Figure 1. Once the masked image (Left panel) has been converter, the Extended Particle Analyzer is ran which finds large round object and traces around it (Middle panel). Next, it eliminates all smaller objects and blends it into the background (Right panel).
Figure 2. The panel shows the area percentage of the xylem within the entire stem and the data comparison between the two manual methods vs. the automated method using the macro.
Figure 3. The averages of the area percentage of the xylem were taken between the two manual methods. This panel shows the comparison between the manual method vs. the automated method through the average percentage of xylem on a bar graph. The error bars show the standard deviation of both processes and since they are relatively the same, the standard deviation show the data is not significant.
Figure 4. The panel shows the result of the two tail t-Test and the P-Value which is greater than 0.05 meaning that the data for the two methods are not significant.
Additional Comments:
The automated macro is a list of instructions that includes multiple plugins within ImageJ-Fiji that is coded to perform together for segmenting the xylem tissue. The image is ran through the macro that first uses Color Deconvolution plugin which separates it into RGB image produced by subtractive mixing (inks, histological dyes) into separate channels corresponding to up to 3 determined colors (Landini, 2007) . In other words, this plugin separates the images into three color vectors which represent: Colour_1: Safranin (The color in which the xylem is stained), Colour_2: Fast Green (staining of the phloem), Colour_3: Cortex (background staining). Once the tissues have been categorized through the color vectors, the macro will then turn the image into a mask image using the Binary option in ImageJ-Fiji. This Binary option converts an image into black and white in which the cross section will be converted as white while the background will be black. Next, the masked image is taken put through an Extended Particle Analyzer plugin. The Extended Particle Analyzer looks specifically for a large object (area > 100000) which has roundness > 0.8 and blends smaller objects with the background. Next, Analyze Particles plugin will start, which will trace alongside the image in white. The tracing of the image is selected and then run through an ROI manager plugin that automatically calculates the area of the white region through tracing the image. The calculated result given from the ROI manager will give the area of the pith and area of the xylem and pith combined. To find the area of just the xylem I used the formula: As a result, the xylem is able to be fully segmented and measured through an automated process.