RootflowRT v.2.8

Making Input Files

In the RootflowRT application within the RootflowRT menu the New Input File option is given to the user.

Once selected, the Make Input File Window will appear. It will allow you to create properly formatted input files for RootflowRT kinematic analysis of your root images.

There are several options for the user to customize in order to get the best performance of the kinematic analysis.

Direction of Root Growth:

The software assumes that the root images are taken such that the direction of growth is horizontal and either left to right or right to left.

Root tip is in:

The order that the software processes images depends on which stack contains the root tip. If you are processing a single stack, you can ignore this option.

Mosaic Method:

If you are processing a single stack, you can ignore this option. If you are processing multiple stacks, then you must either input the exact distance in micrometer motion between stacks or provide background images for the software of each stack.

Ratio of um to pixels:

In order to calculate accurate velocities, the program needs to know the calibrated size of pixels in the images as micrometers.

Seconds Between Frames:

Choose the seconds elapsed between each frame within a stack of root images. If the options here do not reflect your imaging conditions, then editing the resulting input.txt file manually will be necessary.

Coordinate System

One important consideration is that the coordinate system expected by the software assumes that the lower left-hand corner is the origin (0,0) of the coordinate system. ImageJ can be modified such that this is the case using the “Analyze/Set Measurements…” menu by selecting the “Invert Y Coordinates” option.

Choose Diameter File:

This file must contain the left followed by the right diameter length of roots in the fifth frame of each stack. Ideally this file should be made using ImageJ. By selecting the line tool, measurements of length can be made and saved as a separate text file. Otherwise, a tab-delimited file with heading row will be sufficient as long as the column containing diameter length is labeled "Length" and each subsequent line begins with a line number.

Choose Midline Coordinates File

This file must contain the midline coordinates for roots in the fifth frame of each stack. The last coordinate selected in the root tip stack must be that of the Quiescent Center. It is ideally created in ImageJ using the cross-hair tool, but a tab-delimited file with column headings containing "X" and "Y" indicating where in the following lines the coordinate data is located will suffice with each subsequent line being numbered. Below, the cross-hair selected points for the midline are highlighted with yellow circles, while the QC is shown with a red circle.

Choose Timestamps File

This file must contain the timestamps of the fifth frame of the first to last stack in order even if you are only processing one stack.

Run

When ready to make the input.txt file for your root data set, press the Run button. If the run was successful, then in the same directory as the diameter file, an input.txt file will now exist. Final review of this file is advised to ensure content is as desired. If the run was not successful, then the text formatting of the diameter, timestamps and midline coordinates files should be examined.