Fig 1.
Flowchart representing the processing steps in different Colony detection methods.
OpenCFU’s flowchart is not shown here.
Fig 2.
Comparison of different digital colony detection methods with manual measurements for tumorspheres.
The first image in (A) is the original image. The rest are processed images from the colony detection methods. IMJ Cai has the thresholded areas in black, while the detected colonies have a blue outline around them. The inset shows a magnified view of colony that was segmented into 7 parts. Open CFU also has blue bounding boxes around the detected colonies, while Cell Profiler has red outlines. IMJ Edge has the detected colonies in black. (B) shows a plot of average area measured by the different detection methods. The number of colonies detected is shown is given by N below each method. (C) compares the average area (of 5 measurements) of each individual colony from digital methods (y-axis) with manual measurements (x-axis). The equation of the linear least-squared fit and goodness of fit R2 is given for each method. (D) labels each colony with its respective number. (E) plots the average area for each colony (labeled in D) against the colony number. Error bars are standard errors from 5 measurements. (F) is a boxplot showing the measurements of tumorspheres from 12 images measured manually and using IMJEdge.
Fig 3.
Comparison of digital methods for colony detection for bacterial colonies.
(A) shows the original image, and masks/overlays of detected colonies by various methods. (B) Plot of the average of area measurements, while (C) is boxplot showing the distribution of area measurements by various methods. (D) is a plot of the absolute values of difference between digital and manual measurements of each colony. (E) plots the digital measurements of Area against the manual measurements. The equations of the least square fit line is given with R2 value. (F) displays the distribution of bacterial colony area measurements from 12 images by manual and IMJEdge methods. (G) is a plot of average perimeter, and (H) is a boxplot of perimeters measured by different methods. (I) plots the absolute deviations of different methods from manual measurements of perimeter for each colony. (J) plots the digital measurements of Perimeter against the manual measurements.
Fig 4.
Comparison of digital methods for colony detection for bacterial colonies.
(A) shows the original image, and masks/overlays of detected colonies by various methods. Red arrows in IMJ Cai and IMJ Edge mark unsegmented colonies. Black arrows mark undetected colonies. (B) Plot of the number of detected colonies, and (C) is a plot of the average colony areas. (D) plots the digital measurements of Area against the manual measurements. The equations of the least square fit line is given with R2 value. (E) is a boxplot of measurements from 12 Clonogenic assay images by manual and IMJEdge methods
Fig 5.
Application of IMJ Edge for detection of cells.
(A) RGB image showing DAPI staining of nuclei in blue, and phospho-histone3 staining in red. (B) shows the red channel processed with IMJ Edge to calculate the number of dividing cells. (C) shows the blue channel processed by IMJ Edge to identify all the nuclei. (D) shows the blue channel of (A) processed by IMJ Cai, OpenCFU and Cell Profiler. The detected nuclei are outlined in blue boxes by OpenCFU, and in red circles by CellProfiler. (E) shows the original Nomarski image of yeast cells, and the processed images by various methods next to it. (F) shows the Normarski image of a 293 cell and its processed versions next to it. The segmentation step was skipped in IMJ Edge to prevent segmentation of cell processes.
Fig 6.
Application of IMJ Edge on tissue images and cellular assays.
(A) Image of a stained zebrafish heart with heart cells in green (cmcl2:gfp line), nuclei in blue (DAPI), and heart cell membranes stained in red (Alcama antibody). The green channel (B) was processed by IMJ Edge, and the outline overlaid on the red (C) and blue (D) channels (white outline). (C) and (D) show that there are stained cells outside of the outline that will be measured if outlines are not overlaid. (E) Picture of a leaf processed by IMJ Edge. (G) Two time points of a scratch assay processed by IMJ Edge. Black arrow marks the difference between the edges of epithelial cells. (F) Picture of an invasion assay from spheroid processed by IMJ Edge. The white arrow shows the diameter. (H) Images of tube formation assay for endothelial cells processed by IMJ Edge. The fill step was skipped to avoid filling in the areas between tubes.