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Performance and accuracy of the automated measurement software: Simple Online Automated Plant Phenomics (SOAPP)

Ariel M. Hughes

Ariel M. Hughes

Department of Biomedical Engineering and Science, Florida Institute of TechnologyMelbourne,
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Hughes, Ariel M.
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Benjamin E. Jenkins

Benjamin E. Jenkins

Department of Biology, University of North Carolina GreensboroGreensboro,
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Jenkins, Benjamin E.
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Lucas V. Bauer

Lucas V. Bauer

Department of Molecular and Structural Biochemistry, North Carolina State UniversityRaleigh,
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Bauer, Lucas V.
 e 
John Z. Kiss

John Z. Kiss

Department of Biomedical Engineering and Science, Florida Institute of TechnologyMelbourne,
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Kiss, John Z.
  
08 ago 2025
Performance and accuracy of the automated measurement software: Simple Online Automated Plant Phenomics (SOAPP)'s Cover Image
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Categoria dell'articolo: Research Note
Pubblicato online: 08 ago 2025
Pagine: 51 - 64
DOI: https://doi.org/10.2478/gsr-2025-0004
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© 2025 Ariel M. Hughes et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

A sample scan of clinostat-grown 4-day-old A. thaliana seedlings. Scale units are in mm. Foreground noise is due to wrinkles in the membrane upon which the seedlings are grown.
A sample scan of clinostat-grown 4-day-old A. thaliana seedlings. Scale units are in mm. Foreground noise is due to wrinkles in the membrane upon which the seedlings are grown.

Figure 2.

A manual hypocotyl length measurement of a seedling collected using the program ImageJ. To conduct a length measurement, the user manually plots a segmented line (circled, appears as a yellow line connected by manually placed white plotting points) using the included segmented line tool (circled in red in the toolbar).
A manual hypocotyl length measurement of a seedling collected using the program ImageJ. To conduct a length measurement, the user manually plots a segmented line (circled, appears as a yellow line connected by manually placed white plotting points) using the included segmented line tool (circled in red in the toolbar).

Figure 3.

The SOAPP binary masking interface. Prior to image analysis, users must assign and adjust a binary mask to each image for removal of unwanted background or plant tissue areas within the ROIs (Regions of Interest). First, a color space is designated (Box A) for isolation of objects with specific hues and brightness. Color space V was used for all plants in this study. Second, the user selects the masking method (binary by default) and threshold for the color space mask, or how intense the applied color space should be on the image to be analyzed (Box B). Finally, the user selects whether the target objects are darker or lighter than their relative background (Box C).
The SOAPP binary masking interface. Prior to image analysis, users must assign and adjust a binary mask to each image for removal of unwanted background or plant tissue areas within the ROIs (Regions of Interest). First, a color space is designated (Box A) for isolation of objects with specific hues and brightness. Color space V was used for all plants in this study. Second, the user selects the masking method (binary by default) and threshold for the color space mask, or how intense the applied color space should be on the image to be analyzed (Box B). Finally, the user selects whether the target objects are darker or lighter than their relative background (Box C).

Figure 4.

The SOAPP ROI interface. After the construction of a binary mask, users must input the location of ROIs for SOAPP to measure within an image. Circular ROIs are designated by pixel location, with the x axis represented by horizontal inputs and the y axis represented by vertical inputs. The purple circles indicate the area where the ROIs have been designated, and the software will then locate the subject specified in that general area, i.e., so long as part of the subject is within the designated purple ROI area, that subject will be measured. When measuring multiple ROIs at once, indicated by row and column inputs >1, users must indicate the spacing between the ROIs, also on a per-pixel basis.
The SOAPP ROI interface. After the construction of a binary mask, users must input the location of ROIs for SOAPP to measure within an image. Circular ROIs are designated by pixel location, with the x axis represented by horizontal inputs and the y axis represented by vertical inputs. The purple circles indicate the area where the ROIs have been designated, and the software will then locate the subject specified in that general area, i.e., so long as part of the subject is within the designated purple ROI area, that subject will be measured. When measuring multiple ROIs at once, indicated by row and column inputs >1, users must indicate the spacing between the ROIs, also on a per-pixel basis.

Figure 5.

The SOAPP results interface. Following analysis of user-inputted ROIs, SOAPP will present its analysis results both visually (measured plant tissue areas are visualized by purple polygons overlaid upon the original image selected for analysis) and in value table format (Box D). Measurement values provided in the result value table for the designated ROIs include area of the target plant tissue, solidity, and the following are to the right of the pictured columns, accessible by the shown slide bar: Perimeter length, width, height, center of mass, number of convex hull (represented by purple polygons) vertices, major and minor axes of ellipses containing the convex hull and any plant tissue extending beyond the ROI circle (ellipses are not visualized by SOAPP, an external calculation was conducted to determine their shape and orientation), and the ellipse’s eccentricity.
The SOAPP results interface. Following analysis of user-inputted ROIs, SOAPP will present its analysis results both visually (measured plant tissue areas are visualized by purple polygons overlaid upon the original image selected for analysis) and in value table format (Box D). Measurement values provided in the result value table for the designated ROIs include area of the target plant tissue, solidity, and the following are to the right of the pictured columns, accessible by the shown slide bar: Perimeter length, width, height, center of mass, number of convex hull (represented by purple polygons) vertices, major and minor axes of ellipses containing the convex hull and any plant tissue extending beyond the ROI circle (ellipses are not visualized by SOAPP, an external calculation was conducted to determine their shape and orientation), and the ellipse’s eccentricity.

Figure 6.

The SOAPP binary masking interface, image cleanup panel. After the user has selected a color space, masking technique, colorspace threshold, and relative target object brightness, the final step is designating the size of stray unmasked objects to remove from the final binary mask. During the masking process, bright artifacts such as membrane creases can slip through the initial masking steps. This feature removes any unwanted artifacts of a certain size, leaving behind only the desired target objects. This setting was kept at 2000 pixels for all analyses (Box E).
The SOAPP binary masking interface, image cleanup panel. After the user has selected a color space, masking technique, colorspace threshold, and relative target object brightness, the final step is designating the size of stray unmasked objects to remove from the final binary mask. During the masking process, bright artifacts such as membrane creases can slip through the initial masking steps. This feature removes any unwanted artifacts of a certain size, leaving behind only the desired target objects. This setting was kept at 2000 pixels for all analyses (Box E).

Figure 7.

A plant-point ellipse (digitally zoomed in to show the plant about 20X its size). SOAPP generates a plant-point ellipse (shown in blue) for each analyzed plant that is fitted to the identified plant area polygon (shown in purple). In the current iteration of the software, plant-point ellipses are not available as visualization within the graphic user interface (GUI). To resolve this issue, plant-point ellipse values were entered into an Excel sheet that would output the ellipse for each plant. As shown in this example, the major axis would be used to represent hypocotyl length due to its orientation relative to the target plant.
A plant-point ellipse (digitally zoomed in to show the plant about 20X its size). SOAPP generates a plant-point ellipse (shown in blue) for each analyzed plant that is fitted to the identified plant area polygon (shown in purple). In the current iteration of the software, plant-point ellipses are not available as visualization within the graphic user interface (GUI). To resolve this issue, plant-point ellipse values were entered into an Excel sheet that would output the ellipse for each plant. As shown in this example, the major axis would be used to represent hypocotyl length due to its orientation relative to the target plant.

Figure 8.

Mean of manual and normalized SOAPP hypocotyl measurements by ecotype. Ecotypes are divided into clinostat-grown and stationarily grown (control) plant subsets. SOAPP measurements are shown in light blue and yellow, and manual measurements are shown in navy blue and orange. The yellow and orange dots indicate the mean lengths of the statically grown plants as measured by SOAPP and manually, respectively. The navy blue and light blue dots indicate the average lengths of hypocotyls grown on the 2D clinostat as measured manually and using SOAPP, respectively. Asterisks denote the degree of significance. Blue asterisks indicate a significant difference between the SOAPP and manual measurements for the clinostat group and red asterisks indicate significant differences for the control group. A single asterisk indicates a p-value below 0.05, a double asterisk indicates a p-value below 0.001, and a triple asterisk indicates a p-value below 0.0001.
Mean of manual and normalized SOAPP hypocotyl measurements by ecotype. Ecotypes are divided into clinostat-grown and stationarily grown (control) plant subsets. SOAPP measurements are shown in light blue and yellow, and manual measurements are shown in navy blue and orange. The yellow and orange dots indicate the mean lengths of the statically grown plants as measured by SOAPP and manually, respectively. The navy blue and light blue dots indicate the average lengths of hypocotyls grown on the 2D clinostat as measured manually and using SOAPP, respectively. Asterisks denote the degree of significance. Blue asterisks indicate a significant difference between the SOAPP and manual measurements for the clinostat group and red asterisks indicate significant differences for the control group. A single asterisk indicates a p-value below 0.05, a double asterisk indicates a p-value below 0.001, and a triple asterisk indicates a p-value below 0.0001.

Figure 9.

A comparison of the manually measured hypocotyl lengths of the 20 assayed ecotypes grown stationarily (control) and on a rotating 2D clinostat. Mean lengths of the hypocotyls grown statically are shown in orange and those of the plants grown on the 2D clinostat are shown in blue. A single asterisk indicates a p-value below 0.05, a double asterisk indicates a p-value below 0.001, and a triple asterisk indicates a p-value below 0.0001.
A comparison of the manually measured hypocotyl lengths of the 20 assayed ecotypes grown stationarily (control) and on a rotating 2D clinostat. Mean lengths of the hypocotyls grown statically are shown in orange and those of the plants grown on the 2D clinostat are shown in blue. A single asterisk indicates a p-value below 0.05, a double asterisk indicates a p-value below 0.001, and a triple asterisk indicates a p-value below 0.0001.
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