Tracking Root Interactions System (TRIS) Experiment and Quality Control
根相互作用系统的示踪实验和质量控制   

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Proceedings of the National Academy of Sciences of the United States of America
Apr 2017

 

Abstract

Soil organisms are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in shaping the rhizosphere. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited due to methodological intricacy. In this study, we developed a novel microfluidics-based device enabling direct imaging of root-bacteria interactions in real time.

Keywords: Tracking root interactions system (TRIS) (根相互作用系统示踪), Live-imaging microscopy (活体成像显微镜), Root-bacteria interactions (根细菌相互作用), Microbial community dynamics (微生物群落动态), Bacillus subtilis (枯草芽孢杆菌), Arabidopsis thaliana (拟南芥)

Background

Plant roots play a major role in shaping plant-microbe interactions within the rhizosphere, driving a dynamically changing microenvironment. Recent studies revealed multiple beneficial effects of root-associated bacteria (Hardoim et al., 2008; Weyens et al., 2009; Mei and Flinn, 2010; Santhanam et al., 2015), highlighting the important role of rhizosphere interactions. Tracking these interactions with high spatio-temporal resolution is a challenging task that is primarily limited by available methodologies. Several microfluidic approaches have recently been adapted for the use in the plant sciences (reviewed by Sanati Nezhad, 2014 and Stanley et al., 2016) mainly for root development and physiological studies (Englert et al., 2010; Grossmann et al., 2011; Jiang et al., 2014). The TRIS device presented here builds on these developments to provide a robust platform for microscopy-based studies of the interactions between plant roots and associated microorganisms that can be implemented in a typical experimental biology laboratory (Massalha et al., 2017). The TRIS device enables precise control over environmental parameters within the system while allowing direct observation of dynamic biological processes at high spatial and temporal resolutions. Here we discuss in details the TRIS assembly and operation. Studying root-microbe interactions in this device allows the use of endless combinations of bacterial strains and plant genotypes that could be examined with multiple fluorescent reporters. Altogether, we hope and anticipate that the TRIS experimental system, and other platforms building upon it, will open the way for extensive high-resolution studies of the dynamic root microenvironment and interactions within it.

Materials and Reagents

  1. Petri dishes (150 mm x 25 mm) (Corning, catalog number: CLS430597)
  2. Petri dishes (100 mm) (Corning, catalog number: CLS430591)
  3. 200 µl plastic tips
  4. Sterile 14 ml polystyrene culture tubes (Corning, catalog number: 352057)
  5. Glass slides (75 mm x 25 mm) (Thermo, catalog number: 421-004T)
  6. Adhesive tape (Cellophane tape)
  7. Millex-HV Syringe Filter Unit, 0.45 µm, PVDF (Merck, Millipore, catalog number: SLHVM33RS)
  8. 1 ml syringes (BD, catalog number: 309647)
  9. Polyethylene tubing (OD 1.2 mm) (BD, IntramedicTM, Clay Adams®)
  10. Regular bamboo toothpicks (can be obtained from any supermarket)
  11. Parafilm
  12. 1.5 ml tubes (Eppendorf, catalog number: 0030120086)
  13. Blunt end needle (McKESSON, catalog number: 8881202363)
  14. Cotton (can be obtained from any supermarket)
  15. Biohazard bag (the institute warehouse)
  16. Arabidopsis seeds (Col-0)
  17. Bacillus subtilis
  18. Glycerol stock of Bacillus subtilis NCIB 3610 mCherry reporter strain (obtained from a single colony, stored at -80 °C)
  19. Polydimethylsiloxane (PDMS) and Curing agent (Sylgard 184 Silicone Elastomer Kit) (Dow Corning Corporation)
  20. 100% Ethanol (Sigma, catalog number: 34852)
  21. dH2O (Sigma, catalog number: W4502)
  22. KOH (Sigma, catalog number: P5958)
  23. HCl (Sigma, catalog number: H1758) and bleach (Sigma, catalog number: 425044) for seed sterilization.
  24. Half strength plant growth media, basal salt Murashige and Skoog (MS) (Duchefa Biochemie, catalog number: M0221)
  25. Plant agar (Duchefa Biochemie, CAS number: 9002-18-0)
  26. TB buffer (see Recipes)

Equipment

  1. Forceps
  2. Scalpel
  3. 250-ml glass flask
  4. Pipettes (Eppendorf 20, 200, 1,000 µl)
  5. Oven
  6. Hot plate
  7. OD (600 nm) reader
  8. 37 °C shaking incubator
  9. Microfluidic channel mold (see attached CAD file)
  10. SU-8 2150 (MicroChem Corp.)
  11. Desiccator with vacuum line
  12. Harris Unicore hole punch (1 mm) (Ted Pella, catalog number: 15074)
  13. Hand-held corona generator (Laboratory Corona Treater, Electro-Technic Products, model: BD-20AC)
  14. Autoclave
  15. Desiccator
  16. Inverted optical microscope equipped with dark-field objective (e.g., Olympus)
  17. Confocal microscope with automated stage system (e.g., Nikon Ti-eclipse)

Software

  1. Dedicated confocal microscope software
    In this study, we used the Nikon confocal and the NIS-element program.
  2. Fiji (ImageJ)

Procedure

  1. Tracking root interactions system (TRIS) device fabrication and assembly (start a few days before the experiment)
    1. Microchannel molds (Figure 1A) were prepared by depositing the photoresist SU-8 2150 on silicon wafers using soft lithography techniques (Qin et al., 2010).
    2. The procedure was applied based on the manufacture guide sheet to achieve 160 µm depth.
      Note: Read the manufacturer’s recommendations for proper handling of such viscose material (SU-8 2150).
    3. TRIS device assembly:
      1. Place the mold into a 150 mm Petri dish with the patterned mold side of the etched photoresist facing up (Figure 1A). Fix the mold to the plate with sticky tape.
      2. Prepare 25 ml of the PDMS mixture based on the manufacturer’s recommendations. In a plastic weigh-boat, weigh 10 parts silicon elastomer then add 1 part of the curing agent. Mix well the two reagents using a plastic stick.
      3. Place the dish in a desiccator and degas under vacuum for 1 h. It is important to remove all air bubbles from the PDMS mixture before pouring it to the fixed mold on the dish.
      4. Blow nitrogen or air to remove any dust that may have accumulated on the mold. Carefully pour the degassed mixture over the silicon master.
      5. Cure the PDMS for overnight at 60 °C.
        Note: Read the manufacturer’s suggestions for curing PDMS.
      6. Using a sharp blade (Scalpel) carefully cut out each adjacent 8/9 features in a single rectangular piece (Figures 1B and 1C). The patterned microchannels will be visible in the PDMS.
      7. Punch holes at the channel inlets, outlets and the root hole (Figures 1C and 1D) with the 1 mm Harris Unicore hole punch.
      8. After finishing making the holes, cover both sides of the channels (e.g., with adhesive tape) to protect from dust particles and debris.
      9. Before binding the PDMS slab to the glass slide, remove the adhesive tape, place the glass slide and PDMS channel side up on a clean flat surface. In order to activate the surfaces of the PDMS and the glass slide, which will lead to covalent bonding of the two materials, we used the hand-held corona generator approximately 1-2 cm above the surface. The surfaces were treated for 30 s uniformly over each surface.
      10. Carefully place the PDMS device on the treated glass slide with the microchannel pattern facing the glass slide. Apply gentle, uniform pressure by hand to ensure a uniform seal.
      11. Bake the mounted channel with the glass slide at 100 °C for 20 min directly after sealing.
      12. Autoclave the mounted microfluidic devices once at 120 °C for 20 min. Store the autoclaved samples sterilely at room temperature conditions.


      Figure 1. TRIS: a microfluidic device for live tracking of root-bacteria interactions. A. Microfluidic device mask (pattern) design used for the photolithography process to generate an array of nine single microfluidic channels. Left side: Magnification of one single-channel dimensions. B. Illustration of the TRIS device mounted on the microscope stage (dark rim). (Inset) Schematic of a longitudinal section of a microfluidic channel-containing root and bacterial cells (red; not drawn to scale). C. Top view of Arabidopsis seedlings growing in plastic pipette tips attached to the TRIS device. Inlet, plant and outlet ports are attached to the inlet tube, tip with seedling and outlet tube, respectively. Roots are visible as thin white strings extending from the tip ends. (Scale bar: 1 cm). D. Microscopic view of nine Arabidopsis roots growing inside the TRIS microfluidic device captured using bright-field illumination at 10x magnification. Arrows: a, inlet port; b, tip in a root-dedicated port with c, root extending toward the outlet port; d, outlet port. (Scale bar: 5 mm). This figure adapted from Massalha et al. (2017).

  2. Bacterial culture preparation (start one day before the experiment)
    1. Place B. subtilis culture in a sterile polystyrene culture tube containing 3 ml TB medium into a shaking incubator at 37 °C, 165 rpm for overnight.
    2. Four hours before the experiment dilute 1:100 into fresh 3 ml TB medium and incubate again in the same conditions to reach OD600 of 0.6.
    3. Wash cells twice with MS, and resuspend the cells in MS medium to obtain 106 cells/ml (filter the MS before using to eliminate possible small debris using 0.45 µm PVDF syringe filter).
    4. Estimate the ratio of swimming cells to static cells by counting the number of the moving cells from a few frames recorded using dark-field microscopy.

  3. Plant growth (start 5-6 days before the experiment)
    1. Medium and plastic tip preparation:
      1. Prepare 0.5x strength basal salt MS. Adjust the pH to 5.8 using KOH. Divide the prepared media into two portions. Add 1% plant agar to one portion.
      2. Sterilize the two portions of the medium.
      3. Sterilize 200 µl plastic tips, and bamboo toothpicks, by autoclave.
      4. After cooling down the medium melt the solidified medium and fill the sterilized 200 µl tips with 5 µl. Allow medium to solidify again. It is important to use the liquid media right before it solidifies at the room temperature (Figure 2A shows plastic tips with solidified medium).
      5. Before the medium stock solidifies, fill a 90 mm Petri dish with ~30 ml of medium. Allow the medium in the Petri dish to solidify.
      6. Cut the filled tips with a flamed scalpel just above the level of the solidified medium in the tip in a sterile environment. Use flamed forceps to hold the tips in a sterile 150 mm Petri dish.
      7. Collect the filled plastic tips with flamed forceps and put them in the solidified agar (in the 90 mm Petri dish prepared in Step C1e). Filled tips fixed in the solidified agar in a Petri dish can be sealed with parafilm and stored at 4 °C for later use.
    2. In parallel to (1), sterilize Arabidopsis (e.g., Col-0) seeds in chlorine gas for 2 h in a closed desiccator. Place open 1.5 ml tubes with the seeds in a desiccator vessel (in the fume hood). Place a 250-ml glass flask containing 100 ml of bleach into the desiccator and 6 ml of concentrated HCl, immediately close and seal the desiccator with parafilm. After 2 h, open the desiccator in the fume hood and let the chlorine vapor dissipate for several minutes.
    3. Using a sterile toothpick, transfer one seed aseptically to a plastic pipette tip containing half-strength basal salt MS supplemented with 0.8% plant agar (Figure 1C). Prepare as much tips with seeds as needed for your experiment.
    4. Stratify seeds by keeping the plates with tips two days in the cold room covered to avoid light.
    5. Transfer seed-containing tips to a 16-h light/8-h dark period at 23 °C for 3-4 d (or before the growing root penetrates from the end of the plastic tip) (Figure 2A).
    6. Put the mounted microfluidic devices in a box, in a biohazard bag and sterilize it using autoclave (similar to Step ‘A3l’ in TRIS device assembly section).
    7. Fill carefully the autoclaved microfluidic device with liquid 0.5x MS to eliminate air bubbles trapped in the system in sterile conditions.
    8. Place plastic tips with growing seedlings in the autoclaved TRIS device in sterile conditions (Figures 1A and 1B).
    9. Incubate vertically for an additional 4-5 d before imaging (until the growing root reaches the middle of the TRIS channel). Add liquid medium to the vertical plate to cover the output hole and maintain humidity (Figure 2B). Seal the plate with parafilm to prevent any liquid leakage.


      Figure 2. Preparation of Arabidopsis seedlings for tracking root-bacteria interaction using the microfluidics TRIS device. A. Tips of plastic tips filled with solidified agar (the filled agar settled to the same level in all of the tips, indicated by the height of the Arabidopsis seed in the transparent plastic tip) in which Arabidopsis seedlings were germinated placed in agar plates. B. Arabidopsis seedlings in plastic tips fixed to the TRIS device and incubated vertically.

  4. Mount the TRIS system to the microscope stage (start 2 h before imaging)
    1. During bacterial incubation time, wash all tubing that will be attached to the microfluidics chambers. Prepare two tubes per channel (long and short tubings, see next steps to determine the length of each tube). Clean all tubing with 70% ethanol to remove possible manufacture contaminants. Wash the tubings twice with dH2O to remove ethanol remaining using a 1 ml syringe. 
    2. Fix a blunt end needle to 1 ml syringe containing washed bacteria in plant medium. Remove air bubbles from the opening of the syringe. Avoid fast fill to prevent air bubbles.
    3. Connect the long tubing to the blunt end needle and fill the tubes by pushing the piston of the syringe. Make sure the length of the tube is enough to allow flexibility during the assembly of the system on the microscope stage (see Figure 3).


      Figure 3. View of the TRIS device mounted onto a microscope stage. The device contains microfluidics channels with Arabidopsis seedlings and the entire setup includes tubing systems, 1 ml syringes, a medium reservoir, and a humidity chamber made of acrylic plastic (Scale bar: 5 cm). Modified from Massalha et al. (2017).

    4. Keep the short tube without filling with liquid. 
    5. Prepare Eppendorf tubes according to the number of microchannels. Make one hole in the lid of the Eppendorf tube using fine forceps and fill with 1 ml MS.
    6. Quickly, remove the TRIS device from the vertical plate, dry liquid drops with Kimwipe (avoid drying the content of the TRIS device by touching the inlet/outlet holes), fix the TRIS device to the microscope stage holder (preferable slide holder without openings to keep humid conditions).
    7. Insert the long tube connected to the syringe to the inlet hole and the short tube to the output hole of each microchannel. Apply enough pressure to force the tubing roughly half-way from the PDMS surface to the glass slide but carefully to avoid cracks in the PDMS inlet and outlets.
    8. Quickly cover the device with a transparent cover to keep humidity (Figure 3).
    9. Put inside the transparent cover Petri dish a wet cotton to keep the humidity.
    10. Dip the second end of the output tube (short tube) into an Eppendorf tube filled with 1 ml MS media.
    11. Set the microscope parameters:
      1. Scanning (x, y)-coordinates of each channel (stitching parameters should be tested in a preliminary experiment).
      2. Exposure time of all used channels (should be tested in a preliminary experiment).
      3. Number of cycles.
      4. Cycle intervals.
      5. Find the mid-section of the root. Set the ± 40 µm z-stacks.
    12. Introduce the bacteria to the microfluidic channel by pushing the piston of the syringe slowly. Continue pushing the piston until the outlet tube will be filled.
    13. Immediately after finishing Step D9, start scanning that microchannel.
    14. After Step D10 is finished, pause the microscope, introduce the bacteria to the next microchannel, and then continue scanning.
    15. Keep doing Steps D12 to D14 until the last microchannel.

  5. Image analysis
    1. Fiji is used to visualize the results and generate the summarizing movies we used the LOCI package to open bio-microscopy formats) (Schindelin et al., 2012).
    2. Extended depth of field package (Fiji, http://imagej.net/Extended_Depth_of_Field) or FStack function from the File Exchange repository (MathWorks) is used to generate a single EDF image from the different z-stacks.
    3. Matlab is used to segment the EDF images based on the brightness of the bright field channel in each image and then to calculate the relative intensity of the bacterial signal.

Data analysis

The main outcome of the TRIS device is the bacterial density as functions of location and time. The relative intensity of the bacterial fluorescence is used to estimate the density of the bacteria and calculated by subtracting the background for each time point from the region of interest. Other parameters can be calculated using the TRIS device, i.e., root elongation and bacterial dynamics. Data analysis at this stage become highly depended on the biological question.

Notes

The captured volume of the TRIS device is only ~6.4 μl per plant, thus makes plants sensitive to environmental permutations. It is highly recommended to track the growth conditions, especially when moving the plants to the TRIS device.

Recipes

  1. TB buffer
    1% (w/v) tryptone
    0.5% (w/v) NaCl
    Autoclave

Acknowledgments

This protocol is adapted from Massalha et al. (2017). This research was supported as part of a PhD funded by a Planning & Budgeting Committee of the Council of Higher Education of Israel personal grant (to H.M.).

References

  1. Englert, D. L., Manson, M. D. and Jayaraman, A. (2010). A microfluidic device for quantifying bacterial chemotaxis in stable concentration gradients. J Vis Exp(38) pii: 1779.
  2. 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.
  3. Hardoim, P. R., van Overbeek, L. S. and Elsas, J. D. (2008). Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16(10): 463-471.
  4. 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.
  5. 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.
  6. Mei, C. and Flinn, B. S. (2010). The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement. Recent Pat Biotechnol 4(1): 81-95.
  7. Qin, D., Xia, Y. and Whitesides, G. M. (2010). Soft lithography for micro- and nanoscale patterning. Nat Protoc 5(3): 491-502.
  8. Sanati Nezhad, A. (2014). Microfluidic platforms for plant cells studies. Lab Chip 14(17): 3262-3274.
  9. Santhanam, R., Luu, V. T., Weinhold, A., Goldberg, J., Oh, Y. and Baldwin, I. T. (2015). Native root-associated bacteria rescue a plant from a sudden-wilt disease that emerged during continuous cropping. Proc Natl Acad Sci U S A 112(36): E5013-5020.
  10. 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.
  11. 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.
  12. Weyens, N., van der Lelie, D., Taghavi, S. and Vangronsveld, J. (2009). Phytoremediation: plant-endophyte partnerships take the challenge. Curr Opin Biotechnol 20(2): 248-254.

简介

土壤生物在分类学和功能上都是多样的。 该生态系统经历了高度复杂的交互网络,但也可能呈现功能独立的实体。 植物根是土壤中代谢活跃的热点,在根际形成过程中起着重要作用。 由于方法上的复杂性,目前在高空间分辨率下跟踪根 - 微生物相互作用的动态是有限的。 在这项研究中,我们开发了一种新型的基于微流体的装置,可以实时直接成像根 - 细菌相互作用。
【背景】植物根在塑造根际植物 - 微生物相互作用中起主要作用,驱动动态变化的微环境。最近的研究揭示了根相关细菌的多种有益作用(Hardoim et al。,2008; Weyens et al。,2009; Mei and Flinn,2010; Santhanam 等人,,2015),强调了根际相互作用的重要作用。以高时空分辨率跟踪这些交互是一项具有挑战性的任务,主要受可用方法的限制。最近几种微流体方法已被改编用于植物科学(Sanati Nezhad,2014年和Stanley 等人,,2016),主要用于根发育和生理学研究(Englert et al 。,2010; Grossmann et al。,2011; Jiang et al。,2014)。这里介绍的TRIS装置建立在这些发展的基础上,为基于显微镜的植物根与相关微生物之间相互作用的研究提供了一个强大的平台,可以在典型的实验生物学实验室中实施(Massalha et al。 ,2017)。 TRIS装置能够精确控制系统内的环境参数,同时允许以高空间和时间分辨率直接观察动态生物过程。在这里,我们详细讨论TRIS装配和操作。研究该装置中的根 - 微生物相互作用允许使用可以用多种荧光报告子检查的细菌菌株和植物基因型的无限组合。总而言之,我们希望并预期TRIS实验系统以及基于此的其他平台将为动态根微环境及其内部相互作用的广泛高分辨率研究开辟道路。

关键字:根相互作用系统示踪, 活体成像显微镜, 根细菌相互作用, 微生物群落动态, 枯草芽孢杆菌, 拟南芥

材料和试剂

  1. 培养皿(150 mm x 25 mm)(Corning,目录号:CLS430597)
  2. 培养皿(100毫米)(康宁,目录号:CLS430591)
  3. 200μl塑料吸头
  4. 无菌14毫升聚苯乙烯培养管(Corning,目录号:352057)
  5. 玻璃载玻片(75 mm x 25 mm)(Thermo,目录号:421-004T)
  6. 胶带(玻璃纸胶带)
  7. Millex-HV注射器过滤器,0.45μm,PVDF(Merck,Millipore,目录号:SLHVM33RS)
  8. 1毫升注射器(BD,目录号:309647)
  9. 聚乙烯管(OD 1.2 mm)(BD,Intramedic TM ,Clay Adams ®)
  10. 常规竹牙签(可从任何超市获得)
  11. 封口膜
  12. 1.5毫升管(Eppendorf,目录号:0030120086)
  13. 钝头针(McKESSON,目录号:8881202363)
  14. 棉花(可从任何超市获得)
  15. Biohazard包(学院仓库)
  16. 拟南芥种子(Col-0)
  17. 枯草芽孢杆菌
  18. 枯草芽孢杆菌 NCIB 3610 mCherry报告菌株的甘油原液(从单个菌落获得,储存在-80°C)
  19. 聚二甲基硅氧烷(PDMS)和固化剂(Sylgard 184有机硅弹性体套件)(Dow Corning Corporation)
  20. 100%乙醇(西格玛,目录号:34852)
  21. dH 2 O(Sigma,目录号:W4502)
  22. KOH(Sigma,目录号:P5958)
  23. 用于种子灭菌的HCl(Sigma,目录号:H1758)和漂白剂(Sigma,目录号:425044)。
  24. 半强度植物生长培养基,基础盐Murashige和Skoog(MS)(Duchefa Biochemie,目录号:M0221)
  25. 植物琼脂(Duchefa Biochemie,CAS号:9002-18-0)
  26. TB缓冲液(见食谱)

设备

  1. 钳子
  2. 解剖刀
  3. 250毫升玻璃烧瓶
  4. 移液器(Eppendorf20,200,1,000μl)
  5. 烤箱
  6. 热板
  7. OD(600 nm)读数器
  8. 37°C振荡培养箱
  9. 微流体通道模具(参见附带的CAD文件
  10. SU-8 2150(MicroChem Corp.)
  11. 干燥器与真空线
  12. Harris Unicore打孔器(1毫米)(Ted Pella,目录号:15074)
  13. 手持式电晕发生器(Laboratory Corona Treater,Electro-Technic Products,型号:BD-20AC)
  14. 高压灭菌器
  15. 干燥器
  16. 配备暗场物镜的倒置光学显微镜(例如,Olympus)
  17. 带自动舞台系统的共聚焦显微镜(例如,Nikon Ti-eclipse)

软件

  1. 专用共聚焦显微镜软件
    在这项研究中,我们使用了尼康共聚焦和NIS元素程序。
  2. 斐济(ImageJ)

程序

  1. 跟踪根交互系统(TRIS)设备制造和组装(在实验前几天开始)
    1. 通过使用软光刻技术在硅晶片上沉积光致抗蚀剂SU-8 2150来制备微通道模具(图1A)(Qin 等人,,2010)。
    2. 该程序基于制造指南表应用,以达到160微米的深度。
      注意:请阅读制造商关于正确处理此类粘胶材料(SU-8 2150)的建议。
    3. TRIS设备组装:
      1. 将模具放入150mm培养皿中,蚀刻的光致抗蚀剂的图案化模具面朝上(图1A)。用胶带将模具固定在板上。
      2. 根据制造商的建议准备25毫升PDMS混合物。在塑料称重船中,称取10份硅弹性体,然后加入1份固化剂。使用塑料棒将两种试剂充分混合。
      3. 将培养皿置于干燥器中并在真空下脱气1小时。重要的是在将PDMS混合物倒入培养皿上的固定模具之前从PDMS混合物中除去所有气泡。
      4. 吹氮气或空气以去除可能积聚在模具上的任何灰尘。小心地将脱气的混合物倒在硅主体上。
      5. 将PDMS在60°C下固化过夜。
        注意:阅读制造商关于固化PDMS的建议。
      6. 使用锋利的刀片(手术刀)在一个矩形件中仔细切出每个相邻的8/9特征(图1B和1C)。图案化的微通道将在PDMS中可见。
      7. 使用1 mm Harris Unicore打孔器在通道入口,出口和根孔(图1C和1D)上打孔。
      8. 完成制作孔后,盖住通道的两侧(例如,用胶带),以防止灰尘颗粒和碎屑。
      9. 在将PDMS板粘合到载玻片上之前,取下胶带,将载玻片和PDMS通道面朝上放在干净的平面上。为了激活PDMS和载玻片的表面,这将导致两种材料的共价键合,我们在表面上方约1-2cm处使用手持式电晕发生器。在每个表面上均匀地处理表面30秒。
      10. 小心地将PDMS装置放在处理过的载玻片上,微通道图案朝向载玻片。手工施加温和均匀的压力,以确保均匀的密封。
      11. 密封后直接用载玻片在100°C下烘烤已安装的通道20分钟。
      12. 将安装的微流体装置在120℃下高压灭菌20分钟。在室温条件下无菌保存高压灭菌的样品。


      图1. TRIS:用于实时跟踪根 - 细菌相互作用的微流体装置。 A.用于光刻过程的微流体装置掩模(图案)设计,用于生成九个单个微流体通道的阵列。左侧:一个单通道尺寸的放大倍数。 B.安装在显微镜载物台(暗边缘)上的TRIS装置的图示。 (插图)含微流体通道的根和细菌细胞的纵切面示意图(红色;未按比例绘制)。 C.在附着于TRIS装置的塑料移液管尖端中生长的拟南芥幼苗的顶视图。入口,设备和出口分别连接到入口管,尖端有秧苗和出口管。从尖端延伸的细白色弦可见根。 (比例尺:1厘米)。 D.在使用明场照射以10x放大率捕获的TRIS微流体装置内生长的9个拟南芥根的显微镜视图。箭头:a,入口; b,在根专用端口尖端,c,根向出口端延伸; d,出口。 (比例尺:5毫米)。这个数字改编自Massalha et al。(2017)。

  2. 细菌培养准备(在实验前一天开始)
    1. 放置 B。将含有3ml TB培养基的无菌聚苯乙烯培养管中的枯草芽孢杆菌培养物在37℃,165rpm振荡培养箱中培养过夜。
    2. 在实验前4小时将1:100稀释到新鲜的3ml TB培养基中并在相同条件下再次孵育以达到0.6 600 。
    3. 用MS洗涤细胞两次,并将细胞重悬于MS培养基中以获得10 6 细胞/ ml(在使用之前过滤MS以使用0.45μmPVDF注射器过滤器消除可能的小碎片)。
    4. 通过使用暗视野显微镜记录的几帧中的移动细胞数来计算游泳细胞与静态细胞的比例。

  3. 植物生长(实验前5-6天开始)
    1. 中等和塑料尖端准备:
      1. 准备0.5倍强度的基础盐MS。使用KOH将pH调节至5.8。将准备好的媒体分成两部分。将1%植物琼脂加入一份。
      2. 对培养基的两部分进行消毒。
      3. 通过高压灭菌器消毒200μl塑料吸头和竹牙签。
      4. 冷却后,培养基熔化固化的培养基,并用5μl填充灭菌的200μl尖端。让介质再次凝固。重要的是在室温下固化之前使用液体介质(图2A显示了带有固化介质的塑料尖端)。
      5. 在培养基储存固化之前,用~30ml培养基填充90mm培养皿。让培养皿中的培养基固化。
      6. 在无菌环境中,在尖端的固化培养基水平上方用带有火焰的手术刀切割填充的尖端。使用火焰钳将尖端固定在无菌的150 mm培养皿中。
      7. 用带有镊子的镊子收集填充的塑料尖端,并将它们放入固化的琼脂中(在步骤C1e中制备的90mm培养皿中)。固定在培养皿中的固化琼脂中的填充尖端可以用封口膜密封并在4℃下储存以备后用。
    2. 与(1)平行,在封闭的干燥器中将拟南芥(例如,Col-0)种子在氯气中灭菌2小时。将带有种子的1.5ml管子置于干燥器容器中(在通风橱中)。将含有100ml漂白剂的250ml玻璃烧瓶放入干燥器和6ml浓HCl中,立即关闭并用封口膜密封干燥器。 2小时后,打开通风橱中的干燥器,让氯气蒸发几分钟。
    3. 使用无菌牙签,将一粒种子无菌转移到含有半强度基础盐MS的塑料移液管尖端,所述基础盐MS补充有0.8%植物琼脂(图1C)。根据实验需要准备尽可能多的种子提示。
    4. 通过在冷室中保持带有尖端的板两天来对种子进行分层以避免光照。
    5. 将含有种子的尖端转移到23小时的16小时光照/ 8小时黑暗时间中3-4天(或在生长的根部从塑料尖端穿透之前)(图2A)。
    6. 将安装的微流体装置放入盒子中,放入生物危害袋中,并使用高压灭菌器对其进行灭菌(类似于TRIS装置组装部分中的步骤'A3l')。
    7. 用液体0.5x MS小心地填充高压灭菌的微流体装置,以消除在无菌条件下捕获在系统中的气泡。
    8. 在无菌条件下将带有生长幼苗的塑料尖端放入高压灭菌的TRIS装置中(图1A和1B)。
    9. 在成像前垂直孵育4-5天(直到生长的根到达TRIS通道的中间)。将液体介质添加到垂直板上以覆盖输出孔并保持湿度(图2B)。用封口膜密封板以防止任何液体泄漏。


      图2.使用微流体TRIS装置准备拟南芥幼苗以跟踪根 - 细菌相互作用。 A.填充有凝固琼脂的塑料尖端的提示(填充的琼脂沉淀到所有尖端中的相同水平,由透明塑料尖端中拟南芥种子的高度表示,其中拟南芥幼苗发芽放置在琼脂平板中。 B. 拟南芥幼苗在塑料尖端固定在TRIS装置上并垂直培养。

  4. 将TRIS系统安装到显微镜载物台(成像前2小时开始)
    1. 在细菌培养期间,清洗将连接到微流体室的所有管道。每个通道准备两个管(长管和短管,请参见后续步骤以确定每个管的长度)。用70%乙醇清洁所有管道,以去除可能的制造污染物。用dH 2 O洗涤管两次以使用1ml注射器除去残留的乙醇。 
    2. 将钝端针固定在植物培养基中含有洗过的细菌的1ml注射器中。清除注射器开口处的气泡。避免快速填充以防止气泡。
    3. 将长管连接到钝端针,并通过推动注射器的活塞填充管。确保管的长度足以在显微镜载物台上组装系统时具有灵活性(见图3)。


      图3.安装在显微镜载物台上的TRIS装置的视图。该装置包含具有拟南芥幼苗的微流体通道,整个装置包括管道系统,1 ml注射器,培养基水库,以及由丙烯酸塑料制成的湿度室(比例尺:5厘米)。修改自Massalha 等(2017)。

    4. 保持短管不要充满液体。 
    5. 根据微通道的数量准备Eppendorf管。使用细镊子在Eppendorf管的盖子上打一个孔,并用1 ml MS填充。
    6. 快速,从垂直板上取下TRIS设备,用Kimwipe干燥液滴(避免通过触摸入口/出口孔干燥TRIS设备的内容物),将TRIS设备固定到显微镜载物台支架(优选的没有开口的载玻片架)保持潮湿的条件)。
    7. 将连接到注射器的长管插入入口孔,将短管插入每个微通道的输出孔。施加足够的压力迫使管道从PDMS表面到玻璃载片的大约一半处,但要小心,以避免PDMS入口和出口出现裂缝。
    8. 用透明盖快速盖住设备以保持湿度(图3)。
    9. 将透明盖子培养皿放入湿棉布内,以保持湿度。
    10. 将输出管(短管)的第二端浸入装有1ml MS介质的Eppendorf管中。
    11. 设置显微镜参数:
      1. 扫描(x,y) - 每个通道的坐标(缝合参数应在初步实验中测试)。
      2. 所有使用过的通道的暴露时间(应在初步实验中测试)。
      3. 循环次数。
      4. 周期间隔。
      5. 找到根的中间部分。设置±40μmz-stack。
    12. 通过缓慢推动注射器的活塞将细菌引入微流体通道。继续推动活塞,直到出口管充满。
    13. 在完成步骤D9之后,立即开始扫描该微通道。
    14. 在步骤D10完成后,暂停显微镜,将细菌引入下一个微通道,然后继续扫描。
    15. 继续执行步骤D12到D14,直到最后一个微通道。

  5. 图像分析
    1. 斐济用于可视化结果并生成我们使用LOCI包打开生物显微镜格式的总结电影(Schindelin et al。,2012)。
    2. 扩展的景深包(斐济, http://imagej.net/Extended_Depth_of_Field )或来自文件交换的FStack功能repository(MathWorks)用于从不同的z-stack生成单个EDF图像。
    3. Matlab用于根据每个图像中明视场通道的亮度对EDF图像进行分割,然后计算细菌信号的相对强度。

数据分析

TRIS装置的主要结果是细菌密度作为位置和时间的函数。细菌荧光的相对强度用于估计细菌的密度,并通过从感兴趣的区域减去每个时间点的背景来计算。其他参数可以使用TRIS装置计算,即,根伸长和细菌动态。此阶段的数据分析高度依赖于生物学问题。

笔记

每个植物捕获的TRIS装置体积仅为~6.4μl,因此使植物对环境排列敏感。强烈建议跟踪生长条件,特别是在将植物移至TRIS设备时。

食谱

  1. 结核病缓冲区
    1%(w / v)胰蛋白胨
    0.5%(w / v)NaCl
    高压灭菌器

致谢

该协议改编自Massalha 等人(2017)。这项研究得到了由Planning& Co.资助的博士学位的支持。以色列高等教育委员会预算委员会个人补助金(致H.M.)。

参考

  1. Englert,D.L.,Manson,M。D.和Jayaraman,A。(2010)。 用于量化稳定浓度梯度中细菌趋化性的微流体装置。 J Vis Exp (38)pii:1779。
  2. Grossmann,G.,Guo,W.J.,Ehrhardt,D.W。,Frommer,W.B.,Sit,R.V.,Quake,S.R。和Meier,M。(2011)。 RootChip:用于植物科学的集成微流控芯片。 植物细胞 23(12):4234-4240。
  3. Hardoim,P。R.,van Overbeek,L。S.和Elsas,J。D.(2008)。 细菌内生菌的特性及其在植物生长中的作用。 趋势微生物 16(10):463-471。
  4. Jiang,H.,Xu,Z.,Aluru,M。R.和Dong,L。(2014)。 用于拟南芥的高通量表型的植物芯片。 Lab Chip 14(7):1281-1293。
  5. Massalha,H.,Korenblum,E.,Malitsky,S.,Shapiro,O。H.和Aharoni,A。(2017)。 微流体装置中根 - 细菌相互作用的实时成像。 Proc Natl Acad Sci USA 114(17):4549-4554。
  6. Mei,C。和Flinn,B。S.(2010)。 利用有益微生物内生菌对植物生物量和抗逆性的改善。 最近的Pat Biotechnol 4(1):81-95。
  7. Qin,D.,Xia,Y。和Whitesides,G。M.(2010)。 用于微米和纳米级图案化的软光刻。 Nat Protoc 5(3):491-502。
  8. Sanati Nezhad,A。(2014年)。 用于植物细胞研究的微流体平台。 Lab Chip 14 (17):3262-3274。
  9. Santhanam,R.,Luu,V。T.,Weinhold,A.,Goldberg,J.,Oh,Y。和Baldwin,I。T.(2015)。 与根源相关的细菌可以帮助植物免受连作期间出现的突发性枯萎病的影响。< / a> Proc Natl Acad Sci USA 112(36):E5013-5020。
  10. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B 。,Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P。和Cardona,A。(2012)。 斐济:生物图像分析的开源平台。 Nat方法 9(7):676-682。
  11. Stanley,C.E.,Grossmann,G.,i Solvas,X.C。和deMello,A.J。(2016)。 片上土壤:用于环境有机体研究的微流体平台。 实验室芯片 16(2):228-241。
  12. Weyens,N.,van der Lelie,D.,Taghavi,S。和Vangronsveld,J。(2009)。 植物修复:植物内生菌伙伴关系迎接挑战。 Curr Opin Biotechnol 20(2):248-254。
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引用:Massalha, H., Korenblum, E., Shapiro, O. H. and Asaph, A. (2019). Tracking Root Interactions System (TRIS) Experiment and Quality Control. Bio-protocol 9(8): e3211. DOI: 10.21769/BioProtoc.3211.
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