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Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light
红外线下根生长运动学的无创监测实验方案   

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Abstract

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.

Keywords: Hydroponics(水培), Infrared imaging(红外成像), Kinematics(运动学), Root growth(根生长), Time-lapse(延时)

Background

The use of kinematic analyses for the monitoring of growth and tropisms in plants dates back to the end of the 19th century, with early studies from Julius von Sachs and Whilhelm Pfeffer. The widespread use of photography during the 20th century led to easier and continuous monitoring through ‘streak photography’ (List, 1969; Erickson and Silk, 1980). In the 90s, new digital cameras and informatics tools enabled the development of automatic tracking algorithms used for particle image velocimetry. RootFlowRT (Van der Weele et al., 2003), Kineroot (Basu et al., 2007), RootTrace (French et al., 2009), GrowthTracer (Iwamoto et al., 2013) and Kymorod (Bastien et al., 2016) are among many recent examples of software dedicated to the monitoring of root growth. However, all particle image velocimetry methods rely on the use of identifiable image texture patterns in each successive picture. Historically, these patterns were marked on the root using ink or graphite particles (see Sharp et al., 1988 and Merret et al., 2010 for examples). Two difficulties arise from this approach: Firstly, the markings spread during organ (root) growth, inducing loss of resolution after a few hours. Secondly, the physical marking process may stress the organ due to some combination of exposure to small forces, to light or to temperature changes, or to slight drying of the organ surface. In roots such handling effects may cause temporary slowing of growth for a period of minutes to hours. The use of infrared light (840-850 nm) has the double benefit of not influencing root growth, whilst also generating image texture patterns readily tracked using particle image velocimetry.

Materials and Reagents

  1. Clear flexible tubing (VWR, catalog number: 228-0708 )
  2. Plastic sealing tape (Terostat VII, Teroson) (Rubans de Normandie, catalog number: 7TE )
  3. Growing roots of hydroponically-grown plants. Adventitious roots grow on poplar woody cuttings partially immersed in Hoagland half strength nutrient solution (Sigma-Aldrich, catalog number: H2395 )
    Note: The present protocol was initially designed for monitoring the growth of a plagiotropic poplar root (Bizet et al., 2015). The protocol is adaptable for any plant species that tolerates hydroponic or in vitro cultivation (see step D3).

Equipment

  1. Transparent Plexiglass® growth monitoring chamber (specially built, size 12 cm long x 5 cm large x 6 cm height), with holes on one side for nutrient solution inflow and outflow (Figure 1)


    Figure 1. Custom made transparent Plexiglass® growth monitoring chamber

  2. Water pump (e.g., a small aquarium pump, for instance, Newa, model: NJ600 , aquarium shop)
  3. Temperature-controlled dark room (range 21-23 °C)
    Note: The whole system requires about 1 m2 of laboratory working space.
  4. Air pump (e.g., an aquarium pump with airstone, for instance JBL, model: Prosilent a300 , aquarium shop)
  5. Stone diffuser (e.g., JBL, model: Prosilent Aeras Micro S2 , aquarium shop)
  6. Small plastic reservoir tank (buffer tank, few litres capacity, size 12 x 20 x 40 cm3)
    Note: A plastic bucket would suit.
  7. Modified digital camera (see Procedure A, for how to remove the infrared filter)
    Note: Most reflex camera should work fine as long as it is possible to do infrared conversion and to computer-control them using manufacturer’s software or free software such as Digicamcontrol (list of supported cameras: http://digicamcontrol.com/cameras). Light sensitivity and captor size should also be considered for best image quality.
  8. Infrared lamp (840-850 nm) (Pearl, catalog number: KT4243-907 )
  9. Extension tube (Kenko, AF 12/20/36 mm for Nikon)
  10. Optical rail equipped with a translation stage (Edmund Optics, catalog number: 59-263 )
  11. Knuckle with knob and thread adaptor (Edmund Optics, catalog numbers: 53-887 and 58-988 )
  12. Macro objective (Nikkor 60/2.8 D ASF)

Software

  1. Software for image analysis (e.g., Kineroot from Basu et al., 2007)
  2. Fiji (https://fiji.sc)
  3. Rawtherapee (http://rawtherapee.com)
  4. Digicamcontrol (http://digicamcontrol.com)

Procedure

  1. Digital camera modification for infrared detection
    The use of infrared light requires a modified digital camera able to detect wavelengths above 700 nm. Fortunately, the technology behind charged coupled device (CCD) sensors used in digital cameras can detect light up to more than 1,000 nm. The sensitivity of the sensor is however limited by a low-pass filter blocking infrared light above 720-750 nm, depending on manufacturer. Thus, to use a digital camera for infrared imaging, removal of the low-pass filter is necessary−its replacement by a colourless glass filter (e.g., Schott WG280) is desirable to protect against dust particles on the sensor. Filter replacement is widely used for astronomical photography, and tutorials showing how to remove the low-pass filter without damaging the camera CCD sensor are readily available on the internet (https://www.lifepixel.com/tutorials/infrared-diy-tutorials), with specialist companies also offering this service (e.g., above mentioned LifePixel or http://www.eosforastro.com). Figure 2 shows examples of roots from two different species filmed with a modified camera under infrared light.
    Note: The choice of a high-resolution digital camera with good quality (e.g., macro) lens is very important to avoid optical aberration (distortion) of images. This is essential to ensure that tracking of organ growth is accurate across the entire image frame. To check for lens aberrations an accurately drawn square grid should be photographed and the degree of aberration checked–it is possible to digitally correct for aberration, but it is far better to select the camera optics to avoid these problems. The grid can also be used to calibrate the growth measurements from pixels into distance measurements.


    Figure 2. Examples of root apices under infrared light for maize (A) and poplar (B). Pictures were taken with a defiltered Nikon D5200 mounted with a macro objective (Nikkor 60 mm). Scale bars stand for 1 mm. Picture for maize was taken from Bizet et al. (2015).

    Note: Subsequent steps are given for plagiotropic roots obtained from poplar cuttings cultivated in hydroponics. Procedure can be adapted for gravitropic roots from various species using an adapted growth monitoring chamber and material to easily attach the plant.

  2. Root growth monitoring under near-infrared light
    1. Preparation of the growth monitoring chamber
      1. Fill the buffer tank with approx. ten times the volume of the growth monitoring chamber with nutrient solution and place it below the growth monitoring chamber.
      2. Set up circulation of the nutrient solution between the growth monitoring chamber and the buffer tank using a water pump and flexible tubing: add an inflow from the pump to the bottom of the growth monitoring chamber and a passive outflow from the top of the growth monitoring chamber to the buffer tank (Figure 3A). Flow rate should be small, 2.5 to 3 L h-1.
      3. Ensure sealing of the nutrient circulation system using plastic sealing tape.
      4. Add a source of dissolved oxygen inflow in the buffer tank using an air pump, airline tubing and an air stone diffuser (Figure 3B).
        Note: The source of dissolved oxygen should not be added in the growth monitoring chamber to prevent shaking of the root by air bubbles during the monitoring.


        Figure 3. Nutrient solution circuit from the tank to the growth monitoring chamber and back to the tank (A) and the aeration system (B)

      Note: All subsequent steps should be done in a dark room.

    2. Gently place a root in the growth monitoring chamber and attach the plant, e.g., for poplar roots: clamp the cutting.
    3. Add an infrared lamp in front of the root apex.
      Note: A low incidence angle between the root axis and the lamp allow better visualization as long as the lamp is not oriented toward the camera.
    4. Place the defiltered camera for a side view of the root. The camera is attached to a micrometric translation stage mounted on an optical rail (Figure 4).
      Note: The camera should be equipped with a macro objective and extension tubes can be added to obtain higher magnification while keeping a close minimum focusing distance.


      Figure 4. Components of the root growth monitoring system. Note the presence of the buffer tank (T) connected to the growth monitoring chamber (G) in the background of the top picture. Top picture also shows duplication of the system for parallel monitoring (top-right corner). CCD2 arrow indicates the possible position for a second top-view camera (see step D1). Ca: camera; Co: connection to computer; Cu: cutting; ET: extension tube; G: growth monitoring chamber; IR: infrared lamp; O: macro objective; OR: optical rail; T: buffer tank; TS: translation stage.

    5. Set up the focus on the root using the translation stage.
      Notes:
      1. Video streaming on a computer can greatly help obtain a precise focus. Keep the focusing distance on the macro objective at minimum and only use the translation stage during this step to maximize the magnification.
      2. For calibration purposes, take a picture of a ruler (either at the beginning or the end of the monitoring). The scale is valid as long as the focal length and distance between the objective and the root/ruler is conserved (meaning the magnification is identical).
      3. For long term experiment (several days), focus adjustment and framing may be necessary once or twice a day as the root may go out of the camera depth of field and out of the frame.
    6. Launch the intervalometer of the camera. Selecting intervals from 3 min to 30 min between images allows capture of growth at varying resolutions.
      Note: Most cameras can be computer-controlled using manufacturers’ software or free software such as Digicamcontrol (http://digicamcontrol.com).
    7. At the end of the experiment, wash all the materials that were in contact with the nutrient solution.
      Note: Be careful not to scratch the growth monitoring chamber as this degrades image quality and makes organ tracking difficult or impossible.

  3. Image processing
    Depending on the temporal resolution desired, many images may be processed during kinematic analysis. To optimize particle image velocimetry calculation time and resolution (e.g., small roots of Arabidopsis), subsequent steps of image pre-processing should be considered. These pre-processing steps can be done with free software that allows batch processing such as Fiji (https://fiji.sc) or Rawtherapee (http://rawtherapee.com), the latter being able to read raw picture format from various manufacturers (for example .nef for Nikon or .crw for Canon). We used batch processing of Rawtherapee (see the online help files for details), with the following steps:
    1. Crop images to optimize calculation time through deletion of unused information.
    2. Adapt the black threshold to decrease background noise.
    3. Remove the presence of overexposed areas.
    4. Modify local contrasts to blur unused pixel scale details.
    5. Enhance patches of pixels which are more stable between consecutive pictures (Figure 5A).
    Pre-processed pictures are then opened using dedicated software for kinematic analysis of root growth such as Kineroot (Basu et al., 2007; Figure 5B). We worked with the green color channel as it exhibited greater contrast. See http://plantscience.psu.edu/research/labs/roots/methods/computer for a detailed manual of Kineroot. A set of raw images, a set of processed images and a movie of the resulting analysis is provided as Supplementary material.


    Figure 5. Use of pre-processed images for kinematic analysis on Kineroot software. A. Enhanced image texture patterns for kinematic analysis after image pre-processing (bottom) as compared to raw picture (top). Scale bar stands for 1 mm. B. Example of root monitoring with Kineroot. Red points are tracked along the root centerline by particle image velocimetry. Monitoring parameters can be seen in the different panels. Note that R and N parameters (both set here at 40 pixels) must be adapted to the spatial and temporal resolutions (389 pixels mm-1 and 6 min between consecutive pictures here).

  4. Flexibility of the growth monitoring system
    1. 3D monitoring of root shape and deformation: Adding a top view to the initial side view allows the reconstruction of the 3D shape of the root. Multiple cameras can be used simultaneously using Digicamcontrol (http://digicamcontrol.com). See Bizet et al. (2016) for detailed explanations about cameras position and how analysis from two points of view can be merged into a 3D reconstruction of the root apex (Figure 6).


      Figure 6. 3D reconstruction of a root from two orthogonal points of view. Colors indicate relative root curvature.

    2. Stress management during continuous monitoring: The system for root growth monitoring described in Procedure B can be adapted to replace the nutrient solution by another solution whilst monitoring growth continually and without any manipulation of the root (see Bizet et al., 2015, for root exposure to osmotic stress using this system).
      1. Prior to the setup of the root in the growth monitoring chamber, connect a second buffer tank (tank b) in parallel with the initial buffer tank (tank a). Tank b contains the test solution and should be connected to the nutrient inflow using a ball valve (Thermo Fisher) (Figure 7). Check if solution temperature is identical in both tanks.


        Figure 7. Set-up of the double tank with the ball valve for stress application

      2. Close the ball valve from tank b and proceed from step B1 to B6.
      3. To change the circulating solution, turn on pump b, close the ball valve from tank a and open the ball valve from tank b.
      4. Wait five minutes for the solution in the growth monitoring chamber to be replaced and move the outflow flexible tube from buffer tank a to buffer tank b.
      5. At the end of the experiment, wash all the materials that were in contact with the nutrient solution.
    3. The protocol is flexible to suit the species and growth condition requirements. To monitor growth of gravitropic roots, use a vertical growth monitoring chamber. For in vitro plants, for which the root diameter is much finer, higher magnification may be obtained with more extension tubes on the camera, and the agar plate is directly used as the growth monitoring chamber.

Data analysis

Kinematic analyses by Kineroot provide values of velocity along the root apex, namely velocity profiles. Elemental elongation rate along the root apex is the first derivative of the velocity profile. Alternatively, a composite growth function dedicated to root velocity profiles can be fitted on Kineroot output (Peters and Baskin, 2006). See Bizet et al. (2015) for more details about the fitting procedure. Growth parameters such as root growth rate (that equals maximal velocity), growth zone length and maximal elemental elongation rate are extracted from these profiles (Figure 8). Parametric statistics can then be on to analyse these parameters, to compare individuals, or to examine the time course of the response to an environmental stress.


Figure 8. Velocity and elemental elongation rate profiles. Velocity profile (in blue) and elemental elongation rate (EER, in green) profile after fitting of the composite growth function from Peters and Baskin (2006). Among growth parameters, root growth rate (RGR) corresponds to the maximum of the velocity profile. Maximal EER corresponds to the maximal slope of the velocity profile i.e., the maximum of the first derivative. The growth zone length (GZ) is determined at 94% of the maximal velocity to avoid infinite values.

Notes

From our observations, growth rate is very variable between individual roots, varying up to 5 fold between minimal and maximal values. Experimental design should take into account this variability to maximise the power of statistical analyses.

Acknowledgments

FB was supported by a grant from the French National Ministry for Education and Research. The research has received funding from the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-12-LABXARBRE-01) and from Agreenium’s International Research School. The James Hutton Institute receives funding from the Scottish Government. This protocol was adapted from Bizet et al. (2015) and Bizet et al. (2016).

References

  1. Bastien, R., Legland, D., Martin, M., Fregosi, L., Peaucelle, A., Douady, S., Moulia, B. and Hofte, H. (2016). KymoRod: a method for automated kinematic analysis of rod-shaped plant organs. Plant J 88(3): 468-475.
  2. Basu, P., Pal, A., Lynch, J. P. and Brown, K. M. (2007). A novel image-analysis technique for kinematic study of growth and curvature. Plant Physiol 145(2): 305-316.
  3. Bizet, F., Bengough, A. G., Hummel, I., Bogeat-Triboulot, M. B. and Dupuy, L. X. (2016). 3D deformation field in growing plant roots reveals both mechanical and biological responses to axial mechanical forces. J Exp Bot 67(19): 5605-5614.
  4. Bizet, F., Hummel, I. and Bogeat-Triboulot, M. B. (2015). Length and activity of the root apical meristem revealed in vivo by infrared imaging. J Exp Bot 66(5): 1387-1395.
  5. Erickson R. O. and Silk, W. K. (1980). The kinematics of plant growth. Sci Am 242(5): 134–151.
  6. French, A., Ubeda-Tomas, S., Holman, T. J., Bennett, M. J. and Pridmore, T. (2009). High-throughput quantification of root growth using a novel image-analysis tool. Plant Physiol 150(4): 1784-1795.
  7. Iwamoto, A., Kondo, E., Fujihashi, H. and Sugiyama, M. (2013). Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program. J Plant Res 126(1): 187-192.
  8. List, A., Jr. (1969). Transient growth responses of the primary roots of Zea mays. Planta 87(1-2): 1-19.
  9. Merret, R., Moulia, B., Hummel, I., Cohen, D., Dreyer, E. and Bogeat-Triboulot, M. B. (2010). Monitoring the regulation of gene expression in a growing organ using a fluid mechanics formalism. BMC Biol 8: 18.
  10. Peters, W. S. and Baskin, T. I. (2006). Tailor-made composite functions as tools in model choice: the case of sigmoidal vs bi-linear growth profiles. Plant Methods 2: 11.
  11. Sharp, R. E., Silk, W. K. and Hsiao, T. C. (1988). Growth of the maize primary root at low water potentials: I. Spatial distribution of expansive growth. Plant Physiol 87(1): 50-57.
  12. van der Weele, C. M., Jiang, H. S., Palaniappan, K. K., Ivanov, V. B., Palaniappan, K. and Baskin, T. I. (2003). A new algorithm for computational image analysis of deformable motion at high spatial and temporal resolution applied to root growth. Roughly uniform elongation in the meristem and also, after an abrupt acceleration, in the elongation zone. Plant Physiol 132(3): 1138-1148.

简介

对环境线索的根系反应动态进行表型分析是了解植物适应环境的必要条件。 持续监测根系生长是有挑战性的,因为根系通常生长在地下,对其生长环境非常敏感。 该方案将红外成像与水培培养相结合,用于运动学分析。 它允许在精细的时空分辨率下连续成像并最小化干扰根。 提供了如何将程序和材料适用于3D监测以及为实验目的如何操纵环境压力的实例。
【背景】运动学分析用于监测植物的生长和向性可以追溯到19世纪末期,早期研究从朱利叶斯·冯·萨克斯和惠尔普菲弗。 20世纪20年代以前摄影的广泛应用导致了通过“连拍摄影”(List of 1969; Erickson和Silk,1980)更容易和持续的监控。在90年代,新的数码相机和信息学工具使得能够开发用于粒子图像测速的自动跟踪算法。 RootFlowRT(Van der Weele等人,2003),Kineroot(Basu等人,2007),RootTrace(French et al。,et al。,& 2009),GrowthTracer(Iwamoto等人,2013)和Kymorod(Bastien等人,2016)是最近的许多软件专门用于监测根生长的例子。然而,所有粒子图像测速方法都依赖于在每个连续图像中使用可识别的图像纹理图案。历史上,这些图案在根上使用油墨或石墨颗粒标记(参见1988年的Sharp等人,和Merret等人,2010),例如)。这种方法有两个困难:首先,标记在器官(根)生长期间扩散,在几个小时后引起失去分辨率。其次,物理标记过程可能由于暴露于小力,光或温度变化或器官表面的轻微干燥的某些组合而对器官产生压力。在根中,处理效果可能导致生长的暂时放缓数分钟至数小时。使用红外光(840-850nm)具有不影响根生长的双重优点,同时也产生使用粒子图像测速法容易追踪的图像纹理图案。

关键字:水培, 红外成像, 运动学, 根生长, 延时

材料和试剂

  1. 透明柔性管(VWR,目录号:228-0708)
  2. 塑料密封胶带(Terostat VII,Teroson)(Rubans de Normandie,目录号:7TE)
  3. 生长在水培植物的根。不定根生长在部分浸在Hoagland半强度营养液中的杨木木屑上(Sigma-Aldrich,目录号:H2395)
    注意:本议定书最初设计用于监测疟原虫根系的生长(Bizet等,2015)。该方案适用于任何容忍水培或体外培养的植物物种(见步骤D3)。

设备

  1. 透明有机玻璃®生长监测室(专门建造,尺寸12厘米长x 5厘米大x 6厘米高),一侧有孔用于营养液流入和流出(图1)


    图1.定制透明有机玻璃 ® 增长监控室

  2. 水泵(例如,,小型水族馆泵,例如Newa,型号:NJ600,水族馆店铺)
  3. 温度控制暗室(范围21-23°C)
    注意:整个系统需要大约1米的实验室工作空间。
  4. 空气泵(例如,,带有旋转开关的水族馆泵,例如JBL,型号:Prosilent a300,水族馆店铺)
  5. 石头扩散器(例如,JBL,型号:Prosilent Aeras Micro S2,水族馆商店)
  6. 小塑料储罐(缓冲罐,容积不大,尺寸12 x 20 x 40厘米 3 )
    注意:塑料桶适合。
  7. 修改后的数码相机(请参阅程序A,如何清除红外线滤镜)
    注意:只要有可能进行红外线转换,并使用制造商的软件或免费软件,如Digicamcontrol(支持的相机列表: http://digicamcontrol.com/cameras )。还应考虑光敏度和捕获尺寸以获得最佳图像质量。
  8. 红外灯(840-850nm)(珍珠,目录号:KT4243-907)
  9. 延长管(Kenko,AF 12/20/36 mm,尼康)
  10. 配有翻译台的光学轨道(Edmund Optics,目录号:59-263)
  11. 带旋钮和螺纹适配器的转向节(Edmund Optics,目录号:53-887和58-988)
  12. 宏观目标(Nikkor 60 / 2.8 D ASF)

软件

  1. 用于图像分析的软件(例如,来自Basu等人的Kineroot,,2007)
  2. 斐济( https://fiji.sc
  3. Rawtherapee( http://rawtherapee.com
  4. Digicamcontrol( http://digicamcontrol.com

程序

  1. 用于红外线检测的数码相机修改
    使用红外线需要能够检测高于700nm波长的经过修改的数码相机。幸运的是,用于数码相机的电荷耦合器件(CCD)传感器技术可以检测到高达1000nm的光。然而,传感器的灵敏度受到低通滤波器的限制,该滤波器阻挡了高于720-750nm的红外光,这取决于制造商。因此,为了使用数码相机进行红外成像,需要去除低通滤波器 - 其由无色玻璃滤光片(例如,Schott WG280)的替换是期望的,以防止灰尘颗粒传感器。滤波器更换广泛应用于天文摄影,而有关如何清除低通滤波器而不损坏相机CCD传感器的教程便可在互联网上获得( https://www.lifepixel.com/tutorials/infrared-diy-tutorials ),专业公司也提供这项服务例如,上面提到的LifePixel或 http://www.eosforastro。 COM )。图2显示了在红外光下用修改后的相机拍摄的两种不同种类的根的实例 注意:选择高品质(例如,宏观)镜头的高分辨率数码相机对于避免图像的光学像差(失真)非常重要。这对于确保整个图像框架中的器官生长跟踪是准确的至关重要。为了检查镜头像差,应该拍摄精确绘制的方格网格并检查像差度,可以对像差进行数字校正,但是选择相机光学镜件避免这些问题要好得多。网格也可用于校准从像素到距离测量的增长测量。


    图2.玉米(A)和杨树(B)的红外光下根尖的实例。 使用带有宏观物镜(Nikkor 60 mm)的带有滤镜的尼康D5200拍摄照片。比例尺为1 mm。来自Bizet等人的玉米图片(2015)。

    注意:随后的步骤是从在水培中栽培的杨树切片获得的匍匐根。可以使用适应的生长监测室和材料来容易地附着植物,来适应来自各种物种的重力根。

  2. 近红外光照下根生长监测
    1. 生长监测室的准备
      1. 向缓冲罐中加入约生长监测室的体积是营养液的十倍,并置于生长监测室下方
      2. 使用水泵和柔性管道,在生长监测室和缓冲罐之间建立营养液的循环:将来自泵的流入添加到生长监测室的底部,并从生长监测室的顶部被动流出到缓冲罐(图3A)。流量应小,2.5〜3 L h -1。
      3. 使用塑料密封带确保营养循环系统的密封
      4. 使用空气泵,航空管道和空气石头扩散器在缓冲罐中添加溶解氧的来源(图3B)。
        注意:不应在生长监测室中加入溶解氧源,以防止在监测过程中气泡发生根部摇晃。


        图3.从罐到生长监测室和回到罐(A)和曝气系统(B)的营养液回路

      注意:所有后续步骤都应在黑暗的房间内完成。

    2. 轻轻地将根部放在生长监测室中,并将植物(例如)连接到杨树根:夹住切割。
    3. 在根尖前面添加红外灯。
      注意:只要灯不朝向相机,根轴和灯之间的低入射角允许更好的可视化。
    4. 将经过滤镜的相机放在根部的侧视图上。相机连接到安装在光导轨上的微米平移台(图4)。
      注意:相机应配备一个宏观物镜,并且可以添加延长管以获得更高的倍率,同时保持最小的聚焦距离。


      图4.根生长监测系统的组件注意在顶部图片的背景中连接到生长监测室(G)的缓冲罐(T)的存在。顶部图片还显示了并行监控系统的重复(右上角)。 CCD2箭头表示第二顶视图相机的可能位置(参见步骤D1)。 Ca:相机; Co:连接电脑;铜:切割; ET:延长管G:生长监测室红外线:红外灯; O:宏观目标或:光导轨; T:缓冲罐TS:翻译阶段
    5. 使用翻译阶段将重点放在根上。
      注意:
      1. 电脑上的视频流可以极大地帮助您获得精确的焦点。将焦点距离保持在最小的宏观目标上,并且在此步骤中仅使用平移阶段来最大化放大倍数。
      2. 为校准目的,拍摄尺子(监视的开始或结束)。只要目标和根/尺之间的焦距和距离保守(即放大倍率相同),刻度就有效。
      3. 对于长期实验(几天),焦距调整和框架可能需要一天或两次,因为根可能会离开相机的景深并离开帧。
    6. 启动相机的间隔计。在图像间选择3分钟至30分钟的间隔,可以以不同的分辨率捕获生长。
      注意:大多数相机可以使用制造商的软件或免费软件(如Digicamcontrol)进行计算机控制( http://digicamcontrol.com
    7. 在实验结束时,清洗所有与营养液接触的物质。
      注意:注意不要划伤成长监测室,因为这会降低图像质量,并使器官跟踪变得困难或不可能。

  3. 图像处理
    根据期望的时间分辨率,可以在运动学分析期间处理许多图像。为了优化粒子图像测速计算时间和分辨率(例如,,拟南芥的小根),应考虑图像预处理的后续步骤。这些预处理步骤可以使用允许批量处理的免费软件,如斐济( https:// fiji .sc )或Rawtherapee( http://rawtherapee.com ),后者能够从各种制造商处读取原始图像格式(例如,尼康或尼康的.crw为佳能)。我们使用Rawtherapee的批处理(有关详细信息,请参阅在线帮助文件),步骤如下:
    1. 通过删除未使用的信息来裁剪图像以优化计算时间。
    2. 适应黑色阈值以降低背景噪声。
    3. 清除曝光过度的区域。
    4. 修改局部对比度以模糊未使用的像素比例细节。
    5. 增强连续图片之间更稳定的像素补丁(图5A)
    然后使用用于根生长运动学分析的专用软件(例如Kineroot(Basu等人,2007;图5B))打开预处理的图像。我们与绿色通道合作,展现出更大的对比度。请参阅 http://plantscience.psu.edu/研究/实验室/根/方法/计算机,了解Kineroot的详细手册。一组原始图像,一组经处理的图像和所得分析的电影提供为补充材料


    图5.使用预处理的图像进行Kineroot软件的运动分析。 :一种。与原始图片(上图)相比,图像预处理(底部)后的运动学分析的增强图像纹理图案。刻度棒表示1 mm。 B.使用Kineroot进行根监控的示例。通过粒子图像测速法沿着根中心线追踪红点。监控参数可以在不同的面板中看到。请注意,R和N参数(均设置为40像素)必须适应于空间和时间分辨率(389像素mm -1 和连续图像之间6分钟)。

  4. 增长监测系统的灵活性
    1. 根形状和变形的3D监控:将顶视图添加到初始侧视图允许重建根的3D形状。可以使用Digicamcontrol同时使用多台摄像机( http://digicamcontrol.com )。有关摄像机位置的详细说明,以及从两个角度分析如何可以合并到根顶点的3D重建中(见图6),请参阅Bizet 等人(2016)。

      图6.从两个正交的角度对根的三维重建。 颜色表示相对根曲率。

    2. 连续监测期间的压力管理:程序B中描述的根生长监测系统可以适应于通过另一种解决方案替代营养液,同时不间断地监测生长并且不对根进行任何操纵(参见Bizet等人。 >,2015年,根据暴露于渗透胁迫使用该系统)。
      1. 在增长监测室内设置根部之前,先将第二个缓冲罐(tank b )与初始缓冲罐(tank a )并联连接起来。 Tank b 包含测试解决方案,并应使用球阀(Thermo Fisher)连接到营养物流入(图7)。检查两个罐中溶液温度是否相同。


        图7.使用球阀设置双槽以应力应用

      2. 从坦克关闭球阀b ,然后从步骤B1到B6。
      3. 要更换循环溶液,请打开泵,关闭球阀从坦克a ,然后从罐中打开球阀b 。 />
      4. 等待五分钟,以便更换生长监测室中的溶液,并将流出的柔性管从缓冲罐a 移动到缓冲罐b 。
      5. 在实验结束时,清洗所有与营养液接触的物质。
    3. 该协议灵活适应物种和生长条件要求。监测引力根的生长,使用垂直生长监测室。对于体外植物,其根直径更细,可以通过相机上更多的延伸管获得更高的放大倍率,并且琼脂平板直接用作生长监测室。 >

数据分析

Kineroot的运动学分析提供了沿根顶点的速度值,即速度分布。沿根顶点的元素伸长率是速度分布的一阶导数。或者,专用于根速度分布的复合生长函数可以适用于Kineroot输出(Peters和Baskin,2006)。参见Bizet等人(2015)了解更多关于装配程序的细节。从这些轮廓中提取生长参数,如根生长速率(等于最大速度),生长区长度和最大元素伸长率(图8)。然后可以参考参数统计来分析这些参数,比较个人,或者检查对环境压力的响应的时间过程。


图8.速度和元素伸长率曲线。 从Peters和Baskin(2006)拟合复合增长函数后的速度曲线(蓝色)和元素伸长率(EER,绿色)曲线。在生长参数中,根生长速率(RGR)对应于速度分布的最大值。最大EER对应于速度分布的最大斜率,即一阶导数的最大值。在最大速度的94%处确定生长区长度(GZ)以避免无限值。

笔记

根据我们的观察,生长速率在个体根之间变化非常大,在最小值和最大值之间变化高达5倍。实验设计应考虑到这种变异性,以最大限度地发挥统计学分析的力量。

致谢

FB得到法国国家教育和研究部的资助。该研究由法国国家研究机构通过卓越实验室ARBRE(ANR-12-LABXARBRE-01)和Agreenium的国际研究学院获得资助。詹姆斯·哈顿研究所接受苏格兰政府的资助。该协议由Bizet等人(2015)和Bizet等人(2016)进行了改编。

参考

  1. Bastien,R.,Legland,D.,Martin,M.,Fregosi,L.,Peaucelle,A.,Douady,S.,Moulia,B.and Hofte,H。(2016)。< a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/27354251“target =”_ blank“> KymoRod:棒状植物器官的自动运动学分析方法。植物J 88(3):468-475。
  2. Basu,P.,Pal,A.,Lynch,JP and Brown,KM(2007)。  一种用于生长和曲率运动学研究的新型图像分析技术。植物生理学145(2):305-316。
  3. Bizet,F.Bengough,AG,Hummel,I.,Bogeat-Triboulot,MB和Dupuy,LX(2016)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm生长植物根部的3D变形场揭示了对轴向机械力的机械和生物反应。 67(19) ):5605-5614。
  4. Bizet,F.,Hummel,I.和Bogeat-Triboulot,MB(2015)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/25540436 “target =”_ blank“>通过红外成像显示体内根尖分生组织的长度和活动。 66(5):1387- 1395.
  5. Erickson RO and Silk,WK(1980)。  运动学的植物生长。 Sci Am 242(5):134-151。
  6. 法语,A.,Ubeda-Tomas,S.,Holman,TJ,Bennett,MJ和Pridmore,T。(2009)。< a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/19515787“target =”_ blank“>使用新型图像分析工具对根生长进行高通量定量。植物生理学150(4):1784 -1795。
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  8. List,A.,Jr.(1969)。  瞬态Zea mays的主要根源的生长反应。植物 87(1-2):1-19。
  9. Merret,R.,Moulia,B.,Hummel,I.,Cohen,D.,Dreyer,E.和Bogeat-Triboulot,MB(2010)。  使用流体力学形式主义监测生长器官中基因表达的调控。 BMC Biol 8:18.
  10. Peters,WS和Baskin,TI(2006)。裁缝制作复合函数作为模型选择中的工具:S形和双线增长曲线的情况。工厂方法 2:11.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bizet, F., Dupuy, L. X., Bengough, A. G., Peaucelle, A., Hummel, I. and Bogeat-Triboulot, M. (2017). Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light. Bio-protocol 7(14): e2390. DOI: 10.21769/BioProtoc.2390.
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