Extracting Metrics for Three-dimensional Root Systems: Volume and Surface Analysis from In-soil X-ray Computed Tomography Data

Summary

A methodology for obtaining visual and quantitative root structure information from X-ray computed tomography data acquired in-soil is presented.

Abstract

Plant roots play a critical role in plant-soil-microbe interactions that occur in the rhizosphere, as well as processes with important implications to climate change and crop management. Quantitative size information on roots in their native environment is invaluable for studying root growth and environmental processes involving plants. X-ray computed tomography (XCT) has been demonstrated to be an effective tool for in situ root scanning and analysis. We aimed to develop a costless and efficient tool that approximates the surface and volume of the root regardless of its shape from three-dimensional (3D) tomography data. The root structure of a Prairie dropseed (Sporobolus heterolepis) specimen was imaged using XCT. The root was reconstructed, and the primary root structure was extracted from the data using a combination of licensed and open-source software. An isosurface polygonal mesh was then created for ease of analysis. We have developed the standalone application imeshJ, generated in MATLAB¹, to calculate root volume and surface area from the mesh. The outputs of imeshJ are surface area (in mm²) and the volume (in mm³). The process, utilizing a unique combination of tools from imaging to quantitative root analysis, is described. A combination of XCT and open-source software proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information from the 3D data. This methodology of processing 3D data should be applicable to other material/sample systems where there is connectivity between components of similar X-ray attenuation and difficulties arise with segmentation.

Introduction

Roots, as part of the rhizosphere^2-5, represent an "invisible" part of plant biology since soil makes it difficult to image roots non-invasively ^{6, 7}. However, studying root growth and interaction within the soil environment is critical to understanding root/plant growth and nutrient cycling, which in turn affect forestation, food security, and climate. X-ray computed tomography (XCT) has proven to be a valuable tool for noninvasive imaging of plant root samples in their local environments ⁸. In order to measure root development and dimensional changes under different conditions, and be able to compare data from different datas....

Protocol

Caution: The operation of an X-ray tomography scanner requires both general radiation training, and instrument-specific radiation safety training. All corresponding procedures relevant to the experimenter's laboratory should be followed. 

1. Root Imaging

Note: This step describes the imaging of a grass specimen held in its original soil in a tubular plastic pot (a plastic tube with a diameter of 40 mm, a height of 210 mm, and wall thickness of about 2 mm).

1. Place potted plant on the sample manipulator of the instrument at a distance desired for target magnification. For a plant in a 2-inch diameter holder, the sample to source distanc....

Results

The specimen consisting of two stems of the native grass Prairie dropseed (Sporobolus heterolepis) and the original soil around it was taken from a residential area and placed in a small tube-shaped holder seen in Figure 1. The reconstructed data voxel size was approximately 31 µm x 31 µm x 31 µm. The reconstructed volume file was used to create a stack of images from a selected orientation (top view) using the open-source image-processing program ImageJ 1.6^.......

Discussion

A combination of X-ray computed tomography and several open-source programs proved to be a powerful combination to noninvasively image plant root samples, segment root data, and extract quantitative information (surface area and volume) from the 3D data. Our ability to visualize and measure features is always limited by scan resolution, as well as by limitations of the RooTrak software. However, scan resolution was sufficient to capture the majority of the features of the sample in this study, and RooTrak was able to suc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
X-Tek/Metris XTH 320/225 kV	Nikon Metrology		X-ray tomography scanner
Inspect X	Nikon Metrology		Instrument control software
CT Pro 3D	Nikon Metrology		Reconstruction software, version XT 2.2
VG Studio MAX	Visual Graphics GmbH		Visualization software for 3D volumes, version 2.1.5
ImageJ	Open-source		Image processing and analysis software, version 1.6
RooTrak	Open-source		Root segmentation software, version 0.3.1-b1 beta
imeshJ	EMSL		MATLAB script developed by the authors
Prairie dropseed grass sample			Sample obtained from ground in residential area