Accurate root length and diameter measurement using NIH Image: use of Pythagorean distance for diameter estimation

This report describes an image analysis algorithm to estimate the length versus diameter of washed root samples accurately. Image analysis was performed using a Macintosh computer and the public domain NIH Image program. The binary image of the roots was processed to get the thinned image to calculate the length of the roots. The pixels of the root in a binary image were then stripped off from around the periphery based on the pixelʹs Pythagorean distance from the nearest background pixel. The length of the remaining root in each stripping off process was calculated after the image was thinned. Images (300 dpi) of copper wire of 0.23, 0.5, 1.0 mm diameter were analyzed for verification of the usefulness of the procedure. The results showed that more than 93% of the wires in each diameter wire were calculated to be in diameter classes including the true diameter and its adjoining classes: 93.6% of the wires of 0.23 mm diameter appeared in the 0.098-0.38 mm diameter classes, 96.19% of the wires of 0.5 mm diameter appeared in the 0.38-0.61 mm diameter classes, and 96.17% of the wires of 1 mm diameter appeared in the 0.85-1.08 mm diameter classes. The proposed method was tested for primary and secondary roots of water-cultured rice (Oryza sativa L.) and it was proven that the method could provide accurate length and diameter measurements for each root order. In addition, it was found that the method could provide the lengths of the thick primary, thin primary, and secondary roots. The effectiveness of applying sharpening for the grayscale image before making the binary image is also discussed.