After staining the Triticum aestivum leaf section with toluidine blue, place the slide on the microscope stage. Using a 40x objective magnification, focus on the section and adjust the brightness to ensure all cell structures are visible. Then use the navigator function to set the sections location area to ensure the entire section is imaged, then click the stitch button.
After that, click done and run to allow the microscope to image the entire section. Open the image analysis software and the tiles taken by the microscope. Using the align feature, ensure that the tiles do not overlap.
After generating the image, adjust the positioning, brightness, and rotation using the adjust tab. Then print the scale on the image before saving it in TIF format. Open ImageJ software and drag the image into the window to load it.
Using the line tool, draw a line over the scale bar. Choose analyze and click set scale. Change the known distance to the scale bar's length and the unit of length to the correct unit.
To measure the prospective trait, select the line tool, draw a line across the area of interest and press the M key. For area measurements, use the polygon selection tool to outline the tissue of interest. Then press the M key to conclude.
A pop-up window displaying the measurements will appear, which can be copied to a spreadsheet for further data analysis. Box plots illustrated significant anatomical differences between Triticum aestivum and ZMAs, particularly in interveinal distances, bundle sheath diameter, the fraction of bundle sheath cells, non-photo synthetic material, and vein frequency reflecting expected C3 and C4 leaf anatomy distinctions.
