety cabinet or next to a flame).
  9. Growth conditions will vary between laboratories. Therefore, it is important to establish how many days the seeds farms should be incubated in the climate chamber. The plant roots should not grow out of the ends of the pipette tips.
  10. Use syringes of sufficient size to accommodate specific experimental requirements. As a point of reference, 7.2 ml of medium would be used within 24 h period (per syringe) using a flow rate of 5 μl/min.

Recipes

  1. ½x Hoagland's medium (½x HM) (1 L)
    1. Mix 0.8 g of Hoagland’s No. 2 basal salt mixture and 1 g of MES hydrate
    2. Transfer the above reagents into a 1 L beaker and add 900 ml of purified water
    3. Dissolve all the above reagents in purified water with the help of magnetic stirrer
    4. Adjust the pH to 5.7 with KOH
    5. Add purified water to make the total volume of the medium up to 1,000 ml
    6. Autoclave the medium for 20 min at 121 °C
  2. ½x Hoagland's agar medium (1 L)
    1. ½x HM is prepared as described above (see Recipe 1 steps 1a-1d)
    2. Weigh 7 g of plant agar (w/v) and add it to the ½x HM
    3. Make the total volume of the medium up to 1,000 ml with purified water
    4. Autoclave the medium for 20 min at 121 °C
  3. Phosphate rich medium (as in Chandrika et al., 2013)
    1. Mix the following components together to yield these final concentrations in purified water:
      2.5 mM KH2PO4
      2 mM MgSO4•7H2O
      5 mM KNO3
      2 mM Ca(NO3)2•4H2O
      0.04 mM Na-Fe-EDTA
      70 μM H3BO3
      0.5 μM CuSO4•5H2O
      1 μM ZnSO4•7H2O
      14 μM MnCl2•2H2O
      0.2 μM Na2MoO4
      0.01 μM CoCl2•6H2O
      1 g/L MES hydrate
    2. Adjust the pH to 5.5 using 0.5 M KOH
    3. Sterile filter the medium through a 0.2 μm membrane
  4. Phosphate deficient medium (as in Chandrika et al., 2013)
    1. Mix the following components together to yield these final concentrations in purified water:
      0.01 mM KH2PO4
      2.49 mM KCl
      2 mM MgSO4•7H2O
      5 mM KNO3
      2 mM Ca(NO3)2•4H2O
      0.04 mM Na-Fe-EDTA
      70 μM H3BO3
      0.5 μM CuSO4•5H2O
      1 μM ZnSO4•7H2O
      14 μM MnCl2•2H2O
      0.2 μM Na2MoO4
      0.01 μM CoCl2•6H2O
      1 g/L MES hydrate
    2. Adjust the pH to 5.5 using 0.5 M KOH
    3. Sterile filter the medium through a 0.2 μm membrane
  5. Lysogeny broth (LB) medium (1 L)
    1. Add 20 g of LB broth mixture and 5 g of NaCl to 1 L of purified water
    2. Autoclave the medium for 20 min at 121 °C
  6. 100 mM salt solution (100 ml)
    1. Prepare ½x HM, as described in Recipe 1
    2. Dissolve 0.584 g NaCl in 100 ml ½x HM
    3. Sterile filter the solution through a 0.2 μm membrane

Acknowledgments

This protocol has been adapted from Stanley et al. (2018). We acknowledge financial support from the Swiss National Science Foundation in the form of an Ambizione Career Grant (PZ00P2_168005) to CES, a Faculty for the Future Fellowship by the Schlumberger Foundation to JS, the Deutsche Forschungsgemeinschaft (GR4559/3-1) and research group funds from the Heidelberg Excellence Cluster CellNetworks to GG.

Competing interests

DvS represents Wunderlichips GmbH, Switzerland. All other authors declare no competing interests in regard to this publication.

References

  1. Busch, W., Moore, B. T., Martsberger, B., Mace, D. L., Twigg, R. W., Jung, J., Pruteanu-Malinici, I., Kennedy, S. J., Fricke, G. K., Clark, R. L., Ohler, U. and Benfey, P. N. (2012). A microfluidic device and computational platform for high-throughput live imaging of gene expression. Nat Methods 9(11): 1101-1106.
  2. Chandrika, N. N., Sundaravelpandian, K., Yu, S. M. and Schmidt, W. (2013). ALFIN-LIKE 6 is involved in root hair elongation during phosphate deficiency in Arabidopsis. New Phytol 198(3): 709-720.
  3. Crane, M. M., Chung, K., Stirman, J. and Lu, H. (2010). Microfluidics-enabled phenotyping, imaging, and screening of multicellular organisms. Lab Chip 10(12): 1509-1517.
  4. Grossmann, G., Guo, W. J., Ehrhardt, D. W., Frommer, W. B., Sit, R. V., Quake, S. R. and Meier, M. (2011). The RootChip: an integrated microfluidic chip for plant science. Plant Cell 23(12): 4234-4240.
  5. Grossmann, G., Meier, M., Cartwright, H. N., Sosso, D., Quake, S. R., Ehrhardt, D. W. and Frommer, W. B. (2012). Time-lapse fluorescence imaging of Arabidopsis root growth with rapid manipulation of the root environment using the RootChip. J Vis Exp(65): 4290.
  6. Haney, C.H., Samuel, B. S., Bush, J. and Ausubel, F. M. (2015). Associations with rhizosphere bacteria can confer an adaptive advantage to plants. Nature Plants 1: 15051.
  7. Jiang, H., Xu, Z., Aluru, M. R. and Dong, L. (2014). Plant chip for high-throughput phenotyping of Arabidopsis. Lab Chip 14(7): 1281-1293.
  8. Keinath, N. F., Waadt, R., Brugman, R., Schroeder, J. I., Grossmann, G., Schumacher, K. and Krebs, M. (2015). Live cell imaging with R-GECO1 sheds light on flg22- and chitin-induced transient [Ca2+]cyt patterns in Arabidopsis. Mol Plant 8(8): 1188-1200.
  9. Lanquar, V., Grossmann, G., Vinkenborg, J. L., Merkx, M., Thomine, S. and Frommer, W. B. (2014). Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology. New Phytol 202(1): 198-208.
  10. Massalha, H., Korenblum, E., Malitsky, S., Shapiro, O. H. and Aharoni, A. (2017). Live imaging of root-bacteria interactions in a microfluidics setup. Proc Natl Acad Sci U S A 114(17): 4549-4554.
  11. Parashar, A. and Pandey, S. (2011). Plant-in-chip: Microfluidic system for studying root growth and pathogenic interactions in Arabidopsis. Applied Physics Letters 98(26): 740.
  12. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  13. Stanley, C. E. and van der Heijden, M. G. A. (2017). Microbiome-on-a-Chip: New Frontiers in Plant-Microbiota Research. Trends Microbiol 25(8): 610-613.
  14. Stanley, C. E., Grossmann, G., i Solvas, X. C. and deMello, A. J. (2016). Soil-on-a-Chip: microfluidic platforms for environmental organismal studies. Lab Chip 16(2): 228-241.
  15. Stanley, C. E., Shrivastava, J., Brugman, R., Heinzelmann, E., van Swaay, D. and Grossmann, G. (2018). Dual-flow-RootChip reveals local adaptations of roots towards environmental asymmetry at the physiological and genetic levels. New Phytol 217(3): 1357-1369.
  16. Stanley, C. E., Stockli, M., van Swaay, D., Sabotic, J., Kallio, P. T., Kunzler, M., deMello, A. J. and Aebi, M. (2014). Probing bacterial-fungal interactions at the single cell level. Integr Biol (Camb) 6(10): 935-945.
  17. Xing, S., Mehlhorn, D. G., Wallmeroth, N., Asseck, L. Y., Kar, R., Voss, A., Denninger, P., Schmidt, V. A., Schwarzlander, M., Stierhof, Y. D., Grossmann, G. and Grefen, C. (2017). Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance. Proc Natl Acad Sci U S A 114(8): E1544-E1553.

简介

该协议提供了如何制造和使用双流RootChip(dfRootChip)的详细描述,这是一种新型微流体平台,用于研究根管营养,根 - 微生物相互作用以及受控不对称条件下的信号传导和发育。 dfRootChip的开发主要是为了研究植物根系如何通过模拟环境异质性与环境相互作用。 该协议的目标是为希望在其实验室中特别使用dfRootChip或一般微流体装置的生物科学研究人员提供详细资源。

【背景】地下条件是高度异质和动态的,因此植物根部暴露于各种刺激,因此必须适应这种复杂的环境。尽管这些发展适应的重要性,但潜在的机制仍有待阐明。微流体装置已被证明可用于在受控的微环境中培养标本,并有助于从亚细胞到有机物水平的动态过程的实时成像(Crane 等人,,2010)。由于微流体可以以受控方式操纵小流体体积,以高通量进行实验,提取定量信息并进行延时测量,微流体装置已经进入了有机体研究。对于模式植物拟南芥,已经开发了一系列微流体装置,能够在根发育过程中监测基因表达(Busch et al。,2012),信号事件(Keinath et al。,2015)和基于传感器的营养摄取成像(Grossmann et al。,2011; Lanquar et al。, 2014)。此外,使用微流体平台的最新进展包括高分辨率表型分析(Jiang et al。,2014; Xing et al。,2017)和根 - 微生物的调查互动(Parashar和Pandey,2011; Massalha et al。,2017)。虽然可以在这些灌注装置中精确控制根微环境,但环境复杂性(自然根环境的标志)难以模拟(Stanley et al。,2016; Stanley and van der Heijden,2017) 。因此开发dfRootChip是为了能够在细胞水平上研究不对称微环境中的单个拟南芥根,以研究基因表达,信号传导和发育(Stanley et al。,2018) 。重要的是,dfRootChip可以在一系列应用中实施,包括用药物,差异营养素或压力条件进行局部治疗,探测宿主 - 微生物相互作用(例如,致病和有益相互作用,潜在的生物防治剂) ),并调查根生理和根毛发育。

目前的协议旨在为希望实施该平台的研究人员提供基础知识。因此,该协议提供了如何使用光学和软光刻技术制造dfRootChip的详细说明,以及如何在dfRootChip中培养拟南芥幼苗。由于微流体在生物学中的广泛适用性,该协议中的许多步骤也将有助于其他装置设计的制造和处理。此外,该协议说明了如何在三种不同的实验设置中使用dfRootChip。具体而言,我们重点介绍如何进行(i)较长时间段(数小时至数天)的对称和不对称根处理,(ii)用细菌局部接种植物根部和(iii)用dfRootChip快速不对称处理。我们通过分别利用不同的磷酸盐处理,细菌 Pseudomonas fluorescens 和钙诱导剂处理来举例说明这些应用。

关键字:Dual-flow-RootChip, Lab-on-a-chip, 环境感知, 钙信号, 根组织发育, 根毛, 细胞间通讯, 微流体, 植物-微生物互作

材料和试剂

注意:目录号码是为商业非定制产品提供的(见注1)。

  1. 聚酯薄膜光刻掩模(英国微光刻服务有限公司,定制)
  2. 100毫米硅晶圆(硅材料)
  3. SU8 3050光刻胶(MicroChem)
  4. 塑料杯(Semadeni,目录号:8323)
  5. 塑料刮刀(Semadeni,目录号:3340)
  6. 玻璃盖玻片,75 mm x 50 mm,1号(Th.Geyer,目录号:11678524)
  7. 切割刀片(HäberleLabortechnik,目录号:9156110)
  8. Scotch ® Magic TM 隐形胶带(3M)
  9. 微量离心管1.5 ml(Eppendorf Safe-Lock,Eppendorf,目录号:0030120086)
  10. 灭菌过滤嘴100-1,000μl(Pipetman Diamond Tips D1000ST,Gilson,目录号:F171501)
  11. 0.2μm无菌注射器过滤器(Lab Logistic Group,目录号:9.055 511)
  12. 灭菌过滤嘴0.1-20μl(Pipetman Diamond Tips DL10ST,Gilson,目录号:F171201)
  13. 灭菌过滤嘴2-200μl(Pipetman Diamond Tips D200ST,Gilson,目录号:F171301)
  14. 直径94毫米的无菌培养皿(HUBERLAB,目录号:7.663 161)
  15. Parafilm ®(Bemis,HUBERLAB,目录号:15.1550.01)
  16. 硅胶管(TYGON ® 0.020“ID x 0.062”OD;型号ND-100-80)(Th.Geyer,目录号:AAD04103) 
  17. 带Luer锁定配件的23号量针(Gonano Dosiertechnik,目录号:IP423050-EAR-BULK)
  18. 注射器20 ml(VWR,BD Plastipak TM ,目录号:613-3922)
  19. Rotilabo螺旋颈ND24样品瓶(Carl Roth,目录号:LC88.1)
  20. 带钻孔的螺帽(Carl Roth,目录号:LC97.1)
  21. 隔垫Ø22mm,ND24,1.6 mm,55°(Carl Roth,目录号:LC98.1)
  22. 迷你三通旋塞,2 x Luer母头,1 x Luer公头(NeoLab,目录号:270124190)
  23. 公 - 鲁尔连接器(Vygon,目录号:893.00)
  24. 聚苯乙烯比色皿(SARSTEDT,目录号:67.742)
  25. 120 x 120 mm 2 培养皿,无菌(Carl Roth,目录号:PX67.1)
  26. 铝箔(可从任何超市购买)
  27. Arabidopsis thaliana 种子;根据个人需要选择线条
  28. 可选:荧光假单胞菌 WCS365-GFP菌株(Haney et al。, 2015)
  29. mrDev-600开发人员解决方案(Micro Resist Technology)
  30. 异丙醇[(CH 3 ) 2 CHOH](Sigma-Aldrich,目录号:W292907)
  31. 氯三甲基硅烷[(CH 3 ) 3 SiCl](Sigma-Aldrich,目录号:92361)
  32. Sylgard 184 Kit [聚(二甲基硅氧烷),PDMS](Biesterfeld Helvetia,目录号:5498840000)
  33. 丙酮(CH 3 COCH 3 )(Sigma-Aldrich,目录号:00560)
  34. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:71687)
  35. 乙醇(EtOH)(Sigma-Aldrich,目录号:51976)
  36. 去离子水,通过反渗透或超滤净化;以下简称“纯净水”
  37. 次氯酸钠14%Cl 2 水溶液(NaClO)(VWR,目录号:90350.5000)
  38. 微琼脂(Duchefa Biochemie,目录号:M1002.1000) 
  39. Hoagland的2号基础盐混合物(Sigma-Aldrich,目录号:H2395-10L)
  40. MES水合物(C 6 H 13 NO 4 S•xH 2 O)(Sigma-Aldrich,目录号:M8250)
  41. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  42. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  43. 磷酸二氢钾(KH 2 PO 4 )(AppliChem,目录号:A3620)
  44. 硫酸镁七水合物(MgSO 4 •7H 2 O)(默克,目录号:1.05886.1000)
  45. 硝酸钾(KNO 3 )(Sigma-Aldrich,目录号:31263)
  46. 乙二胺四乙酸二钠盐二水合物(Na 2 EDTA•2H 2 O)(AppliChem,目录号:A3553)
  47. 硝酸钙四水合物(Ca(NO 3 ) 2 •4H 2 O)(Sigma-Aldrich,Fluka,目录号:21197)
  48. 硼酸(H 3 BO 3 )(Sigma-Aldrich,目录号:B6768)
  49. 硫酸铜(II)五水合物(CuSO 4 •5H 2 O)(Grüssing,目录号:12079)
  50. 硫酸锌(ZnSO 4 )(Sigma-Aldrich,目录号:Z1001)
  51. 钼酸钠(Na 2 MoO 4 )(Sigma-Aldrich,目录号:243655)
  52. 氯化锰二水合物(MnCl 2 •2H 2 O)(Grüssing,目录号:12097)
  53. 氯化钴(II)六水合物(CoCl 2 •6H 2 O)(Carl Roth,目录号:T889)
  54. 氯化钾(KCl)(AppliChem,目录号:A3582)
  55. Luria Bertani肉汤(Sigma-Aldrich,目录号:L3022)
  56. 硫酸卡那霉素(Carl Roth,目录号:T832.3)
  57. ½xHoagland的中等(½xHM)(见食谱)
  58. ½xHoagland的琼脂培养基(参见食谱)
  59. 富含磷酸盐的培养基(见食谱)
  60. 磷酸盐缺乏培养基(见食谱)
  61. Lysogeny肉汤(LB)培养基(见食谱)
  62. 100 mM盐溶液(见食谱)

设备

  1. Duran ®实验室瓶500毫升(DWK Life Sciences,DURAN,目录号:21 801 44 5)
  2. Duran ®实验室瓶1,000 ml(DWK Life Sciences,DURAN,目录号:21 801 54 5)
  3. 金属销(New England Small Tube,目录号:NE-1310-02)
  4. Schott ®培养管,160 mm x 16 mm(DWK Life Sciences,DURAN,目录号:26 135 21 5)
  5. 压缩空气(无油压缩机,9 L,8 bar)(例如,IMPLOTEX,目录号:NEW-325)
  6. 玻璃烧杯(HUBERLAB,目录号:9.0112.43)
  7. 镊子(VWR,RGS Solingen,目录号:232-0078)
  8. 精密空气调节器(Ashcroft,Ingersoll-Rand,目录号:PR4021200)
  9. 250毫升Erlenmeyer烧瓶(Sigma-Aldrich,DWK Life Sciences,DURAN,目录号:Z232815)
  10. 抹刀(HUBERLAB,目录号:13.1556.05)
  11. Vortex(HUBERLAB,目录号:17.1378.01)
  12. 移液器(Pipetman classic P1000,Gilson,目录号:F123602)
  13. 移液器(Pipetman classic P200,Gilson,目录号:F123601)
  14. 移液器(Pipetman classic P10,Gilson,目录号:F144802)
  15. pH计(Mettler-Toledo International,型号:FiveEasyT M FE20)
  16. 高压灭菌器(Systec,型号:VX-75)
  17. 干燥箱(SalvisLab,型号:VC 20)
  18. 标准冰箱(4-6°C)
  19. 配备紫外线的生物安全柜(Kojair,型号:Kojair ® SilverLine BlueSeries 200)
  20. 精密天平(Mettler-Toledo International,型号:PB3001)
  21. 分析天平(Mettler-Toledo International,型号:AB104-S / FACT)
  22. 旋涂机(SAWATEC,型号:SM-180-BM)
  23. 热板(SAWATEC,型号:HP 160 III BM)
  24. 125毫米x 125毫米x 2毫米钠钙玻璃板(WilliMöller)
  25. 面膜对准器(KarlSüss,型号:MA6)
  26. 定制塑料芯片支架(带有用于RootChip的孔径的框架和适合显微镜载物台的外部尺寸)
  27. 孵化器(Memmert,型号:UM400)
  28. 带有烧瓶摇床的培养箱(Eppendorf,New Brunswick TM ,型号:Innova ® 44) 
  29. 带过滤层流气流的湿式工作台(Goller Reinraum)
  30. 旋涂机(Laurell Technologies,型号:WS-650-23) 
  31. 工具刀
  32. 打孔器(Syneo,型号:Accu-Punch MP10)
  33. 打孔器(切削刃直径,0.71 mm)(Syneo,目录号:CR0350255N20R4)
  34. 打孔器(切削刃直径,1.02 mm)(Syneo,目录号:CR0500355N18R4)
  35. 打孔器(切削刃直径,4.75 mm)(Syneo,目录号:HS1871730P1183S)
  36. 超声波清洗器(BANDELIN electronic,目录号:301)
  37. 等离子清洁器(Diener electronic,型号:FEMTO40kHZ) 
  38. 真空泵(Pfeiffer Vacuum,目录号:PK D56 712)
  39. 真空干燥器(Thermo Fisher Scientific,目录号:5311-0250)
  40. 离心机(Thermo Fisher Scientific,型号:Heraeus TM Pico TM 21,目录号:75002553)
  41. 气候室(Panasonic,PHC,型号:MLR-352H)
  42. Stratalinker ® UV交联剂(Stratagene,型号:1800,目录号:400071)
  43. 注射泵(World Precision Instruments,型号:AL-6000)
  44. 立体显微镜(尼康,型号:SMZ1270)
  45. 分光光度计(GE Healthcare,Novaspec TM Plus,目录号:80-2117-50)
  46. 倒置显微镜(任何型号,特定于用户和应用)
  47. 可选:加压,清洁,干燥空气源

软件

  1. AutoCAD Mechanical 2011(AutoDesk,USA)
  2. 斐济(Schindelin et al。,2012)

程序

  1. 主模具制造(图1中概述的过程;见注1)
    1. 使用旋涂机(SAWATEC)用SU-8 3050光刻胶涂覆100mm直径的硅晶片。根据光刻胶制造商的规格调整旋转速度,可以获得大约115μm的薄膜厚度(见注2)。
    2. 将涂有光致抗蚀剂的晶片放在95℃的热板上并烘烤20分钟(软烘烤,见注2)。可以使用较大的硅晶片或铝箔片来保护加热板免受光致抗蚀剂的污染。
    3. 将光刻薄膜掩模贴在125毫米的钠钙玻璃板上,使用带有内置紫外光源的掩模对准器,通过将抗蚀剂曝光到紫外光源,将dfRootChip设计图案化到光刻胶上。曝光能量为500 mJ / cm 2 (λ= 365 nm)(见注2和3)。钠钙玻璃板的功能是承载掩模并使其保持刚性以确保与抗蚀剂的良好接触。
    4. 将光刻胶在95°C的热板上烘烤20分钟(曝光后烘烤,见注2)。
    5. 为了开发结构(即,去除未曝光的光刻胶),在搅拌下将硅晶片完全浸入SU-8显影剂中<15分钟(mr-Dev 600)开发人员解决方案然后,用新鲜的显影剂溶液冲洗结构 ca。 10秒(见注2)。
    6. 用异丙醇冲洗主模具 ca。 10秒并彻底风干。为确保结构完全干燥,请使用过滤的压缩空气(见注2)。如果在冲洗时产生白色薄膜,则主模具已经开发出来。如果是这种情况,请将母模浸入新鲜的显影剂溶液中,直至胶片被取下并再次用IPA冲洗。
    7. 将母模和含有50μl氯三甲基硅烷的开口玻璃瓶放入真空干燥室内。接下来,通过施加50毫巴的真空压力抽空腔室,然后关闭阀门以密封腔室并使主模具在氯三甲基硅烷蒸气中孵育至少1小时。

    图1. dfRootChip制造。 A.说明了制造dfRootChip所涉及的光刻和软光刻工艺。首先,用SU8光致抗蚀剂(1)涂覆硅晶片,之后使用光刻掩模(2)来曝光光致抗蚀剂的特定区域以准直UV光(3)。在曝光期间,光致抗蚀剂聚合(4)然后显影。显影去除未聚合的光致抗蚀剂区域,产生具有图案化微结构(<5)的母模。然后将聚(二甲基硅氧烷)(PDMS)浇铸并固化(6),之后可将其从母模(7)中取出并切割,冲压并粘合到涂有薄层PDMS的玻璃盖玻片上以形成 PDMS设备(8)。 B.为了补充A中的“侧视”图示,为了清楚起见,已包括光刻掩模的“顶视图”图示,主模和 PDMS设备。 C-即示例性光刻掩模(安装在钠钙玻璃板上),主模和PDMS装置的照片分别以C,D和E显示。 F.为清楚起见,显示了入口和出口的具体尺寸和位置。

  2. PDMS器件制造(有关PDMS器件制造的详细视频说明,请参见视频1;图1和图2中概述的过程;见注1和4)
    1. 使用10:1比例的碱与固化剂(Sylgard 184 Kit)制备聚(二甲基硅氧烷)(PDMS)&nbsp;
    2. 用刮刀彻底混合。
    3. 使用50毫巴的真空压力将混合物脱气1小时。
    4. 将PDMS倒在主模上(见注5)。&nbsp;
    5. 涂上五层干净的盖玻片(用丙酮冲洗,IPA和吹干),涂上一层薄薄的PDMS(Laurell Technologies,旋转涂层,转速约为1,200转,持续25秒)。&nbsp;
    6. 将主模和涂覆的盖玻片在70℃的烘箱中固化过夜。
    7. 使主模冷却至室温并除去固化的PDMS。
    8. 使用美工刀切割PDMS设备。在图2E所示的切割装置中,刀片固定在定制的金属框架中并向下推动以切割固化的PDMS板坯。
    9. 打孔以形成根入口(Ø= 1.02 mm),溶液入口和出口(Ø= 0.71 mm)和储液器(Ø= 4.75 mm)。根部入口应在 ca处打孔。 30-45°角。&nbsp;
    10. 使用超声波浴清洗以下每种溶液中的装置和盖玻片5分钟:0.5mM NaOH,70%EtOH和纯净水。在每个清洗步骤之间用纯净水冲洗设备。&nbsp;
    11. 用压缩空气干燥设备和盖玻片,并在70°C的烘箱中放置1小时。
    12. 使用等离子清洁器将干燥的装置粘合到盖玻片上(见注6)。在粘合之前,透明胶带可用于去除PDMS表面的灰尘颗粒。

    图2. PDMS设备制造。说明了准备PDMS设备所涉及的过程(详见过程B)。通过将碱和固化剂混合在一起(A)并将混合物(B)脱气来制备PDMS。然后将脱气的PDMS倒在母模(C)的顶部,然后在烘箱(D)中固化后除去。在切割出(E)并将孔(F)冲入PDMS后,将其洗涤(G),干燥(H)并粘合到玻璃基板(I-L)上。有关PDMS设备制造的详细视频说明,请参见视频1.


    视频1. PDMS设备制造。该视频显示了协议程序B和图2中概述的PDMS设备制造的整个过程。

  3. 准备用于片上植物栽培的双流RootChip(见注1)
    1. 粘接后,应立即用半强度的Hoagland介质(½xHM,参见配方1)填充设备。要做到这一点,手动将½xHM通过根入口吸入每个微通道。当介质通过入口和出口排出时,微通道被填充。检查气泡,并根据需要使用更多介质以去除气泡。使用½xHM填充进样口/出口和储液器以确保它们完全充满。
    2. 使用UV交联装置使用紫外线对设备进行灭菌30分钟。

  4. 表面种子灭菌(见注1和7)&nbsp;
    1. 将一个装有拟南芥种子(约100个)的小抹刀放入2ml微量离心管中。
    2. 向微量离心管中加入1ml无菌过滤的次氯酸钠溶液(5%)。
    3. 用手摇动种子使种子悬浮,然后在中等水平上涡旋3-5分钟。
    4. 将带有种子的试管放入 ca。 100 x g 的离心机中几秒钟以使种子旋转。在无菌条件下(生物安全柜)继续操作。
    5. 移取上清液。&nbsp;
    6. 将种子重悬于1ml高压灭菌的纯净水中。
    7. 置于涡旋上20秒。
    8. 重复步骤D4-D7,直至种子用水洗涤三次。
    9. 将种子储存在冰箱(4°C,在纯净水中)中三天进行分层。

  5. 种子农场准备(参见图3;参见注释1和7;种子农场准备程序的视频已发表在Grossmann 等人, 2012)。
    注意:本节中的步骤应在无菌条件下进行(生物安全柜)。
    1. 准备含有0.7%植物琼脂的培养基(例如,见配方2)。此步骤中使用的介质取决于要进行的实验。&nbsp;
    2. 在温暖的情况下( ca。 50°C),将含琼脂的培养基倒入无菌培养皿(例如,10)中,并加入高压灭菌的0.1-20μl移液器吸头( Gilson DL10ST)与5μl相同的培养基(每个种子农场 ca。 48个吸头)。让介质完全冷却。未用于种子农场准备的培养皿可用Parafilm ®密封,并存放在冰箱中备用。&nbsp;
    3. 使用加热灭菌的切割刀片将移液管尖端切割成约5mm的最终长度。
    4. 将移液器吸头竖直放入琼脂板(每块板 ca。 48个吸头)。
    5. 取以前用表面灭菌的拟南芥种子,用移液管将一粒种子放在每个移液管尖端上。
    6. 用Parafilm ®密封平板并将它们放入气候室中。在长日照条件下连续生长植物(在100μEm -2 sec -1 ,22°C,50-70%相对湿度下16小时光照)。 />

    图3.拟南芥种子农场。 A.这张照片说明了一个“种子农场”,它可以容纳5天大的拟南芥幼苗,该幼苗在琼脂填充的切割移液器吸头上发芽。通过将拟南芥种子置于保持在琼脂平板中的中等填充的移液管尖端上来产生种子场。 B.在转移到芯片上之前,在解剖显微镜下选择具有适当根长度的幼苗(在A培养皿底部标有蓝点)。

  6. 植物选择(见注1;选择程序的视频已发表在Grossmann 等, 2012)
    1. 将一个5天大的种子农场(见注8)放在立体显微镜下。
    2. 调整培养皿的光照水平和方向,直到根部可见。&nbsp;
    3. 确定每dfRootChip 8-10株植物,其中根系处于相同的发育阶段。优选地,根刚刚到达移液管尖端的末端,但没有生长通过开口。如果一个设备中的所有根处于相同的发育阶段,则稍微短的根可以起作用。&nbsp;
    4. 标记选定的植物以供以后识别。

  7. 将植物转移到双流RootChip上(参见注释1和7;转移程序的视频已发表在Grossmann 等, 2012)。
    注意:这些步骤需要无菌条件(生物安全柜)。&nbsp;
    1. 对两对镊子进行消毒,并采用预先粘合并填充的dfRootChip(参见程序C)。
    2. 使用镊子,将含有植物的预先确定的移液器吸头轻轻插入植物入口(参见程序F)。为防止将气泡引入微通道,首先在植物入口上放置少量培养基。
    3. 确保将移液器吸头插入植物入口,直到它们几乎接触通道底部。这将有助于确保微通道中的根生长始终如一。
    4. 在填充所有植物入口后,将dfRootChip放入无菌培养皿中。
    5. 用 ca.填充培养皿。 15毫升½倍HM(参见配方1)。
    6. 用Parafilm ®密封培养皿,放置在气候室中 ca。 3-4天,或直到微通道中可见几个根尖。

  8. 使用双流RootChip进行对称和非对称处理:以磷酸盐处理为例(见图4和注1)
    1. 准备管道:对于dfRootChip上的每个工厂准备两个相同长度的管道,一端带有鲁尔锁定剂量针,另一端带有金属连接器针脚(通过入口将“注射器”连接到设备,“入口”管道) ,加上另一长度的管道,仅在一端带有金属连接器针脚(将设备出口连接到废液容器,“出口”管道)(图4C)。在选择合适的入口管长度时,需要考虑安装在显微镜载物台上的芯片完全横向移动所需的距离。
    2. 高压灭菌所有管道,然后放入生物安全柜中。&nbsp;
    3. 获取含有拟南芥植物的dfRootChip(参见程序G)并放入生物安全柜中。使用放大镜或解剖显微镜,确保根已经生长到微通道中并且已经达到足够的长度,优选地在通道的开始处。此外,确保植物本身看起来健康,带有绿色子叶。
    4. 通过金属针将“插座”管连接到dfRootChip插座。&nbsp;
    5. 在生物安全柜内,用所需的培养基填充无菌注射器(见注9)。例如,为了用5个植物进行实验,将需要10个注射器。要执行不对称治疗,请使用第一个感兴趣的介质(例如,参见食谱3)填充一半注射器,并使用第二个感兴趣的介质填充一半(例如,请参阅食谱4)。
    6. 使用剂量针将每个“入口”管连接到注射器。手动将介质推入每根管道,避免产生气泡。如果气泡留在腔室内,一旦灌注开始,气泡就会被推出。


    图4.使用dfRootChip进行对称和非对称处理的实验装置。芯片支架(A)用于在进行实验时容纳dfRootChip(B)。 dfRootChip连接到注射泵(C-E),以根据所选择的处理灌注根部。盖子和湿纸巾用于保持植物周围的湿度。然后可以将设置转移到显微镜上以对根进行成像(F,比例尺=250μm)。

    1. 将注射器装载到注射泵上,输入注射器直径并以高初始流速(,例如,100μl/ min)开始灌注。请注意,注射泵可以保留在生物安全柜外面。&nbsp;
    2. 启动泵并使其保持运行,直到介质被推出松散的管端。&nbsp;
    3. 将流速设置为5μl/ min(图4D)。
    4. 当泵仍在运行时,使用无菌镊子通过金属连接器针将“入口”管连接到入口。这确保了没有气泡进入微通道。&nbsp;
    5. dfRootChip现在可以进行成像了。将dfRootChip放入芯片支架(图4A)并将设置传送到倒置显微镜。

  9. 用微柱阵列捕获的细菌对拟南芥根进行局部接种:以荧光假单胞菌菌株WCS365-GFP(Haney 等, 2015)为例进行概念验证(参见注释) 1)
    1. 在28℃下在100ml补充有50μg/ ml卡那霉素的LB培养基中振荡(200rpm)培养荧光假单胞菌。
    2. 检查细菌培养物的OD(600nm)。对于处于对数期的细菌,OD应在0.1-0.2的范围内。
    3. 将培养物转移到20ml Falcon管中,并以12,000 x g 离心5分钟。
    4. 弃去上清液,将沉淀重悬于20ml的1/2x HM中。
    5. 再次以12,000 x g 离心5分钟。
    6. 重复步骤I4和I5以去除任何痕量的LB和卡那霉素。
    7. 轻轻地将细菌沉淀重新悬浮在½xHM中并用它来填充注射器。
    8. 用 P接种拟南芥根。荧光灯,将注射器连接到双流RootChip,如程序H中所述。

  10. 在双流 - RootChip中进行快速不对称处理:使用100 mM NaCl溶液进行钙诱导剂处理,作为概念验证(参见图5和注释1)
    1. 准备可加压的小瓶(螺旋瓶小瓶; dfRootChip上每个植物两个):将隔垫放入瓶盖,用金属针刺穿,将针留在隔垫中。将一段管子连接到瓶子的底部,到达销子的内端,并将一段管子连接到销子的外端。高压灭菌瓶子和附加管道。此外,高压灭菌器,每个工厂,两个额外长度的入口管道,一端带有金属销,另一个带有一个出口管道。&nbsp;
    2. 将含有拟南芥植物的dfRootChip放入生物安全柜中(参见程序G)。确保根已经生长到微通道中并且已经达到足够的长度,优选地在通道的开始处(参见步骤H3)。
    3. 用对照培养基(例如,见配方1)填充其中一个无菌小瓶,用处理培养基(例如,100mM NaCl,见配方6)填充另一个。
    4. 将“luer-lock stopcock阀组”(乙醇灭菌)以“关闭”配置添加到来自每个样品瓶的管道末端。将额外长度的管道添加到旋塞阀出口。
    5. 通过将额外长度的管道连接到加压,清洁,干燥的空气源并用销钉刺穿隔垫来加压样品瓶。打开每个旋塞,用介质完全填充管道。一旦管道填满,再次关闭旋塞阀。
    6. 将装有控制介质的样品瓶连接到其中一个介质入口并打开旋塞阀。在我们的设置中,5 PSI的压力导致体积流速为20μl/ min。最初,根被对称灌注,并且对照介质将流出介质出口和第二介质入口。
    7. 将含有处理溶液的小瓶连接到第二个介质入口。来自对照介质的活性流将阻止处理溶液进入微流体通道。
    8. 准备好进行治疗时,打开与治疗瓶相关的旋塞,开始快速不对称灌注。
    9. 要停止不对称治疗,请关闭与治疗瓶相关的旋塞。在几秒钟内,灌注将恢复到对称控制条件。


    图5.在dfRootChip中进行快速不对称处理的实验装置。此图说明了气压源,可加压的样品瓶和旋塞阀是如何连接并与dfRootChip连接的。

数据分析

  1. 关于实验设计,每个dfRootChip可以并行执行多个实验(即,技术复制)。此外,每个数据集应至少进行三次生物学重复。分析使用dfRootChip获取的数据集的示例可以在原始文章Stanley et al。(2018)中找到。
  2. 关于数据分析,我们建议使用斐济的手绘线工具和kymograph功能来分析例如初生根和根毛生长速率。要访问有关图像分析的大量信息,我们建议您访问以下网站: http://www.plant -image-analysis.org

笔记

  1. 请联系我们获取更多信息和建议。
  2. 该协议已用于我们之前发表的工作(Stanley et al。,2018)。所描述的程序中与dfRootChip的使用不相关的部分与以前发表的作品相似或改编(Grossmann et al。,2011和2012; Stanley et al。,2014)但概括为提供全面而完整的协议。&nbsp;
  3. 为获得最佳效果,我们建议整个制造过程在1000级洁净室内进行。主模制造程序必须在黄光下进行。我们还建议所有湿式化学操作都在湿式工作台中进行,并使用过滤层流气流。如果没有洁净室设施,可以与研究小组或其他能够完成制作的商业服务合作实现。请联系我们获取更多信息和建议。
  4. 使用AutoCAD Mechanical 2011绘制dfRootChip的通道图案。然后可以由商业提供商将设计文件打印为聚酯薄膜光刻掩模。掩模布局被布置成使得几个dfRootChip复制品可以装配在单个100mm硅晶片中。请联系我们获取更多信息和建议。
  5. PDMS装置制造中涉及的过程应在具有过滤层流气流的工作台中进行。
  6. 主模具可以放置在3D打印的塑料支架中,如图2所示。或者,铝箔可以围绕玻璃培养皿成形,并用于在将PDMS浇注到微结构顶部时保持母模。
  7. 当使用等离子体清洁剂时,采用以下条件:功率,50%;治疗时间,1分钟。但是,这可能会因产品而异,因此必须确定每种等离子清洁剂的理想条件。&nbsp;
  8. 在无菌条件下工作(例如,在生物安全柜中或在火焰旁边)。
  9. 实验室之间的生长条件会有所不同。因此,重要的是确定种子农场应在气候室中孵化多少天。植物根部不应长出移液管尖端。
  10. 使用足够大小的注射器以满足特定的实验要求。作为参考,使用5μl/ min的流速在24小时内(每个注射器)使用7.2ml培养基。

食谱

  1. ½xHoagland的中等(½xHM)(1升)
    1. 混合0.8克Hoagland的2号基础盐混合物和1克MES水合物
    2. 将上述试剂转移到1L烧杯中并加入900ml纯净水
    3. 在磁力搅拌器的帮助下将所有上述试剂溶解在纯净水中
    4. 用KOH将pH调节至5.7
    5. 加入纯净水使培养基的总体积达到1000毫升
    6. 在121℃下将培养基高压灭菌20分钟
  2. ½xHoagland的琼脂培养基(1升)
    1. ½xHM如上所述制备(参见配方1步骤1a-1d)
    2. 称取7 g植物琼脂(w / v)并将其加入½xHM
    3. 用纯净水使介质总体积达到1000毫升
    4. 在121℃下将培养基高压灭菌20分钟
  3. 富磷培养基(如Chandrika et al。,2013)
    1. 将以下组分混合在一起,在纯净水中产生这些最终浓度:
      2.5mM KH 2 PO 4
      2mM MgSO 4 •7H 2 O
      5 mM KNO 3
      2 mM Ca(NO 3 ) 2 •4H 2 O
      0.04mM Na-Fe-EDTA
      70μMH 3 BO 3
      0.5μMCnSO 4 •5H 2 O
      1μMZnSO 4 •7H 2 O
      14μMMnCl 2 •2H 2 O
      0.2μMNa 2 MoO 4
      0.01μMCoCl 2 •6H 2 O
      1克/升MES水合物
    2. 使用0.5M KOH将pH调节至5.5
    3. 通过0.2μm膜对培养基进行无菌过滤
  4. 磷酸盐缺乏培养基(如Chandrika et al。,2013)
    1. 将以下组分混合在一起,在纯净水中产生这些最终浓度:
      0.01mM KH 2 PO 4
      2.49 mM KCl
      2mM MgSO 4 •7H 2 O
      5 mM KNO 3
      2 mM Ca(NO 3 ) 2 •4H 2 O
      0.04mM Na-Fe-EDTA
      70μMH 3 BO 3
      0.5μMCnSO 4 •5H 2 O
      1μMZnSO 4 •7H 2 O
      14μMMnCl 2 •2H 2 O
      0.2μMNa 2 MoO 4
      0.01μMCoCl 2 •6H 2 O
      1克/升MES水合物
    2. 使用0.5M KOH将pH调节至5.5
    3. 通过0.2μm膜对培养基进行无菌过滤
  5. Lysogeny肉汤(LB)培养基(1 L)
    1. 将20g LB肉汤混合物和5g NaCl加入1L纯净水中
    2. 在121℃下将培养基高压灭菌20分钟
  6. 100 mM盐溶液(100 ml)
    1. 准备½xH3,如配方1中所述
    2. 将0.584g NaCl溶解在100ml½xHM中
    3. 通过0.2μm膜无菌过滤溶液

致谢

该协议改编自Stanley 等人(2018)。我们感谢瑞士国家科学基金会提供的资金支持,以Ambizione Career Grant(PZ00P2_168005)的形式提供给CES,斯伦贝谢基金会为JS,Deutsche Forschungsgemeinschaft(GR4559 / 3-1)和研究组提供未来奖学金。从海德堡卓越集群CellNetworks到GG的资金。

利益争夺

DvS代表瑞士Wunderlichips GmbH。所有其他作者均未就本出版物声明任何竞争利益。

参考

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引用:Stanley, C. E., Shrivastava, J., Brugman, R., Heinzelmann, E., Frajs, V., Bühler, A., van Swaay, D. and Grossmann, G. (2018). Fabrication and Use of the Dual-Flow-RootChip for the Imaging of Arabidopsis Roots in Asymmetric Microenvironments. Bio-protocol 8(18): e3010. DOI: 10.21769/BioProtoc.3010.
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