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Micro-computed Tomography to Visualize Vascular Networks in Maize Stems
玉米茎杆中维管网络的微型计算机断层摄影术观察   

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本实验方案简略版
The Plant Cell
Jun 2017

Abstract

Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.

Keywords: Micro-CT scanning (微型CT扫描), Maize (玉米), Stem (茎), Vascular networks (维管网络), 3D modeling (三维建模)

Background

Monocot stems have a characteristic vascular network in which veins remain separate and independent. Despite its importance to supporting growth and development, the pattern of vascular networks in monocot stems has been poorly studied. Visualization of stem vascular networks is quite challenging because veins are deeply embedded in tissues. Conventional tissue sectioning can be applied to observe the networks, however, it is a laborious and time-consuming process which requires observation of hundreds of sections. In addition, the size of crop stems, which is much larger than the field of view under the microscopes, makes it difficult to capture the whole system.

Recently, we reported that maize transcription factor BEL1-like homeobox (BLH) 12 and BLH14 play important roles in the stem development and vein network formation (Tsuda et al., 2017). To obtain a comprehensive view of the vascular systems, we adopted the micro-CT scanning described previously and optimized it for maize stems (Metscher, 2009a and 2009b, Degenhardt et al., 2010, Staedler et al., 2013, Gignac et al., 2016). By combining this method with image analyses, we were able to reconstruct 3D models of inner stem structures in an efficient and reliable manner. This protocol can be used to visualize inner structures such as veins in various species and tissues.

Materials and Reagents

  1. 50 ml Polypropylene tube (Greiner Bio One International, catalog number: 227261 )
  2. Kimwipes
  3. 1.5 ml micro-tube (Eppendorf, catalog number: 0030125150 )
  4. Filter foams MOLTOFILTER MF-13 thickness 5 mm (INOAC Corp.)
  5. Maize (B73)
  6. Formalin (Wako Pure Chemical Industries, catalog number: 061-00416 )
  7. Acetic acid (Wako Pure Chemical Industries, catalog number: 017-00256 )
  8. Ethanol (Wako Pure Chemical Industries, catalog number: 057-00451 )
  9. Lugol stock solution
  10. Potassium iodide (Wako Pure Chemical Industries, catalog number: 166-03971 )
  11. Iodine (Wako Pure Chemical Industries, catalog number: 094-05421 )
  12. Fixative FAA solution (see Recipes)
  13. Iodine staining (Degenhardt et al., 2010) (see Recipes)
  14. 25% Lugol working solution (see Recipes)

Equipment

  1. X-ray micro-CT imaging system (Comscantechno, model: ScanXmate-E090S105 ) (Figure 1)
    1. X-ray tube: Microfocus X-ray source (Hamamatsu Photonics K.K., model: L9421-02 )
    2. Detector: Flat panel detector (Varex Imaging, model: PaxScan 1313DX )


      Figure 1. X-ray micro-CT imaging system used in this protocol

Software

  1. coneCTexpress (built in the micro-CT imaging system. Comscantechno Co. Ltd., Kanagawa, Japan)
  2. OsiriX MD v8.5 (http://www.osirix-viewer.com, Pixmeo SARL, Swiss)
  3. Imaris 8.2 (http://www.bitplane.com, Bitplane, UK)
  4. Adobe Premiere Pro CC (http://www.adobe.com, Adobe, US)

Procedure

  1. Sample fixation and staining with contrasting reagents
    1. Maize stem dissection and fixation
      1. Grow maize plants for 6-8 weeks. Our growth condition is at 26 °C under 14-h-light and 10-h-dark condition in the green house. At 6 weeks, the floral transition of the shoot apical meristem has already occurred and the tassel primordium is developing.
      2. Dissect maize shoot into ~10 cm long sections (Figure 2). Make sure to include the shoot apex in this region. As internodes become shorter toward the apex, we can assume the approximate position of the shoot apex.


        Figure 2. A maize stem sample fixed in FAA

      3. Put the sample into a 50 ml conical tube and fill the tube with FAA solution (see Recipes).
      4. Slowly vacuum the sample until small air bubbles begin to come out from the samples, then seal the chamber. After 15 min, slowly release the vacuum pressure.
      5. After releasing the vacuum, replace the solution with new FAA. Vacuum the sample for 15 min again.
      6. Replace the solution with new FAA and store the sample at 4 °C for at least 1 week. g. Replace FAA with 70% EtOH and rotate the sample tube at 4 °C for 3 h. Replace 70% EtOH with new 70% EtOH. Store the sample in 70% EtOH at 4 °C until observation.
        Note: FAA and 70% EtOH containing formaldehyde must be disposed of according to regulations of your institute/university and local governments.
    2. Sample staining with contrasting reagents
      1. Replace 70% EtOH with 35% EtOH and incubate for 2 h at room temperature.
      2. Discard 35% EtOH and incubate the samples in distilled water at room temperature for 1.5 h. Repeat this step two more times with fresh distilled water.
      3. Replace the water with 25% Lugol solution (see Recipes) and vacuum for 15 min. Stain the sample for 6 days at room temperature in the dark.
        Note: Staining duration depends on the type of tissues and their size. Check the staining before detailed observation (please see Step C2). For maize stems, it takes 6 days (Figure 3).


        Figure 3. Time-course observation of a maize stem stained with Lugol solution. A maize stem stained in 25% Lugol solution for 1 day (A), 2 days (B), 4 days (C) and 6 days (D) was scanned using micro-CT. These images were processed and displayed in OsiriX. Scale bar = 5 mm.

  2. Mounting samples
    1. Place wet Kimwipes and filter foams at the bottom of 50 ml conical tube.
    2. Put the stained maize stem in the center of the tube. Make sure to insert the sample so that the lower side of the sample comes to the bottom of the tube (Figure 4A).
      Notes:
      1. Softwares used in the downstream data analysis (OsiriX and Imaris) display the imported data upside down. To avoid cumbersome data processing to correct this, we simply mount the samples upside down.
      2. As Lugol staining is rapidly released from tissue into water, just remove excess solution with paper towels. Do not rinse the sample with water.
      3. Samples will be rotated on the stage in micro-CT equipment during the data acquisition. It is important to place samples in the center of the stage to obtain high resolution images.
    3. Place filter foams and wet Kimwipes on the other end of the sample and close the tube.
      Note: Maize stems become softer after Lugol staining. Handle the sample gently to avoid deformation. At the same time, make sure to place enough wet Kimwipes and filter foams so that the sample doesn’t move in the tube. The movement of samples during data acquisition will result in a blurred image. Also, moisture in the tube is important to avoid sample drying and deformation.


      Figure 4. Mounting a maize stem for micro-CT scanning. A. The stained maize stem was mounted in a 50 ml polypropylene tube using wet Kimwipes and filter foams. B. The tube was attached on the stage in micro-CT scanner with a piece of clay.

  3. Data acquisition
    1. Put a piece of clay on the sample stage in the micro-CT equipment and fix the sample tube tightly on the clay (Figure 4B).
    2. Set the voltage at 90 kV and the current at 85 µA. Close the door of the equipment and turn on the X-ray. Now you can see the sample displayed in the fluoroscopic image monitor. To make the stem inner structure visible in the monitor, narrow down the luminance window of the fluoroscopic image. If the sample is stained enough, you can see the conical shape of the shoot apex in the monitor at this point (Figure 5). Determine the position of the shoot apex.
    3. Check the sample by rotating the stage. Adjust the tube angle so that the sample stands upside down in the center at a right angle to the stage (Figure 5A).


      Figure 5. X-ray fluoroscopic imaging before scanning. A. The stem sample mounted on the stage. Note that the stem sample is upside down. A yellow box represents region magnified in (B). B. A magnification of the boxed region in (A). Dashed lines and circle depict a young stem and tassel primordium, respectively. The image was rotated 180° from (A).

    4. Return the luminance window to its full range.
      Note: We adjusted the luminance window just to make the sample visible on the monitor. In data acquisition, the luminance window should be the full range.
    5. Determine the scanning area and set scanning parameters (e.g., Table S1) depending on the purpose of your experiment. It is a trade-off between data acquisition area and resolution as shown in (Figure 6).


      Figure 6. Maize stem images scanned using micro-CT at various resolution. A. A vertical section of the stem. Dashed boxes (c) (d) and (e) represent the magnified regions in (C), (D) and (E), respectively. B-E. Transverse sections of the stem. The resolutions are 67.7, 30.0, 17.0 and 10.0 µm/pixel in (A and B), (C), (D) and (E), respectively. The detailed settings in the scanning are provided in Table S1.

    6. Scan the samples.
      Note: When the sample size is larger than the area of data acquisition, the surrounding tissue outside of the field of view causes various noise (Figure 6E). If higher resolution is necessary, trimming the sample to remove surrounding tissue/organs will reduce noise in the data (see below).
    7. If necessary, remove leaves and stems to expose the part of your interest. To mount smaller samples, we use micro-tubes of appropriate size. Put wet Kimwipes and filter foams to fix the sample inside of the tube (Figure 7).


      Figure 7. Mounting trimmed maize stems in 1.5 ml tubes. A. Tenth and eleventh leaves from the tassel were removed from the stem sample shown in Figure 2. B. Ninth and eighth leaves were further removed. C. Diagrams showing the trimmed maize stem mounted in 1.5 ml tubes. Red arrows indicate supporting points of samples.

    8. Scan the samples with the setting for higher resolution (Table S2). Figure 8 shows that trimming the samples greatly reduces the noise and enables to increase the resolution.
      Note: The resolution of micro-CT images could be further improved by increasing frame average and number of projections (and hence, scanning time as well) (Table S2 and Figure 9).


      Figure 8. Micro-CT images at improved resolution achieved by trimming samples. A, C and E. Transverse sections of untrimmed (A) and trimmed (C and E) stem. B, D and F. Longitudinal sections of untrimmed (B) and trimmed (D and F) stem. Dashed green lines represent planes shown in transverse sections. Stem samples in Figures 2, 7A and 7B correspond to the micro-CT images in (A and B), (C and D) and (E and F), respectively. Scale bars = 1 mm.


      Figure 9. Micro-CT images at further improved resolutions achieved by increasing frame average and number of projections. Micro-CT images of the maize shoot apex scanned for 9.5 min (A-C) and 26 min (D-E). A, B, D and E. Transverse sections. C and F. Longitudinal sections. Note that provascular bundles enclosed by yellow dashed lines became more distinguishable in (D-E) compared to (A-C). Green lines (a), (b), (d) and (e) represent the plane of transverse sections shown in (A), (B), (D) and (E), respectively. The detailed settings of frame average and number of projections are shown in Table S2. Scale bars = 1 mm.

    9. The raw data will be saved as .raw files. Import the raw data into a built-in reconstruction software in the micro-CT system (e.g., coneCTexpress). Trim blank spaces, determine axes for coronal/sagittal sections, reduce noises using the noise reduction filter and convert the raw data into 8-bit Tiff images. Save the reconstructed Tiff image dataset.
      Note: To compare multiple samples, a uniform setting in this step will be important.
    10. Before importing the Tiff files into an image analysis software OsiriX, we rename them as following: Date_sample_resolution (µm)_number.tiff. This is important so that the image files are read by OsiriX in the correct order.

  4. Constructing scanning movie from micro-CT data
    Here, we introduce how to make a scanning movie using the software OsiriX to explore the micro-CT data (Video 1).

    Video 1. A 2D movie showing transverse sections of a maize stem. Green bars represent the plane shown in the transverse window. Structural annotations were added using Adobe Premiere Pro CC.

    1. To import Tiff image data, disable ‘Import DICOM file format only’ option in the preferences of OsiriX. (Top Menu ‘OsiriX MD’ → ‘Preferences’ → ‘Database’ icon → ‘File Management window’ → uncheck the box of ‘Import DICOM file format only’.)
    2. Import Tiff images. (‘Import’ icon → Select the data folder. → ‘Copy Links’)
    3. To display the imported data, select the data file shown in ‘Documents DB window’ and click ‘2D Viewer’ icon. Then, select ‘3D Volume Rendering’ from the ‘3D Viewer’ in the top menu and enter resolution information in the fields of pixel X, Y and slice interval (Figure 10A).
      Notes:
      1. If OsiriX gives an error message, click ‘Continue’.
      2. The unit of the resolution is mm/pixel. Convert the resolution in µm/pixel into mm/pixel (e.g., 30 µm/pixel → 0.030 mm/pixel).
    4. After clicking ‘OK’, a new window will open. Close this window.
    5. At this point, ‘Orientation’ icon has become active (Figure 10B). Now we can set the direction of 2D sections shown in the display using this icon and explore the sections using the slider located on the upper side of the 2D window.
    6. Open additional two windows by clicking ‘Window’ icon and selecting ‘3 window’ (Figure 10C). Enter the resolution information for these two windows as described in the Steps D3 and D4. Display coronal, sagittal and transverse sections in each window using ‘Orientation’ icon (Figures 10B and 9D).
    7. Select settings for window appearance using ‘WL/WW & CLUT’ and ‘Thick Slab’ functions. Our setting is below:
      WL/WW & CLUT (Figure 10E (a))
      WL/WW (window level/window width): Other
      CLUT (color look up table): B/W Inverse
      Opacity: Linear Table
      Thick Slab (Figure 10E (b))
      Mode: MIP
      Slider: 5
    8. Adjust image WL/WW, position, magnification and angle in the window using tools shown in Figure 10E (c), (d), (e) and (f), respectively.
    9. To adjust size and arrangement of each window for the movie preparation, disable ‘Magnetic Windows for move & resize’. (Top Menu ‘OsiriX MD’ → ‘Preferences’ → ‘Viewers’ icon → uncheck ‘Magnetic Windows for move & resize’.) Adjust size and arrangement of windows (e.g., Figure 10F). Also, you can change the magnification of sections in each window using icon shown in Figure 10E (e).
    10. Export a movie file. (Select a window of transverse sections → ‘Movie Export’ icon → Set the data area exported as a movie and check the box of ‘Include all displayed 2D viewers’ (Figure 10G). Select the directory, set the format and frame rate. → Save the movie.
      Note: As default, ‘Basic’ option is selected in ‘Annotations’ function area and various information about source data will be displayed in the 2D windows. You can disable this setting by checking ‘Graphic’.


      Figure 10. Movie preparation of the maize stem micro-CT data using OsiriX for easy observation. Magenta boxes and circles highlight icons, check boxes and numeric input fields selected in each step. A. A window to set the resolution; B. Orientation icons; C. Window icon to select ‘3 windows’; D. Three windows displaying different sections of the same sample. Left, middle and right windows display coronal, sagittal and transverse sections, respectively. E. Tools and icons used to modify window appearances. (a) WL/WW & CLUT, (b) Thick slab, (c) WL/WW, (d) position, (e) magnification and (f) angle. F. Arranged and resized windows to produce a movie. Note that the red-framed window displaying transverse sections is selected and active. Green bars in coronal and sagittal windows represent planes shown in transverse sections. G. A window to set parameters to export a movie.

  5. Creating 3D model of maize stem vascular networks
    Here, we extract vein traces and reconstruct vascular networks in maize stems using Imaris, a software which can be used in various analysis of 3D data including the extraction of filamentous structure such as veins. As this procedure includes many steps of manual operation, we provide videos to explain how to determine vein traces in 3D data (Videos 2 and 3).
    1. Preparation of a 3D model of the maize stem in Imaris
      1. Import the sequential Tiff data into Imaris (Top Menu ‘Surpass’ icon → ‘File’ → ‘open’ → Select the first Tiff image in the data folder. → ‘Open’. Video 2: 0~10 sec).
      2. Enter the scale information (Top menu ‘Edit’ → ‘Image Properties’ → ‘Geometry’ (Figure 11A) → Enter the resolution (µm/pixel) → ‘OK’ → The 3D object often becomes very small after entering the resolution. Resize and fit the object to the window using ‘Fit’ icon at the bottom of the screen. Video 2: 12~27 sec).
      3. Change the model color. We selected white in this example. (Top menu ‘Edit’ → ‘Show Display Adjustment’ → ‘Channel 1’ (Figure 11B) → ‘Base color’ tab (Figure 11C) → Select color. Video 2: 33~38 sec).
      4. Change the model size and angle. (Video 2: 38~43 sec)
        Note: You can rotate, move and enlarge/reduce the imported 3D model using left-dragging, right-dragging and wheeling actions of your computer mouse, respectively.
      5. Crop 3D model to determine the data area analyzed. In this example, we cropped the stem into a quarter cylinder (‘Setting’ tab (Figure 11D) → ‘Blend’ → Top menu ‘Edit’ → ‘Crop 3D’ → Crop the 3D model (Figure 11E). → ‘OK’. Video 2: 44 sec~1 min 13 sec).
      6. Save the cropped 3D model. (Top menu ‘Export’ icon → Enter file name → ‘Save’. Video 2: 1 min 14 sec~1 min 35 sec).


        Figure 11. Preparation of a 3D model of the maize stem in Imaris. Magenta boxes highlight icons, check boxes and numeric input fields selected in each step. A. Geometry window to set resolution; B. Display adjustment window; C. Color selection window; D. Setting tab in the property area of the selected volume object; E. Crop 3D window.

        Video 2. A movie introducing procedures to prepare the maize stem 3D model in Imaris

    2. Tracing veins to create 3D model of vascular networks
      Vein traces are the combination of relatively straight lines in internodes and winding lines in nodes. To make a vein trace from the micro-CT data, we divide a vein into a few segments depending on the position (internode or node), extract them independently and join them into a single trace.
      1. Change settings of FilamentTracer to extract veins manually (Top menu ‘3D view’ icon → ‘Filament’ icon (Figure 12A) → Click ‘Skip automatic creation, edit manually’ (Figure 12B) → ‘Draw’ tab → Check ‘Manual’, ‘Automatic Placement’ and ‘XZ Plane’ (magenta boxes in Figure 12C). Video 3: 0~14 sec).
        Note: Setting ‘XZ Plane’ is to extract a vertical segment of a vein in internodes in the Step E2d.
      2. As default, the volume object shown as ‘Volume’ in the object list area is selected. To display slices of the XZ plane, uncheck ‘Volume’, otherwise the slices hide behind the volume object. Determine a vein to be traced using the slider (the bottom magenta box in Figure 12C and Video 3: 15~24 sec).
      3. Before starting to trace a vein, check ‘Select’ in ‘Camera/Labels’ window and then adjust the line width to that of the vein by wheeling the computer mouse. The size of square around the mouse pointer represents the line width. (The magenta box in Figure 12D and Video 3: 26~31 sec.)
        Note: This step is important for Imaris to recognize a vein efficiently based on the brightness of the 3D data.
      4. Manually trace a segment of the vein in an internode. (Check ‘Select’ in ‘Camera/Labels’ window (the magenta box in Figure 12D) → Trace the vein by left-dragging while pressing ‘Shift’ key. Video 3: 32~43 sec).
      5. Edit the width and color of the traced line (Line width: ‘Filament’ tab → ‘cylinder’ → Enter the width (Figure 12E, e.g., 150). Line color: ‘Color’ tab → ‘Base’ → Change the color (Figure 12F). Video 3: 44~55 sec).
      6. Correct the traced line based on the brightness of the 3D data (‘Edit’ tab → ‘Segment’ → Click and select the traced line. (The line color will be yellow.) → ‘Center’ (Magenta boxes in Figure 12G). Video 3: 56 sec~1 min 04 sec).
      7. Manually trace a segment of the vein running horizontally in the node (‘Draw’ tab → Check ‘Manual’ and ‘Automatic Placement’ and ‘XY Plane’ (a blue box in Figure 12C) → Confirm the start and end of the segment of the vein in the node using a slider (Figure 12C and 12H). → Trace the segment and edit the line as described in Steps E2c-E2f. Video 3: 1 min 05 sec ~1 min 55 sec).
      8. Manually trace a segment of the vein in the lower internode as described in Steps E2c-E2f (Video 3: 1 min 58 sec~2 min 18 sec).
      9. Join the upper two segments (‘Camela/Labels’ window → Check ‘Navigate’ (A blue box in Figure 12D) → Enlarge the joint between upper two segments → Check ‘Select’ in ‘Camera/Labels’ window (a magenta box in Figure 12D). → ‘Edit’ tab (blue boxes in Figure 12G) → ‘Point’ → While pressing ‘Ctrl’ key, click the lower end of the upper segment and the upper end of the lower segment. Then both ends turn into yellow. → ‘Join’ → Click a blue joint point. → ‘Delete’ → Click newly formed ends while pressing ‘Ctrl’ key. → ‘Join’. Video 3: 2 min 19 sec~2 min 42 sec).
      10. Join the lower two segments as described in the Step E2i. (Video 3: 2 min 42 sec~3 min 00 sec)
      11. Correct the whole vein trace based on the brightness of the 3D data as described in the Step E2f (Video 3: 3 min 05 sec~3 min 14 sec).
      12. Repeat Steps E2c-E2k to extract traces of other veins. An example of the extracted vein network is shown in Figure 12I.


        Figure 12. Tracing veins to create 3D model of vascular networks. Magenta boxes and circles highlight icons, check boxes and numeric input fields selected in each step. Blue boxes indicate those used in later steps. A. Filament icon; B. A button to skip automatic creation in the creation wizard window; C. Draw tab; D. Camera/Labels window; E. Setting tab; F. Color tab; G. Edit tab; H. Confirming the start and end of the segment running horizontally in the node. Top, middle and bottom panels represent the start, middle and end of the segment, respectively. Arrows indicate the vein to be traced shown in the node slices. I. A vascular network extracted from the micro-CT data of the maize stem.

        Video 3. A movie introducing procedures to extract vascular networks in the maize stem

Data analysis

The raw data of micro-CT scanning was saved as .raw files and converted to 8-bit Tiff images using coneCTexpress as described in section C9. The 3D model of micro-CT scanning data was reconstructed from these tiff images using OsiriX (see Procedure D). The vascular network information was extracted using Imaris as described in Procedure E.
In our original research, we observed three independent biological replicates. Student’s t-test was applied for statistical analysis (Tsuda et al., 2017).

Recipes

  1. Fixative FAA solution
    Formalin:acetic acid:50% ethanol = 5:5:90
  2. Iodine staining (Degenhardt et al., 2010)
    Note: Tips for iodine staining using Lugol solution are well described by Gignac et al. (2016).
    100% Lugol stock solution
    Potassium iodide 10 g
    Iodine 5 g
    Distilled water (DW) to 100 ml
  3. 25% Lugol working solution
    Dilute 100% stock solution to 25% with DW upon use

Acknowledgments

We thank Mitsuhiko Kurusu (National Institute of Genetics, Office for Research Development) for organizing imaging seminars which led us to collaborate in this project and Akatsuki Kimura (National Institute of Genetics, Cell Architecture Laboratory) for introducing and maintaining Imaris. We also thank Sarah Hake (University of California, Berkeley) for providing maize stem samples. This work was supported by JSPS KAKENHI Grant Number 17H00440 to A.M. and JP16K18637 to K.T. This protocol was adapted from our recent work (Tsuda et al., 2017). The authors declare no conflicts of interest or competing interest.

References

  1. Degenhardt, K., Wright, A. C., Horng, D., Padmanabhan, A. and Epstein, J. A. (2010). Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining. Circ Cardiovasc Imaging 3(3): 314-322.
  2. Gignac, P. M., Kley, N. J., Clarke, J. A., Colbert, M. W., Morhardt, A. C., Cerio, D., Cost, I. N., Cox, P. G., Daza, J. D., Early, C. M., Echols, M. S., Henkelman, R. M., Herdina, A. N., Holliday, C. M., Li, Z., Mahlow, K., Merchant, S., Muller, J., Orsbon, C. P., Paluh, D. J., Thies, M. L., Tsai, H. P. and Witmer, L. M. (2016). Diffusible iodine-based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-resolution, 3-D imaging of metazoan soft tissues. J Anat 228(6): 889-909.
  3. Metscher, B. D. (2009a). MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol 9: 11.
  4. Metscher, B. D. (2009b). MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions. Dev Dyn 238(3): 632-640.
  5. Staedler, Y. M., Masson, D. and Schonenberger, J. (2013). Plant tissues in 3D via X-ray tomography: simple contrasting methods allow high resolution imaging. PLoS One 8(9): e75295.
  6. Tsuda, K., Abraham-Juarez, M. J., Maeno, A., Dong, Z., Aromdee, D., Meeley, R., Shiroishi, T., Nonomura, K. I. and Hake, S. (2017). KNOTTED1 cofactors, BLH12 and BLH14, regulate internode patterning and vein anastomosis in maize. Plant Cell 29(5): 1105-1118.

简介

茎中的植物血管系统与空中器官连接根部以移动含有矿物质,营养素和信号分子的溶质,因此它们在植物生长和发育中起关键作用。 然而,由于它们深深地嵌入组织中,所以干燥的血管系统,特别是在作物种类中,描述得很差。 在这里我们描述一个协议,利用微型计算机断层扫描(微CT)扫描可视化玉米茎中的血管网络。 该协议涵盖样品固定和对比试剂染色,数据采集使用显微CT,重建三维(3D)模型的茎内部结构和提取血管网络模型。 该协议可以很容易地适用于各种类型的物种和器官/组织。

【背景】单子叶茎具有独特和独立的静脉的特征性血管网络。尽管支持生长发育的重要性,但单子叶植物的血管网络模式研究甚少。干血管网络的可视化是相当具有挑战性的,因为静脉深深地嵌入组织中。常规的组织切片可以用来观察网络,但是这是一个费时费力的过程,需要观察数百个部分。此外,作物茎的尺寸比显微镜下的视野大得多,因此很难捕捉整个系统。

最近,我们报道了玉米转录因子BEL1样同源异形体(BLH)12和BLH14在茎发育和静脉网络形成中起重要作用(Tsuda等人,2017)。为了全面了解血管系统,我们采用了先前描述的微型CT扫描并针对玉米茎杆进行了优化(Metscher,2009a和2009b,Degenhardt等人,2010,Staedler< et al。,2013,Gignac 等人,2016)。通过将这种方法与图像分析相结合,我们能够以有效和可靠的方式重建内部结构的三维模型。这个协议可以用来可视化内部结构,如各种物种和组织的静脉。

关键字:微型CT扫描, 玉米, 茎, 维管网络, 三维建模

材料和试剂

  1. 50毫升聚丙烯管(Greiner Bio One International,目录号:227261)
  2. Kimwipes
  3. 1.5毫升微管(Eppendorf,目录号:0030125150)
  4. 过滤泡沫MOLTOFILTER MF-13厚度5毫米(INOAC Corp.)
  5. 玉米(B73)
  6. 福尔马林(Wako Pure Chemical Industries,目录号:061-00416)
  7. 乙酸(Wako Pure Chemical Industries,目录号:017-00256)
  8. 乙醇(Wako Pure Chemical Industries,目录号:057-00451)
  9. 卢戈股票解决方案
  10. 碘化钾(Wako Pure Chemical Industries,目录号:166-03971)
  11. 碘(Wako Pure Chemical Industries,目录号:094-05421)
  12. 固定FAA解决方案(见食谱)
  13. 碘染色(Degenhardt等人,2010)(参见食谱)
  14. 25%卢戈尔工作解决方案(见食谱)

设备

  1. X射线微CT成像系统(Comscantechno,型号:ScanXmate-E090S105)(图1)
    1. X射线管:微焦点X射线源(Hamamatsu Photonics K.K.,型号:L9421-02)
    2. 检测器:平板检测器(Varex Imaging,型号:PaxScan 1313DX)


      图1.本协议中使用的X射线显微CT成像系统

软件

  1. coneCTexpress(内置于Micro-CT成像系统,Comscantechno Co. Ltd.,神奈川县,日本)
  2. OsiriX MD v8.5( http://www.osirix-viewer.com ,Pixmeo SARL,瑞士)
  3. Imaris 8.2(英国Bitplane)( http://www.bitplane.com
  4. Adobe Premiere Pro CC( http://www.adobe.com ,Adobe,美国)

程序

  1. 固定样品并用对比试剂染色
    1. 玉米茎解剖和固定
      1. 种玉米植物6-8周。我们的生长条件是在温度为26°C,温室14小时,10小时的条件下。在6周时,已经发生了茎尖分生组织的花转变,并且流苏原基正在发育。
      2. 解剖玉米芽〜10厘米长的部分(图2)。确保在这个地区包括芽尖。随着节间变得越来越短,我们可以假设茎尖的近似位置。


        图2.固定在FAA的玉米茎样品

      3. 将样品放入50ml锥形管中,并用FAA溶液填充管(见食谱)。
      4. 缓慢抽真空,直到样品中出现小气泡,然后密封反应室。 15分钟后,缓慢释放真空压力。
      5. 释放真空后,用新的FAA更换溶液。将样品再次抽真空15分钟。
      6. 用新的FAA替换溶液,并将样品在4°C保存至少1周。 G。用70%乙醇替换FAA,并在4℃下旋转样品管3小时。用新的70%乙醇代替70%乙醇。
        将样品储存在4°C的70%乙醇中直至观察 注意:含有甲醛的FAA和70%EtOH必须按照您所在学院/大学和当地政府的规定进行处理。
    2. 样品用对比试剂染色

      1. 用35%EtOH替代70%EtOH,并在室温下孵育2小时
      2. 弃去35%EtOH,并将样品在室温下在蒸馏水中孵育1.5小时。用新鲜的蒸馏水重复这个步骤两次。
      3. 用25%的Lugol溶液替换水(参见食谱)并抽真空15分钟。
        在室温下在黑暗中将样品染色6天 注:染色持续时间取决于组织的类型和大小。在详细观察之前检查染色(请参阅步骤C2)。对于玉米茎,需要6天(图3)。


        图3.用Lugol溶液染色的玉米茎的时间过程观察将玉米茎在25%卢戈溶液中染色1天(A),2天(B),4天(C)和6天(D)使用显微CT扫描。这些图像被处理并显示在OsiriX中。比例尺= 5毫米。

  2. 安装样品
    1. 将湿Kimwipes和过滤泡沫放在50毫升锥形管底部。
    2. 把染色的玉米茎放在管的中心。确保插入样品,使样品的下侧到达管的底部(图4A)。
      注意:
      1. 下游数据分析中使用的软件(OsiriX和Imaris)将导入的数据上下颠倒显示。为了避免繁琐的数据处理来纠正这个问题,我们只需将样品倒挂。
      2. 由于Lugol染色从组织迅速释放到水中,只需用纸巾除去多余的溶液即可。不要用水冲洗样品。
      3. 在数据采集过程中,样品将在微型CT设备的舞台上旋转。将样品置于舞台中央以获得高分辨率的图像很重要。

    3. 在过滤器泡沫和湿Kimwipes放在样品的另一端,并关闭管。
      注意:Lugol染色后,玉米茎变软。轻轻处理样品以避免变形。同时,确保放置足够湿的Kimwipes和过滤泡沫,使样品不会在管中移动。数据采集过程中样本的移动会导致图像模糊。此外,管中的水分对于避免样品干燥和变形非常重要。


      图4.安装用于显微CT扫描的玉米杆。 :一种。使用湿Kimwipes和过滤泡沫将染色的玉米茎放置在50ml聚丙烯管中。 B.用一块粘土将管子连接在微型CT扫描仪的台上。

  3. 数据采集
    1. 在微型CT设备的样品台上放一块粘土,并将样品管紧紧地固定在粘土上(图4B)。
    2. 将电压设置为90 kV,电流设置为85μA。关闭设备的门并打开X光。现在,您可以看到荧光透视图像监视器中显示的示例。为了使显示器内的显像管内部结构可见,请缩小透视图像的亮度窗口。如果样本染色不够,可以在此处看到显示器顶端的圆锥形状(图5)。确定茎尖的位置。
    3. 旋转舞台检查样品。调整管角度,使样品在中心与台上成直角(图5A)。


      图5.扫描前的X射线透视成像A.安装在平台上的茎样品。请注意,茎样本是倒挂的。 (B)中的黄色框表示放大的区域。 B.(A)中的盒装区域的放大。虚线和圆圈分别描绘了一个年轻的茎和流苏原基。图像从(A)旋转了180°。

    4. 将亮度窗口返回到其全部范围。
      注:我们调整了亮度窗口,使样品在显示器上可见。在数据采集中,亮度窗口应该是全范围。
    5. 确定扫描区域并设置扫描参数( ,表S1 ),这取决于您的实验目的。这是数据采集区域和分辨率之间的一个折衷(如图6所示)。


      图6.使用不同分辨率的微CT扫描的玉米茎图像。 :一种。茎的垂直部分。 (c)(d)和(e)分别表示(C),(D)和(E)中的放大区域。是。茎的横切面。 (A和B),(C),(D)和(E)中的分辨率分别为67.7,30.0,17.0和10.0μm/像素。扫描中的详细设置在表S1中提供

    6. 扫描样品。
      注意:当样本量大于数据采集的面积时,视野外的周围组织会产生各种噪声(图6E)。如果需要更高的分辨率,修剪样品以去除周围的组织/器官将减少数据中的噪音(见下文)。
    7. 如果有必要,删除叶和茎揭露你感兴趣的部分。为了安装更小的样品,我们使用适当尺寸的微管。把湿的Kimwipes和过滤泡沫固定在试管内(图7)。


      图7.在1.5ml试管中固定修剪过的玉米杆A.A。从雄穗样品中取出第十和第十一片叶,从图2所示的茎中取出。B.第九和第八片叶被进一步去除。 C.显示装在1.5ml管中的修剪的玉米茎的图。红色箭头表示样本的支持点。

    8. 使用高分辨率设置扫描样本( Table S2 )。图8显示,修整样本大大降低了噪音,并能够提高分辨率。
      注:微CT图像的分辨率可以通过增加帧平均数和投影数来进一步改善(因此也可以是扫描时间)( Table S2 和图9) em>


      图8.通过微调样品获得的改进分辨率的微CT图像A,C和E.未修剪的(A)和修剪的(C和E)茎的横向切片。 B,D和F。未修剪的(B)和修剪的(D和F)茎的纵向切片。绿色的虚线表示以横截面显示的平面。图2,7A和7B中的茎样品分别对应于(A和B),(C和D)和(E和F)中的微CT图像。比例尺= 1毫米。


      图9.通过增加帧平均数和投影数量获得的进一步提高的分辨率的微CT图像扫描玉米梢尖的微CT图像9.5分钟(AC)和26分钟(DE) 。 A,B,D和E.横向部分。 C和F纵向部分。注意,与(A-C)相比,用黄色虚线包围的维管束在(D-E)中变得更加可区分。绿线(a),(b),(d)和(e)分别表示(A),(B),(D)和(E)所示横断面。平均帧数和投影数量的详细设置显示在表S2 。比例尺= 1毫米。

    9. 原始数据将被保存为.raw文件。将原始数据导入到micro-CT系统中的内置重建软件(例如,coneCTexpress)中。修剪空白处,确定冠/矢状切面的轴,使用降噪滤波器降低噪声,并将原始数据转换为8位Tiff图像。保存重建的Tiff图像数据集。
      注意:为了比较多个样本,在这一步中统一的设置将是非常重要的。
    10. 在将Tiff文件导入图像分析软件OsiriX之前,我们将它们重命名为:Date_sample_resolution(μm)_number.tiff。这很重要,以便OsiriX以正确的顺序读取图像文件。

  4. 从微CT数据构建扫描电影
    在这里,我们介绍如何使用OsiriX软件制作扫描电影,探索微CT数据(视频1)。

    视频1

    1. 要导入Tiff图像数据,请在OsiriX的首选项中禁用“仅导入DICOM文件格式”选项。 (顶部菜单“OsiriX MD”→“首选项”→“数据库”图标→“文件管理窗口”→取消选中“仅导入DICOM文件格式”框)。
    2. 导入Tiff图像。 (“导入”图标→选择数据文件夹→“复制链接”)
    3. 要显示导入的数据,请选择“文档数据库窗口”中显示的数据文件,然后单击“2D查看器”图标。然后,从顶部菜单的“3D查看器”中选择“3D体绘制”,并在像素X,Y和切片间隔(图10A)的字段中输入分辨率信息。
      注意:
      1. 如果OsiriX发出错误信息,请点击“继续”。
      2. 分辨率的单位是毫米/像素。将以μm/像素为单位的分辨率转换为mm /像素(例如30μm/像素→0.030 mm /像素)。
    4. 点击“确定”后,将打开一个新窗口。关闭这个窗口。
    5. 此时,“方向”图标变为活动状态(图10B)。现在,我们可以使用这个图标来设置显示屏中显示的2D部分的方向,并使用位于2D窗口上方的滑块来浏览部分。
    6. 点击“窗口”图标并选择“3窗口”打开另外两个窗口(图10C)。按照步骤D3和D4所述输入这两个窗口的分辨率信息。使用“方向”图标在每个窗口中显示冠状,矢状和横切面(图10B和9D)。
    7. 选择“WL / WW& CLUT“和”厚板“功能。我们的设置如下:
      WL / WW& CLUT(图10E(a))
      WL / WW(窗口级别/窗口宽度):其他
      CLUT(颜色查找表):B / W反转
      不透明度:线性表格
      厚板(图10E(b))
      模式:MIP
      滑块:5
    8. 使用图10E(c),(d),(e)和(f)分别显示的工具,调整图像的WL / WW,位置,放大倍率和角度。
    9. 要调整电影准备的每个窗口的大小和排列,请禁用“Magnetic Windows for move&调整”。 (顶部菜单“OsiriX MD”→“首选项”→“查看者”图标→取消选中“磁性窗口移动和调整大小”)调整窗口大小和排列(如图10F所示)。另外,您可以使用图10E(e)所示的图标来更改每个窗口中的部分的放大率。
    10. 导出电影文件。 (选择一个横切面的窗口→'电影导出'图标→设置导出的数据区域为电影,并勾选'包括所有显示的2D查看器'(图10G),选择目录,设置格式和帧率。 →保存电影。
      注意:默认情况下,在“注释”功能区中选择“基本”选项,关于源数据的各种信息将显示在2D窗口中。您可以通过选中“图形”来禁用此设置。


      图10.利用OsiriX电影准备玉米干微CT数据以便于观察。品红框和圆圈突出显示在每个步骤中选择的图标,复选框和数字输入字段。 A.设置分辨率的窗口; B.方向图标; C.窗口图标选择“3个窗口”; D.三个窗口显示同一样本的不同部分。左,中,右窗分别显示冠状,矢状和横断面。 E.用于修改窗口外观的工具和图标。 (a)WL / WW& CLUT,(b)厚板,(c)WL / WW,(d)位置,(e)放大率和(f)角度。 F.安排和调整窗口大小来制作电影。请注意,显示横断面的红框窗口被选中并激活。冠状面和矢状面窗口中的绿色条代表横断面所示的平面。 G.设置参数导出电影的窗口。

  5. 创建玉米茎血管网络的三维模型
    在这里,我们使用Imaris提取静脉痕迹并重建玉米茎中的血管网络,Imaris是一种可用于各种3D数据分析(包括提取静脉等丝状结构)的软件。由于此程序包含许多手动操作步骤,因此我们提供视频来解释如何确定3D数据中的静脉痕迹(视频2和3)。
    1. 在Imaris中制备玉米茎的3D模型
      1. 将连续的Tiff数据导入Imaris(Top Menu'Surpass'图标→'文件'→'打开'→在数据文件夹中选择第一个Tiff图像→'打开'视频2:0〜10秒) >
      2. 输入比例尺信息(顶部菜单'编辑'→'图像属性'→'几何'(图11A)→输入分辨率(μm/像素)→'OK'→输入分辨率后3D对象经常变得非常小调整并使用屏幕底部的“飞度”图标将对象贴在窗口上。视频2:12〜27秒)。
      3. 更改模型颜色。我们在这个例子中选择了白色。 (顶部菜单'编辑'→'显示调整'→'频道1'(图11B)→'基本颜色'标签(图11C)→选择颜色视频2:33〜38秒)。
      4. 更改模型的大小和角度。 (视频2:38〜43秒)
        注意:您可以分别使用鼠标左键,右键拖动和滚动操作来旋转,移动和放大/缩小导入的3D模型。
      5. 裁剪3D模型以确定分析的数据区域。在这个例子中,我们将茎切成四分之一圆柱('设置'选项卡(图11D)→“混合”→顶部菜单“编辑”→“裁剪3D”→裁剪3D模型(图11E)→“确定”视频2:44秒〜1分13秒)。
      6. 保存裁剪的三维模型。 (顶部菜单“导出”图标→输入文件名→“保存”视频2:1分14秒〜1分35秒)。


        图11.在Imaris中制作玉米茎的3D模型。品红框突出显示在每个步骤中选择的图标,复选框和数字输入字段。 A.几何窗口设置分辨率; B.显示调整窗口; C.颜色选择窗口; D.在选定的卷对象的属性区域中设置选项卡; E.裁剪3D窗口。

        视频2

    2. 追踪静脉创建血管网络的三维模型
      静脉痕迹是节点中相对直线和节点上绕组线的组合。为了从micro-CT数据中做出静脉追踪,我们根据位置(节间或节点)将静脉分成几段,独立地提取它们并将它们合并成单一的迹线。
      1. 更改FilamentTracer的设置以手动提取静脉(顶部菜单'3D view'图标→'Filament'图标(图12A)→点击'跳过自动创建,手动编辑'(图12B)→'绘制'选项卡→检查'手动' “自动放置”和“XZ平面”(图12C中品红色盒子)视频3:0〜14秒)。
        注意:设置“XZ平面”是在步骤E2d的节间中提取静脉的垂直段。
      2. 默认情况下,选择对象列表区域中显示为“Volume”的卷对象。要显示XZ平面的切片,请取消选中“卷”,否则切片隐藏在卷对象后面。使用滑块(图12C和视频3:15〜24秒中的底部洋红色框)确定要跟踪的静脉。
      3. 在开始追踪静脉之前,请在“相机/标签”窗口中选择“选择”,然后通过转动电脑鼠标将线宽调整为静脉的宽度。鼠标指针周围的大小代表线宽。 (图12D中的品红色盒子和视频3:26〜31秒)
        注意:这一步对Imaris来说很重要,可以根据3D数据的亮度有效识别静脉。
      4. 手动追踪节间中的一段静脉。 (在“摄像机/标签”窗口(图12D中品红色框)中选择'选择'→按住'Shift'键的同时左键拖动来跟踪静脉。视频3:32〜43秒)。
      5. 编辑被跟踪线的宽度和颜色(线宽:'细丝'标签→圆柱体→输入宽度(图12E,例如,150)线条颜色:“颜色”标签→'基地“→改变颜色(图12F)。视频3:44〜55秒)。
      6. 根据三维数据的亮度校正曲线(“编辑”选项卡→“段”→单击并选择描迹线(线颜色为黄色)→“中心”(图12G中的品红色框)。视频3:56秒〜1分04秒)。
      7. 在节点(“绘图”选项卡→检查“手动”,“自动放置”和“XY平面”(图12C中的蓝色框)中手动跟踪横向运行的一段静脉)→确认段的开始和结束使用一个滑块(图12C和12H)在节点上的静脉→按照步骤E2c-E2f中所描述的那样跟踪片段并编辑线段视频3:1分05秒〜1分55秒)
      8. 如步骤E2c-E2f(视频3:1分58秒〜2分18秒)中所述手动跟踪下节间中的一段静脉。
      9. 加入上面两个部分('Camela / Labels'窗口→检查'Navigate'(图12D中的蓝色框)→放大上部两个部分之间的连接点→在'Camera / Labels'窗口中选择'Select'图12D)→'编辑'选项卡(图12G中的蓝色框)→'点'→在按住'Ctrl'键的同时点击上部分的下端和下部分的上端,然后两端变成黄色→“加入”→点击一个蓝色的连接点→“删除”→按住“Ctrl”键点击新形成的结束→“加入”视频3:2分19秒〜2分42秒。 />
      10. 如步骤E2i所述加入下面两个段。 (视频3:2分42秒〜3分00秒)
      11. 根据步骤E2f(视频3:3分05秒〜3分14秒)中所述的3D数据的亮度校正整个静脉轨迹。
      12. 重复步骤E2c-E2k来提取其他静脉的痕迹。提取的静脉网络的一个例子如图12I所示。


        图12.追踪静脉以创建血管网络的三维模型。品红框和圆圈突出显示在每个步骤中选择的图标,复选框和数字输入字段。蓝色框表示在后面的步骤中使用的那些。 A.长丝图标; B.在创建向导窗口中跳过自动创建的按钮; C.绘制标签; D.相机/标签窗口; E.设置标签; F.彩色标签; G.编辑标签; H.确认在节点中水平运行的段的开始和结束。顶部,中部和底部分别表示分段的开始,中间和结束。箭头指示要在节点切片中显示的要跟踪的静脉。 I.从玉米茎的微CT数据中提取的血管网。

        视频3

数据分析

微CT扫描的原始数据保存为.raw文件,并使用coneCTexpress转换为8位Tiff图像,如C9部分所述。使用OsiriX从这些tiff图像重建微CT扫描数据的3D模型(参见程序D)。如程序E所述,使用Imaris提取血管网信息。
在我们最初的研究中,我们观察到三个独立的生物学重复。应用学生t检验进行统计分析(Tsuda et al。,2017)。

食谱

  1. 固定FAA解决方案
    福尔马林:醋酸:50%乙醇= 5:5:90
  2. 碘染色(Degenhardt et al。 ,2010)
    注意:使用Lugol溶液进行碘染色的技巧在Gignac等人的文章中有详细描述。 (2016)。
    100%卢戈储备液
    碘化钾10克
    碘5克
    蒸馏水(DW)至100毫升
  3. 25%卢戈工作解决方案
    使用DW将100%储备溶液稀释至25%

致谢

我们感谢Mitsuhiko Kurusu(研究开发办公室国立遗传学研究所)组织的成像研讨会,这个研讨会促成了我们在这个项目中的合作,以及Akatsuki Kimura(国家遗传学研究所细胞建筑实验室)介绍和维护Imaris。我们也感谢Sarah Hake(加利福尼亚大学伯克利分校)提供的玉米茎样品。这项工作得到了JSPS KAKENHI Grant No. 17H00440至A.M.的支持。和K.K.的JP16K18637。这个协议是根据我们最近的工作(Tsuda et。,2017)改编的。作者声明不存在利益冲突或利益冲突。

参考

  1. Degenhardt,K.,Wright,A.C。,Horng,D.,Padmanabhan,A。和Epstein,J.A。(2010)。 用碘染色显微CT检测小鼠胚胎心血管发育快速3D表型
    Circ Cardiovasc成像 3(3):314-322
  2. Gignac,PM,Kley,NJ,Clarke,JA,Colbert,MW,Morhardt,AC,Cerio,D.,Cost,IN,Cox,PG,Daza,JD,Early,CM,Echols,MS,Henkelman,RM,Herdina AN,Holliday,CM,Li,Z.,Mahlow,K.,Merchant,S.,Muller,J.,Orsbon,CP,Paluh,DJ,Thies,ML,Tsai,HP和Witmer,LM(2016)基于扩散碘的造影增强计算机断层扫描(diceCT):一种新兴的快速,分辨率,后生动物软组织的三维成像。 J Anat 228(6):889-909。
  3. Metscher,B. D.(2009a)。 MicroCT用于比较形态学:简单染色方法允许各种非矿化动物组织的高对比度3D成像。 BMC Physiol 9:11。
  4. Metscher,B. D.(2009b)。 MicroCT for developmental biology:一种用于组织学分辨率高对比度3D成像的多功能工具。 Dev Dyn 238(3):632-640。
  5. Staedler,Y.M。,Masson,D。和Schonenberger,J。(2013)。 通过X射线断层扫描植入3D组织:简单的对比方法可以进行高分辨率成像。
  6. Tsuda,K.,Abraham-Juarez,M. J.,Maeno,A.,Dong,Z.,Aromdee,D.,Meeley,R.,Shiroishi,T.,Nonomura,K. I.和Hake,S.(2017)。 KNOTTED1辅助因子,BLH12和BLH14,调节玉米节间格局和静脉吻合。 < (Plant Cell) 29(5):1105-1118。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Maeno, A. and Tsuda, K. (2018). Micro-computed Tomography to Visualize Vascular Networks in Maize Stems. Bio-protocol 8(1): e2682. DOI: 10.21769/BioProtoc.2682.
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