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Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes
对根进行自动跟踪以进行细胞过程的共焦延时成像   

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参见作者原研究论文

本实验方案简略版
Nature Plants
Jun 2016

Abstract

Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amenable to automatically track non-plant structures with an apical growth.

Keywords: Cell division (细胞分裂), Mitosis (有丝分裂), Cytokinesis (细胞因子), Root (根), Microscopy (显微镜检查), Tracking (跟踪), Arabidopsis (拟南芥), Phosphoinositide (磷酸肌醇)

Background

Cytokinesis is the last step of cell division, when the mother cell cytoplasm is partitioned between two daughter cells (Lipka et al., 2015). In plants, it is achieved through the centrifugal expansion of a cell plate in the division plane, which eventually becomes the newly synthetized cell wall between the cells that underwent mitosis (Buschmann and Zachgo, 2016; Müller and Jürgens, 2016). Plant cells, being embedded in a stiff cell wall, cannot migrate. Orientation of cell division together with elongation is therefore critical for organ morphogenesis. Root meristems are a good model to study cell division because they are easily amenable to microscopy techniques without the need of dissection. However, roots undergoing cell division grow in length, and therefore require manual adjustment of the observation field over time. This protocol allows easy time-lapse imaging of cytokinesis, and of other cellular processes.

Materials and Reagents

  1. 12-well microplates (Corning, Costar®, catalog number: 3513 )
  2. Microscope slides 76 x 26 x 1.1 mm (RS Components, catalog number: ISO 8037 )
  3. Microscope coverslips 22 x 60 mm (Thermo Fisher Scientific, Menzel-Gläser, catalog number: 630-2102 )
  4. Observation chambers, Lab-Tek II Chambered Coverglass W/Cover #1.5 Borosilicate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 155360 )
  5. Strait scalpel blades (e.g., Swann Morton, straights mounted BS EN 27740 blades)
  6. Arabidopsis thaliana, 4 to 7 days-old seedlings of wild-type genotype, or expressing membrane and/or cell plate-localized fluorescent fusion proteins (e.g., 2xp35S::MP:YFP in Col-0, where MP is a myristoylation and palmitoylation signal sequence [Martinière et al., 2012; Simon et al., 2016]). Seedling can be grown vertically in squared plates (90 x 90 x 15) or round plates to get intact growing roots
  7. Murashige and Skoog (MS) medium (Sigma-Aldrich, catalog number: M5519 )
  8. Agar (Sigma-Aldrich, catalog number: A7921-1KG )
  9. FM4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T-3166 )
  10. Suitable fluorescent dyes (alternative) or fluorescent proteins for registration
    1. Plasma membrane or cell wall dyes such as FM4-64 or propidium iodide (Sigma-Aldrich, catalog number: P4864 )
    2. Nuclei dyes such as Hoechst (Thermo Fisher Scientific, InvitrogenTM, catalog number: H21486 )
    Note: The registration step consists in analysing two successive images and superimposing them using relative position registration (thereafter called registration). For this registration purpose, ImageJ needs to find common features in both images. It is therefore required to have in one of the channel imaged a fluorescent staining that is relatively stable over time. This will allow the imageJ plugin Turboreg to compare two successive images and accommodate the displacement (see Note 1). As stable fluorescent marker with easily recognizable features, we successfully used: 1) plasma membrane localised fluorescent proteins such as myristoylated and palmitoylated YFP (Martinière et al., 2012; Simon et al., 2016), or vital plasma membrane dyes such as FM4-64 (alternatively, other dyes such as propidium iodide [Sigma-Aldrich, catalog number: P4864 ] can be used), and 2) nucleus localised fluorescent proteins such as H2B-RFP (Federici et al., 2012; Larrieu et al., 2015), or vital nuclei dyes such as Hoechst (Thermo Fisher Scientific, InvitrogenTM, catalog number: H21486 ). However, any other fluorescent protein or dye might be used, providing that it labels stable structure(s) over time and is photostable during the time-lapse acquisition (see Note 2).

Equipment

  1. Inverted confocal microscope (e.g., Carl Zeiss, model: AxioObserver Z1 ), equipped with a spinning disk module (e.g., Yokogawa Electric, model: CSU-W1 [T3 model])
  2. Metamorph and ImageJ software on the computer connected to the microscope
  3. 20 °C plant growth chamber with 24 h daylight (e.g., SANYO Electric, model: MLR-351 )
  4. Small tweezers (e.g., straight fine tweezers)
  5. Sterile hood

Software

  1. ImageJ (https://imagej.nih.gov/ij/; Schneider et al., 2012; this protocol was tested with version 1.49p but should be compatible with newer versions) with the plugins Turboreg (P. Thévenaz, http://bigwww.epfl.ch/thevenaz/turboreg/) and Stackreg (P. Thévenaz, http://bigwww.epfl.ch/thevenaz/stackreg/) installed are required. Fiji (http://fiji.sc/; Schindelin et al., 2012) could be used instead of ImageJ because it bundles the required plugins. To add the plugins, simply download them and copy them in ImageJ/plugins folder.
  2. Download the three files (CL_ini-Pos-Ch-finished.JNL; CL_ini-Pos-Ch.JNL; CL-root-track-MM_63multiD.ijm) attached as a zip file (named Files_BioProtocol_Doumane, as well available at this url http://www.ens-lyon.fr/RDP/SiCE/METHODS.html) and extract them in the folder C:\MM\app\mmproc\journal\root-tracking\. The ImageJ macro file CL-root-track-MM_63multiD.ijm should be edited according to the calibration of the objective used. Open the file in ImageJ (use the menu Open>File...), search for the line with ‘objective’, and replace the number 0.2063 with the real size of the pixel in microns, on this line and the next one (when using our 63x objective, 1 pixel is 0.2063 microns wide; Figure 1; Note 5). Save the file. Install the macro by selecting it in Plugins > Macro > Install. CL-root-track-MM_63multiD should appear in the Plugins > Macros > lower panel (using a 63x objective).


    Figure 1. Macro file .ijm in the Fiji editor. Replace the number 0.2063 (here in lines 81 and 82) by the real size of the pixel (in microns) on your microscope, with the objective that will be used. 

Procedure

  1. Automated imaging of growing Arabidopsis roots stained with a vital dye (see Video 1 and Figure 2)

    Video 1. Preparation of the Lab-Tek® observation chamber for FM4-64 staining. Pour 3 ml of ½ MS-0.8% agar with 5 μl of 100 mg/ml FM4-64 in the Lab-Tek® observation chamber.


    Figure 2. Preparation of the Lab-Tek® observation chamber for FM4-64 staining. A. Drop 5 μl of 100 mg/ml FM4-64 into the observation chamber; B. Add 3 ml of pre-heated ½ MS-0.8% agar; C. Mix gently to homogenize; D. Let cool down and solidify.

    1. Incubate seedlings in the FM4-64 bath (1 μg/ml FM4-64 in liquid ½ MS) for 5 to 10 min (time required for the preparation of the Lab-Tek® observation chamber).
    2. Prepare ½ MS-0.8% agar-FM4-64 in the Lab-Tek® observation chamber (see Recipes section and Video 2 and Figure 3). While seedlings incubate in the FM4-64 bath, pour ½ MS-0.8% agar-FM4-64 in the Lab-Tek® chamber, wait until it has solidified and dried.

      Video 2. Transfer of seedlings in the observation chamber. Cut out a piece of ½ MS-agar, add ½ MS and align carefully seedlings prior to putting back the squared piece of ½ MS-agar. Proceed to observation (FM4-64 staining) or let it stand several hours in a growth chamber so that seedlings acclimate to the Lab-Tek® chamber.


      Figure 3. Transfer of seedlings in the observation chamber. If working without FM4-64, start the procedure at panel (C). In this case, prefer working under sterile conditions. A. Prepare a solution of ½ MS-FM4-64 at 1 μg/ml final concentration; B. Transfer the seedlings into the solution and incubate them for 5 min; C. In the meantime, cut a squared block of agar in the Lab-Tek® observation chamber; D. Then remove it from the chamber using a coverslips; E. Add 200 μl of ½ MS in the hole left in the chamber; F. Transfer the seedlings into the Lab-Tek® observation chamber. If you plan to leave Lab-Tek® observation chamber in a growth chamber overnight (when working without FM4-64), make sure seedlings are roughly aligned. G. Put the block of agar back inside the Lab-Tek® chamber, on top of either the whole seedlings, or only of their root system. H. Gently chase air bubbles with a gloved fingertip; I. Seedlings are ready for observation or to be grown overnight.

    3. Cut a squared area of agar in the chamber. The piece of agar should go from one side of the chamber to the other, and be as wide as a coverslip (20 x 24 mm). Using a coverslip, remove the agar block from the observation chamber and keep it aside for later use (see step A6).
    4. Pour 200 µl of liquid ½ MS in the hole left by the removal of the agar block.
    5. Carefully align up to 4 seedlings in the Lab-Tek® observation chamber.
    6. Put back the cube of ½ MS-agar-FM4-64 on top of the seedling(s), chasing air bubbles manually (gently using a gloved fingertip).
    7. Place the sample under a microscope. Locate the first root tip by direct bright field observation under the microscope.
    8. Go to step B8.

  2. Automated imaging of growing Arabidopsis roots expressing fluorescent proteins (see Naramoto et al., 2015; Aki and Umeda, 2016 for detailed alternatives procedure)
    1. Under the sterile hood, pour 3 ml of pre-heated ½ MS-0.8% agar in the observation chamber and let it cool down.
    2. Use the scalpel to cut a squared area of agar (20 x 24 mm) from one side to the other of the chamber, and as wide as a coverslip. Use a coverslip to remove the piece of agar from the observation chamber and keep it aside for later use (see step B5).
    3. Pour 200 µl of liquid ½ MS in the hole left by the removal of the agar block.
    4. Carefully align up to 4 seedlings in the observation chamber.
    5. Gently put back the cube of ½ MS-0.8% agar-FM4-64 on top of the seedling(s). Whole small/young seedlings can be positioned between the agar block and the bottom of the chamber. For longer/older seedlings, only the root system can be positioned between the agar and the bottom, with the aerial part of the seedling rising out of the agar. Chase air bubbles manually, gently using a gloved fingertip.
    6. Leave the plants to grow in their new environment, with an inclination of ± 70° of angle, in a growth chamber overnight, so that seedlings acclimate to the observation chamber and keep growing.
    7. Place the sample under a microscope. Locate the first root tip by bright field observation under the microscope.
    8. In Metamorph, use the menu ‘Apps’ > ‘Multi Dimensional Acquisition’ (MDA).
    9. In the ‘Main’ tab, choose the dimensions ‘Timelapse’, ‘Multiple Stage Positions’, ‘Multiple Wavelengths’ (even if you have only one position and/or one channel) and ‘Run journals’. These modalities are required. You can use the options ‘Z series’ and ‘Stream’ too if needed (Figure 4): these modalities are optional, but compatible with each other.


      Figure 4. The Multi Dimensional Acquisition Main tab: Timelapse, Multiple Stage Positions (even for only one), Multiple Wavelengths (even for only one) and Run Journals are required. Z series and Stream are optional.

    10. Choose an empty folder to save the images. You must not use ‘_w’ or ‘_s’ in the base name of the images.
    11. Change the other settings of the selected tabs of the MDA menu as needed (use the same settings as for a regular time-lapse acquisition): you could use the settings displayed in Figure 5 as a starting point. If you use the ‘Z series’ option, make sure you remember slice number that should be used for the registration (we use the central slice of the stack most of the time; Figure 5).


      Figure 5. An example of the settings we often use for the tabs of the MDA module. In the Z series, make sure you remember the number of steps (here, 121) in E: if you want to use the central slice for the registration, you need to use the number 60 for the ‘Slice used for registration’ in step B15.

    12. In the ‘Journal’ menu, choose ‘CL_ini-Pos-Ch.jnl’ for ‘end of stage position’, ‘CL_ini-Pos-Ch-finished.jnl’ for ‘end of acquisition’, ‘CL_ini-Pos-Ch-finished.jnl’ for ‘on cancel’ (Figure 6).


      Figure 6. The Multi Dimensional Acquisition Journal tab. Choose the proper journal for each category.

    13. You should save the settings of the MDA in a file (‘save state...’ in the Main tab) for the next time. Do not start the acquisition with Metamorph yet.
    14. Start ImageJ/Fiji and run the macro CL-root-track-MM_63multiD.ijm.
    15. A window pops up. Fill in the ‘Number of positions’ (as in the MDA, 1 or more), the ‘Channel used for registration’ (the channel number in the MDA, 1 or more), and the ‘Slice used for registration’ (as explained in step B11; Figure 7)


      Figure 7. Fill in the macro pop-up window with the appropriate numbers, according to the settings chosen in the MDA module. Here is an example that is compatible with Figure 5.

    16. Another window pops up. Select the folder used in the MDA to save the images.
    17. Do not close ImageJ or any of its opened windows. Go back to Metamorph. Select in the menu ‘Journal’ > ‘Journal control’ > ‘Stopwatches’: let the new window opened.
    18. Start the acquisition with the ‘Acquire’ button in the MDA (see Note 4).

Data analysis

After making a movie of your stacks, you obtain stable image over time (Video 3).

ttom:0px;font-family:Arial;font-size:10pt;"> Video 3. Example of video obtained using automated root-tracking. 1 h 18 min time-lapes of root tip of seedlings expressing CENH3-RFP (Simon et al., 2016). Colour is coded by signal intensity (min black/blue, max yellow/white)

Notes

  1. You can check that X and Y coordinates change overtime for each root, to make sure registration is occurring. Variations in positions should be observed from the third time-point.
  2. The suitability of the dye for relative position registration could be tested with short regular time-lapse acquisition (e.g., 3-5 time-points and without real-time tracking): if the plugin Stackreg is able to correctly register the slices of this acquisition, we can consider that the dye is appropriate. It is also a good way to test the correct interval of time to use to do the time-lapse acquisition.
  3. If you want to perform long time-lapse analysis (e.g., 4-24 h), it is better to work in sterile conditions under a laminar flow hood. For short time lapses, this is optional.
  4. At the end of the acquisition, you will have the images or stacks, with the roots that will seem almost stationary over the time (a registration post-acquisition could be necessary, if the movement of the root was not linear enough). You will find a csv file with the displacements of the stage (in microns) recorded as a log, that you will need if you want to measure the speed of objects in the image, or if you need to troubleshoot the process. If a problem appears with ImageJ during the acquisition, Metamorph will continue to do the acquisition, and will use the last calculated displacements in X and Y for all the next time points (no additional coordinates will be recorded in the csv file).
  5. This protocol has been tested on our microscope with objective's magnifications ranging from 10x to 63x. But there is no limitation on this factor, as long as the macro file is edited as described in the second paragraph of the ‘Software’ section.

Recipes

  1. FM4-64 stock solution
    1. Resuspend FM4-64 powder in sterile distilled water and make a stock solution at 100 mg/ml (add 100 μl of sterile distilled water to 100 μg of FM4-64 powder)
    2. FM4-64 can be stored away from light at -20 °C as a powder, and up to 3 months at 100 mg/ml at -20 °C
  2. ½ MS-FM4-64 bath
    1. In 12 wells microplates, dilute FM4-64 stock solution to 1 μg/ml final concentration in 1 ml of half strength MS (½ MS)
    2. Mix gently
  3. Lab-Tek® chamber with ½ MS-0.8% agar-FM4-64 dye (see Video 2)
    1. Prepare ½ MS-0.8-% agar medium and let it cool down to ± 60 °C
    2. Under a sterile hood, drop 5 μl of FM4-64 stock solution in the observation chamber (see Note 3)
    3. Add 3 ml of ½ MS-0.8% agar medium and mix gently
    4. Let it cool down until it solidifies

Acknowledgments

We thank Dr. Laia Armengot and Dr. Antoine Larrieu for critical comments on the manuscript. M.D. is funded by a fellowship from the French Ministry of Higher Education and Research, Y.J. by ERC No. 3363360-APPL under FP/2007-2013, M-C.C by a group leader starting package «fond de recherche» from ENS Lyon.

References

  1. Aki, S. S. and Umeda, M. (2016). Cytrap marker systems for in vivo visualization of cell cycle progression in Arabidopsis. Methods Mol Biol 1370: 51-57.
  2. Buschmann, H. and Zachgo, S. (2016). The evolution of cell division: from streptophyte algae to land plants. Trends Plant Sci 21(10): 872-883.
  3. Federici, F., Dupuy, L., Laplaze, L., Heisler, M. and Haseloff, J. (2012). Integrated genetic and computation methods for in planta cytometry. Nat Methods 9(5): 483-485.
  4. Larrieu, A., Champion, A., Legrand, J., Lavenus, J., Mast, D., Brunoud, G., Oh, J., Guyomarc’h, S., Pizot, M., Farmer, E. E., Turnbull, C., Vernoux, T., Bennett, M. J. and Laplaze, L. (2015). A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants. Nat Commun 6: 6043.
  5. Lipka, E., Herrmann, A. and Mueller, S. (2015). Mechanisms of plant cell division. Wiley Interdiscip Rev Dev Biol 4(4): 391-405.
  6. Martinière, A., Lavagi, I., Nageswaran, G., Rolfe, D. J., Maneta-Peyret, L., Luu, D. T., Botchway, S. W., Webb, S. E., Mongrand, S., Maurel, C., Martin-Fernandez, M. L., Kleine-Vehn, J., Friml, J., Moreau, P. and Runions, J. (2012). Cell wall constrains lateral diffusion of plant plasma-membrane proteins. Proc Natl Acad Sci U S A 109(31): 12805-12810.
  7. Müller, S. and Jürgens, G. (2016). Plant cytokinesis-No ring, no constriction but centrifugal construction of the partitioning membrane. Semin Cell Dev Biol 53: 10-18.
  8. Naramoto, S., Dainobu, T. and Otegui, M. (2015). A bioimaging pipeline to show membrane trafficking regulators localized to the golgi apparatus and other organelles in plant cells. Bio-protocol 5(17): e1583.
  9. 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.
  10. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  11. Simon, M. L., Platre, M. P., Marques-Bueno, M. M., Armengot, L., Stanislas, T., Bayle, V., Caillaud, M. C. and Jaillais, Y. (2016). A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat Plants 2: 16089.

简介

在这里,我们描述一个协议,可以自动执行增长根尖的延时成像几个小时。表达荧光蛋白或用染料染色的植物根据显影阶段的自动移动而成像,其补偿根生长,并允许随着时间跟随根部的给定区域。该协议使图像采集多个生长根尖成为可能,因此增加了给定实验中记录的有丝分裂事件的数量。该协议还允许多个荧光蛋白或染料同时显示,使用多通道采集。我们特别关注在拟南芥根尖分生组织中细胞分裂的成像,但是这种方案也适用于在各种植物物种中追随根毛发生长,花粉管生长和其他根部区域。也可以修改为自动跟踪非植物结构的根尖增长。

细胞因子是细胞分裂的最后一步,当母细胞细胞质在两个子细胞之间分配(Lipka等人,2015)时。在植物中,通过分裂平面中的细胞板的离心扩张实现,其最终成为经历有丝分裂的细胞之间的新合成的细胞壁(Buschmann和Zachgo,2016;Müller和Jürgens,2016)。植物细胞嵌入僵硬的细胞壁,不能迁移。因此细胞分裂与伸长的取向对于器官形态发生至关重要。根分生组织是研究细胞分裂的一个很好的模型,因为它们容易适用于显微技术,而不需要解剖。然而,进行细胞分裂的根长度增长,因此需要随时间手动调整观察场。该协议允许容易地延迟细胞分裂成像和其他细胞过程。

关键字:细胞分裂, 有丝分裂, 细胞因子, 根, 显微镜检查, 跟踪, 拟南芥, 磷酸肌醇

材料和试剂

  1. 12孔微孔板(Corning,Costar ®,目录号:3513)
  2. 显微镜幻灯片76 x 26 x 1.1 mm(RS组件,目录号:ISO 8037)
  3. 显微镜盖玻片22 x 60 mm(Thermo Fisher Scientific,Menzel-Gläser,目录号:630-2102)
  4. 观察室Lab-Tek II Chambered Coverglass W/Cover#1.5硼硅酸盐(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:155360)
  5. 海峡手术刀片(例如,,Swann Morton,直升安装BS EN 27740刀片)
  6. 拟南芥,野生型基因型的4至7天龄的幼苗,或表达膜和/或细胞板定位的荧光融合蛋白(例如, 2-p35S :: MP:YFP 在Col-0中,其中MP是肉豆蔻酰化和棕榈酰化信号序列[Martinièreet al。,2012; Simon等人。 ,2016])。幼苗可以垂直生长在平板(90×90×15)或圆盘上,以获得完整的生长根
  7. Murashige和Skoog(MS)培养基(Sigma-Aldrich,目录号:M5519)
  8. 琼脂(Sigma-Aldrich,目录号:A7921-1KG)
  9. FM4-64(3-三乙基铵丙基)-4-(6-(4-(二乙基氨基)苯基)己三烯基)吡啶鎓二溴化物)(Thermo Fisher Scientific,Molecular Probes < sup>,目录号:T-3166)
  10. 用于注册的合适的荧光染料(替代品)或荧光蛋白
    1. 血浆膜或细胞壁染料如FM4-64或碘化丙锭(Sigma-Aldrich,目录号:P4864)
    2. 核染料如Hoechst(Thermo Fisher Scientific,Invitrogen,目录号:H21486)
    注意:注册步骤包括分析两个连续的图像,并使用相对位置注册(此后称为注册)叠加它们。为了注册,ImageJ需要在两个图像中找到共同的特征。因此,需要在一个通道中成像一段时间相对稳定的荧光染色。这将允许imageJ插件Turboreg比较两个连续的图像并适应位移(见注1)。作为具有易识别特征的稳定荧光标记物,我们成功地使用了:1)质膜局部荧光蛋白如肉豆蔻酰化和棕榈酰化YFP(Martinièreet al。,2012; Simon et al。,2016)或重要的质膜染料如FM4 -64(或者,可以使用其它染料如碘化丙锭[Sigma-Aldrich,目录号:P4864]),和2)核定位荧光蛋白如H2B-RFP(Federici等,2012; Larrieu et al。 ,2015)或重要的核染料如Hoechst(Thermo Fisher Scientific,Invitrogen,目录号:H21486)。然而,可以使用任何其他荧光蛋白质或染料,只要它在一段时间内标记稳定的结构,并且在延时获取过程中是稳定的(见注2)。

设备

  1. 装配有旋转盘模块(例如,,横河电机,型号:CSU-W1 [T3]的倒车共焦显微镜(例如,卡尔蔡司,型号:AxioObserver Z1)模型])
  2. 连接显微镜的计算机上的Metamorph和ImageJ软件
  3. 20℃的植物生长室,具有24小时日光(例如,三洋电机,型号:MLR-351)
  4. 小镊子(例如,直镊子)
  5. 无菌罩

软件

  1. ImageJ( https://imagej.nih.gov/ij/;使用插件Turboreg(P.Thévenaz, http://bigwww.epfl.ch/thevenaz/turboreg/)和Stackreg(P.Thévenaz, http://bigwww.epfl.ch/thevenaz/stackreg/)需要安装斐济( http://fiji.sc/; Schindelin等人< em>。,2012)可以用来代替ImageJ,因为它捆绑了所需的插件。要添加插件,只需将其下载并复制到ImageJ/plugins文件夹中即可。
  2. 下载作为zip文件(名为Files_BioProtocol_Doumane)的三个文件(CL_ini-P-Ch-finished.JNL; CL_ini-Pos-Ch.JNL; CL-root-track-MM_63multiD.ijm),也可以在此url http://www.ens-lyon.fr/RDP/SiCE/METHODS.html ),并将其解压缩到C:\ MM \ app \ mmproc \ journal \ root-tracking \文件夹中。应根据所使用目标的校准编辑ImageJ宏文件CL-root-track-MM_63multiD.ijm。在ImageJ中打开文件(使用菜单Open> File ...),搜索带有"objective"的行,并将这个行和下一行的像素的实际大小(以微米为单位)替换为0.2063使用我们的63x物镜,1像素宽0.2063微米;图1;注5)。保存文件。通过在插件>中选择宏来安装宏宏>安装。 CL-root-track-MM_63multiD应该出现在插件>宏>>下图(使用63x物镜)。


    图1.斐济编辑器中的宏文件.ijm。将显示器上像素的实际大小(以微米为单位)替换为数字0.2063(第81和82行),目标是将被使用。 

程序

  1. 用生物染料染色的拟南芥生长的自然成像(见视频1和图2)

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    视频1.准备用于FM4-64染色的Lab-Tek ®观察室。将5μl100毫克/毫升的3毫升1/2 MS-0.8%琼脂FM4-64在Lab-Tek ®观察室。
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    图2.用于FM4-64染色的Lab-Tek ®观察室的制备A.将5μl的100mg/ml FM4-64滴入观察室; B.加入3ml预加热的½MS-0.8%琼脂; C.轻轻混合均质;让我们冷静下来,巩固。

    1. 在FM4-64浴(1μg/ml的FM4-64液体½MS)中培育幼苗5至10分钟(制备Lab-Tek观察室所需的时间)。
    2. 在Lab-Tek ®观察室中准备½MS-0.8%琼脂FM4-64(参见食谱部分和视频2和图3)。当幼苗在FM4-64浴中孵育时,在Lab-Tek ®室中倒入1/2 MS-0.8%琼脂FM4-64,等到固化并干燥。

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      视频2.将幼苗转移到观察室中。切下一块1/2 MS琼脂,加入1/2 MS,然后放回½MS琼脂平方厘米。继续进行观察(FM4-64染色)或让其在生长室中放置数小时,以使幼苗适应于Lab-Tek ®。
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      图3.在观察室中转移幼苗。如果在没有FM4-64的情况下工作,请在面板(C)开始步骤。在这种情况下,更喜欢在无菌条件下工作。 A.以1μg/ml终浓度制备½MS-FM4-64的溶液; B.将幼苗转移到溶液中孵育5分钟; C.同时,在Lab-Tek ®观察室中切下一块平方的琼脂块; D.然后用盖玻片将其从室中取出; E.在室内留下的孔中加入200μl的½MS; F.将幼苗转移到Lab-Tek ®观察室。如果您计划将Lab-Tek ®观察室放置在生长室中(无FM4-64工作时),确保幼苗大致对齐。 G.将琼脂块放回Lab-Tek ®室内,放在整个幼苗上,或者仅在根系中。 H.用手套指尖轻轻追一下气泡;一,幼苗准备观察或一夜之间种植。

    3. 在室内切成琼脂平方面。琼脂片应该从室的一侧到另一侧,并且与盖玻片一样宽(20×24mm)。使用盖玻片,从观察室中取出琼脂块,并将其放在一边以备后用(参见步骤A6)。
    4. 将200μl液体½MS倒入离开琼脂块的孔中。
    5. 仔细对准Lab-Tek ®观察室中的4个幼苗。
    6. 将½MS-agar-FM4-64的立方放在幼苗的顶部,手动追逐气泡(轻轻用手套指尖)。
    7. 将样品放在显微镜下。在显微镜下通过直接明场观察找到第一根根尖。
    8. 转到步骤B8。

  2. 表达荧光蛋白的拟南芥生长的自动成像(参见Naramoto等人,2015; Aki和梅田,2016年详细的替代程序)
    1. 在无菌罩下,在观察室中倒入3ml预加热的1/2 MS-0.8%琼脂,使其冷却。
    2. 使用手术刀从室的一侧切割成平方的琼脂区域(20×24mm),并且与盖玻片一样宽。使用盖玻片从观察室中取出琼脂,并将其放在一边供以后使用(参见步骤B5)。
    3. 将200μl液体½MS倒入离开琼脂块的孔中。
    4. 仔细对准观察室中的4棵幼苗。
    5. 轻轻地将½MS-0.8%琼脂FM4-64的立方放在幼苗顶部。整个小/幼苗可以位于琼脂块和腔室底部之间。对于较长/较老的幼苗,只有根系可以位于琼脂和底部之间,幼苗的地上部分从琼脂中升出。手动追逐气泡,轻轻用手套指尖。
    6. 让植物在新的环境中生长,倾斜角度为±70°,在生长室过夜,使幼苗适应观察室,并保持增长。
    7. 将样品放在显微镜下。在显微镜下通过明场观察找到第一根根尖。
    8. 在Metamorph中,使用菜单'Apps'> "多维采集"(MDA)。
    9. 在"主"选项卡中,选择维度"Timelapse","多级位置","多波长"(即使只有一个位置和/或一个通道)和"运行日志"。这些模式是必需的。如果需要,也可以使用"Z系列"和"流"选项(图4):这些模式是可选的,但彼此兼容。


      图4.多尺度采集主选项卡:需要Timelapse,多级位置(即使只有一个),多波长(甚至只有一个)和运行日志。 Z系列和Stream是可选的。

    10. 选择一个空文件夹来保存图像。您不得在图像的基本名称中使用"_w"或"_s"。
    11. 根据需要更改MDA菜单中所选选项卡的其他设置(使用与常规延时采集相同的设置):可以使用图5中显示的设置作为起点。如果您使用"Z系列"选项,请确保记住应该用于注册的切片编号(大部分时间使用堆叠的中心切片);


      图5.我们经常用于MDA模块选项卡的设置示例。在Z系列中,确保记住E中的步数(这里为121):如果你想要使用中央切片进行注册,您需要在步骤B15中使用第60号"用于注册的切片"。

    12. 在"日记"菜单中,选择"结束阶段位置"的"CL_ini-Pos-Ch.jnl","收购结束","CL_ini-P-Ch-finished.jnl","CL_ini-Pos-Ch- done.jnl'为'取消'(图6)。


      图6."多维采集日志"选项卡。为每个类别选择适当的日志。

    13. 您应该将MDA的设置保存在下一次的文件(主选项卡中的"保存状态...")中。不要用Metamorph开始收购。
    14. 启动ImageJ/Fiji并运行宏CL-root-track-MM_63multiD.ijm。
    15. 一个窗口弹出。填写"位置数"(如MDA,1或更多),"用于注册的通道"(MDA中的通道号,1或更多)和"用于注册的切片"(如所述在步骤B11;图7)


      图7.根据MDA模块中选择的设置,使用相应的数字填充宏弹出窗口。下面是与图5兼容的示例。

    16. 另一个窗口弹出。选择MDA中使用的文件夹来保存图像。
    17. 不要关闭ImageJ或其任何打开的窗口。回到Metamorph。选择菜单'日志'> "日记帐控制"> '秒表':让新窗口打开。
    18. 通过MDA中的"Acquire"按钮开始采集(见注4)。

数据分析

制作电影后,您可以随时间获得稳定的影像(视频3)。

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视频3.使用自动根跟踪获得的视频示例。 1小时18分钟表达CENH3-RFP的幼苗根尖18分钟(Simon等人,2016年) )。颜色由信号强度(最小黑/蓝,最大黄/白)编码
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笔记

  1. 您可以检查X和Y坐标是否改变每个根的加班时间,以确保注册正在发生。应从第三个时间点观察位置变化。
  2. 用于相对位置配准的染料的适用性可以通过短时间的定时延时采集(例如,,3-5个时间点进行测试,无需实时跟踪):如果插件Stackreg可以要正确注册此次采集的片段,我们可以认为染料是合适的。这也是测试正确使用时间间隔进行延时采集的好方法。
  3. 如果要进行长时间推移分析(例如,4-24小时),最好在层流罩下的无菌条件下工作。对于短时间的丢失,这是可选的。
  4. 在收购结束时,您将有图像或堆叠,根本看起来几乎是静止的(如果根的运动不够线性,则可能需要注册后采集)。您将找到一个csv文件,其中记录为日志的阶段位移(以微米为单位),如果要测量图像中对象的速度,或者需要对进程进行故障排除,则需要该文件。如果在采集过程中出现ImageJ问题,Metamorph将继续进行采集,并将在下一个时间点使用X和Y中的最后计算的位移(在csv文件中不会记录附加坐标)。 />
  5. 该协议已经在我们的显微镜上进行了测试,目标范围从10x到63x。但是,只要宏文件按"软件"部分的第2段所述进行编辑,这个因素就没有限制。

食谱

  1. FM4-64库存解决方案
    1. 将FM4-64粉末重悬在无菌蒸馏水中,制成100毫克/毫升的储备溶液(加入100微升无菌蒸馏水至100微克FM4-64粉末)
    2. FM4-64可以在-20°C的光照条件下作为粉末储存,最多可在-20℃下以100 mg/ml的速度储存3个月
  2. ½MS-FM4-64浴
    1. 在12孔微量培养板中,将稀释的FM4-64储备溶液稀释至1毫升半强度MS(½MS)中的1μg/ml终浓度
    2. 轻轻混合
  3. 具有½MS-0.8%琼脂FM4-64染料的Lab-Tek ®腔(见视频2)
    1. 准备½MS-0.8-%琼脂培养基,让其冷却至±60°C
    2. 在无菌罩下,在观察室中放入5μl的FM4-64储备溶液(见注3)
    3. 加入3 ml½MS-0.8%琼脂培养基,轻轻混匀
    4. 让它冷却,直到它固化

致谢

我们感谢Laia Armengot博士和Antoine Larrieu博士对手稿的批评性评论。 M.D.由法国高等教育研究部的研究员资助,由ERC编号3363360-APPL根据FP/2007-2013,M-C.C由集团领导人从ENS里昂开始包装«fond de recherche»的Y.J.。

参考文献

  1. Aki,SS和梅田,M。(2016)。用于体内 在拟南芥中细胞周期进程的可视化的Cytrap标记系统。 Methods Mol Biol 1370:51-57。 />
  2. Buschmann,H.和Zachgo,S.(2016)。细胞分裂的演变:从链球菌藻类到土地植物。 趋势植物科学 21(10):872-883。
  3. Federici,F.,Dupuy,L.,Laplaze,L.,Heisler,M。和Haseloff,J.(2012)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/22466793"target ="_ blank">植物细胞计数的综合遗传和计算方法。 Nat方法 9(5):483-485。 />
  4. Larrieu,A.,Champion,A.,Legrand,J.,Lavenus,J.,Mast,D.,Brunoud,G.,Oh,J.,Guyomarc'h,S.,Pizot,M.,Farmer,EE ,Turnbull,C.,Vernoux,T.,Bennett,MJ and Laplaze,L。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25592181"target ="_ blank">荧光激素生物传感器揭示植物中茉莉酮酸信号的动态。 6:6043.
  5. Lipka,E.,Herrmann,A.和Mueller,S。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25809139" target ="_ blank">植物细胞分裂的机制。 Wiley Interdiscip Rev Dev Biol。4(4):391-405。
  6. Martinière,A.,Lavagi,I.,Nageswaran,G.,Rolfe,DJ,Maneta-Peyret,L.,Luu,DT,Botchway,SW,Webb,SE,Mongrand,S.,Maurel, Fernandez,ML,Kleine-Vehn,J.,Friml,J.,Moreau,P.and Runions,J.(2012)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/22689944"target ="_ blank">细胞壁限制植物血浆膜蛋白的横向扩散。美国Proc Natl Acad Sci USA 109(31): 12805-12810。
  7. Müller,S.和Jürgens,G.(2016)。植物细胞分裂 - 无环,无收缩但分离膜的离心构建。 Semin Cell Dev Biol 53:10-18。
  8. Naramoto,S.,Dainobu,T.和Otegui,M。(2015)。生物成像管道,用于显示位于植物细胞中的高尔基体和其他细胞器的膜运输调节物。 5(17):e1583。
  9. 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:用于生物图像分析的开源平台。 Nat方法 9(7 ):676-682。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Doumane, M., Lionnet, C., Bayle, V., Jaillais, Y. and Caillaud, M. (2017). Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes. Bio-protocol 7(8): e2245. DOI: 10.21769/BioProtoc.2245.
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