L, Pluripotin biological activity imclearborder). The image was smoothed and filtered to remove any noise (imerode, medfilt2) and the area enclosed by the detected leading edge was estimated (regionprops). Before we 10781694 analyzed the experimental images, we undertook a preliminary step where we Fruquintinib applied a wide range of threshold values to our experimental images, S[?:001,0:5. We found that thresholds in the range S[?:01,0:08 produced visually reasonable results.0.2.2 Automatic edge detection using the MATLAB Image Processing Toolbox. The manual edge detection methoddescribed in section 0.2.1 can be implemented in an automated mode by allowing the MATLAB Image Processing toolbox to automatically determine the threshold, S, for each individual image [25]. The following procedure was used to detect the location of the leading edge. The image was imported (imread) and converted from color to grayscale (rgbtogray). The Sobel method was applied in the automatic mode (edge[grayscale image, `Sobel’]). The lines in the resulting image were dilated (strel(7), imdilate). Remaining empty spaces were filled and all objects disconnected from the leading edge were removed (imfill, imclearborder). The image was smoothed and filtered (imerode, medfilt2) and the area enclosed by the detected leading edge was estimated (regionprops). 0.2.3 Automatic edge detection using ImageJ. 16985061 ImageJ software [24] was used to automatically detect the position of the leading edge. For all images, the image scale was set (Analyze-Set scale) and color images were converted to grayscale (Image-Type32bit). The Sobel method was used to enhance edges (Process-Find Edges). The image was sharpened (Process-Find Edges) and anSensitivity of Edge Detection Methodsautomatically determined threshold was applied (Image-AdjustThreshold-B W-Apply). After applying the Sobel method again (Process-Find Edges), the wand tracing tool, located in the main icons box, was used to select the detected leading edge. The area enclosed by the detected leading edge was calculated (Analyze-Set Measurements-area, Analyze-Measure).Results 0.4 Locating the Leading EdgeTo demonstrate the sensitivity of different image processing tools, we apply the manual edge detection method, with different threshold values, to images showing the entire spreading populations in several different barrier assays. Images in Fig. 1A and Fig. 1G show the spreading population in a barrier assay with 30,000 cells at t 0 and t 72 hours, respectively. Visually, the leading edge of the cell population at t 0 (Fig. 1A) appears to be relatively sharp and well-defined. In contrast, the leading edge of the cell population at t 72 hours (Fig. 1G) is diffuse and less welldefined. This indicates that is it difficult to visually identify the location of the leading edge after the barrier has been lifted and the cell population spreads outwards, away from the initiallyconfined location. Our visual interpretation of the images indicate that the precise location of the leading edge is not always straightforward to define. To explore this subjectivity, we use the manual edge detection method (section 0.2.1) by specifying different values of the Sobel threshold, S. Results in Fig. 1B and Fig. 1C show the detected leading edges at t 0 hours using a high threshold (S 0:0800) and a low threshold (S 0:0135), respectively. For both thresholds, the detected leading edges appear to be appropriate representations of the leading edge of the spreading population, and are very similar to ea.L, imclearborder). The image was smoothed and filtered to remove any noise (imerode, medfilt2) and the area enclosed by the detected leading edge was estimated (regionprops). Before we 10781694 analyzed the experimental images, we undertook a preliminary step where we applied a wide range of threshold values to our experimental images, S[?:001,0:5. We found that thresholds in the range S[?:01,0:08 produced visually reasonable results.0.2.2 Automatic edge detection using the MATLAB Image Processing Toolbox. The manual edge detection methoddescribed in section 0.2.1 can be implemented in an automated mode by allowing the MATLAB Image Processing toolbox to automatically determine the threshold, S, for each individual image [25]. The following procedure was used to detect the location of the leading edge. The image was imported (imread) and converted from color to grayscale (rgbtogray). The Sobel method was applied in the automatic mode (edge[grayscale image, `Sobel’]). The lines in the resulting image were dilated (strel(7), imdilate). Remaining empty spaces were filled and all objects disconnected from the leading edge were removed (imfill, imclearborder). The image was smoothed and filtered (imerode, medfilt2) and the area enclosed by the detected leading edge was estimated (regionprops). 0.2.3 Automatic edge detection using ImageJ. 16985061 ImageJ software [24] was used to automatically detect the position of the leading edge. For all images, the image scale was set (Analyze-Set scale) and color images were converted to grayscale (Image-Type32bit). The Sobel method was used to enhance edges (Process-Find Edges). The image was sharpened (Process-Find Edges) and anSensitivity of Edge Detection Methodsautomatically determined threshold was applied (Image-AdjustThreshold-B W-Apply). After applying the Sobel method again (Process-Find Edges), the wand tracing tool, located in the main icons box, was used to select the detected leading edge. The area enclosed by the detected leading edge was calculated (Analyze-Set Measurements-area, Analyze-Measure).Results 0.4 Locating the Leading EdgeTo demonstrate the sensitivity of different image processing tools, we apply the manual edge detection method, with different threshold values, to images showing the entire spreading populations in several different barrier assays. Images in Fig. 1A and Fig. 1G show the spreading population in a barrier assay with 30,000 cells at t 0 and t 72 hours, respectively. Visually, the leading edge of the cell population at t 0 (Fig. 1A) appears to be relatively sharp and well-defined. In contrast, the leading edge of the cell population at t 72 hours (Fig. 1G) is diffuse and less welldefined. This indicates that is it difficult to visually identify the location of the leading edge after the barrier has been lifted and the cell population spreads outwards, away from the initiallyconfined location. Our visual interpretation of the images indicate that the precise location of the leading edge is not always straightforward to define. To explore this subjectivity, we use the manual edge detection method (section 0.2.1) by specifying different values of the Sobel threshold, S. Results in Fig. 1B and Fig. 1C show the detected leading edges at t 0 hours using a high threshold (S 0:0800) and a low threshold (S 0:0135), respectively. For both thresholds, the detected leading edges appear to be appropriate representations of the leading edge of the spreading population, and are very similar to ea.
