Automated Tracking of Root for Confocal Time-lapse Imaging of 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 Scientific^TM, 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 Probes^TM, 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, Invitrogen^TM, 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, Invitrogen^TM, 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