This tool allows to analyze morphological characteristics of complex roots. While for young roots the root system architecture can be analyzed automatically, this is often not possible for more developed roots. The tool is inspired by the Sholl analysis used in neuronal studies. The tool creates a binary mask and the Euclidean Distance Transform from the input image. It then allows to draw concentric circles around a base point and to extract measures on or within the circles. Instead of circles, which present the distance from the base point, horizontal lines can be used, which present the distance in the soil from the base-line. The following features are currently implemented:

• The area of the root per distance/depth.
• The number of border pixel per distance/depth, giving an idea of the surface in contact with the soil.
• The maximum radius per distance/depth of a root, measured at the crossing points with the circles or lines.
• The number of crossings of roots with the circles or lines.
• The maximum distance to the left and the right from the vertical axis at crossing points with the circles or lines.

Plot of the area [cm^2] per distance [cm] for different roots of the same condition.

The source code in git-hub can be found here.

To install the tool, save the file analyze_complex_roots_tools.ijm into the folder macros/toolsets of your FIJI installation and restart FIJI.

Select the "analyze_complex_roots_tools" toolset from the >> button of the ImageJ launcher.

• The first button (the one with the image) opens this help page
• The m-button creates the binary mask and the distance transform of the image.
• The c-button activates the circle tool. With the tool active, click on the base point of the root.
• The l-button activates the lines tool. With the tool active, click in the image at the base-height of the root.
• Plot the selected features per distance or depth.
• Export the selected features in batch-mode.

Open the image of the root. Per default the height of the image is assumed to be 29.7 cm. You can change this in the options of the m-button (right-clisk on the button). First create the mask and the Euclidean Distance Transform (edt) using the m-button. Then activate the mask image and select the circle- or the line- tool. Click into the mask at the base point of the root. This will add the lines or the concentric circles to the mask. You can transfer them from one image to another, for example from the mask to the edt-image, using the key f3 (circles) or f4 (lines). Right-click on the p-button to select the features you want to plot. Press the p-button and wait until the processing finishes.

To use the batch-mode, add the circles or lines on all images beforehand. Select the features to export in the options of the p-button and start the batch-processing by pressing the b-button.

The root must not touch the borders to the right, left or at the bottom of the image. If this is the case and you want to analyze the image nevertheless, draw a black line with an adequate width to cut the root from the border. The tool takes care of the top of the image on its own.

height of the image in cm

the tool will assume that the height of the image corresponds to the given length in cm for the spatial calibration

enhance contrast

if activated the contrast of each channel is enhanced independently before the segmentation

distance to border

the distance from the border at which a black line is drawn at the top of the image, so that the segmented object will not touch the top border of the image

thresholding method

the thresholding method that is used to create the binary mask

min. area

isolated objects with a smaller area will be removed from the binary mask

Select the tool and click at the base-point in the image to create the circles. Parts of the root above the base-point will be ignored. The circles can either grow by a constant distance or the distance between two consecutive circles can grow with growing radius. Once you created the circles on one image you can transfer them to another image by selecting the new image and pressing F3. The parameters of the circles will be stored in the image information and will be used when you run the tool in batch-mode.

initial radius

the radius of the first (the innermost) circle in pixel

initial delta

the initial length by which the radius of the circles grows

factor

factor by which the difference of radius between two circles grows

Select the tool and click at the depth of the base-line in the image. As for the circles, the distance between the lines can either be constant or growing with growing depth. Once you created the lines on one image you can transfer them to another image by selecting the new image and pressing F4. The parameters of the lines will be stored in the image information and will be used when you run the tool in batch-mode.

initial y

the distance of the first line from the top-border of the image, the value will be set by clicking in the image when the tool is active

initial distance

the initial distance between to consecutive lines

factor

the factor by which the distances between consecutive lines will grow

You can use the tool's resource id (see scicrunch.org):

(Analyze Complex Roots Tool, RRID:SCR_021019)

1. Ikram MADANI, Thanyakorn Rongsawat, Volker Baecker, Anne-Aliénor Véry, Hervé Sentenac, et al.. A phenotyping procedure for imaging root hair on whole root systems reveal responses to nutrient availability and inoculation with PGPR-like bacteria. 11th Symposium of the International Society of Root Research, May 2021, the University of Missouri Columbia, Missouri, US, United States. hal-03812295

2. Rongsawat, Thanyakorn. Root developmental responses to nutrient shortage and biotic conditions in wheat: identification of beneficial bacteria from wheat rhizosphere and new procedures for phenotyping root and root hair development. (2019), Montpellier, SupAgro.