The plugin will ask you to choose the desired postfix for all the names of processed images and result files, plus the directory where you want every resultant file to be saved.After you have completed the “Image straightening and ROI selection” steps, go to the plugins menu and select “ColonyArea” (Go to _Plugins -> ColonyArea -> Colony area).Thresholding and colony quantification using Colony_area: This option allows the user to analyze only a subsection of the plate if desired. The selection should be done in such a way that the sides of the rectangle touch the outer walls of the wells. (With an acceptable error of about 1-1.5 degrees max.) Then click on “OK”.Īfter the image has been straightened, use the “Rectangular Selection” tool from the ImageJ toolbar to make a selection of a region of interest (ROI) containing the wells you want to process (See Fig. Based on visual inspection, select an angle such that the image is nearly horizontal.1).įigure 1: Use the rotate command to straighten the image.įigure 2: Use the rectangular selection tool to choose the wells to be analyzed. Set the angle to 0, gridlines to anywhere from 20-100 depending on image sizeĪnd your discretion.Close all images opened in ImageJ and open the “.tiff” image of the colony formation assay that you want to process.Sample image files used in the manual can be downloaded here. Usageĭetailed usage instructions and examples here. In the Plugins dropdown of the Fiji menu, ColonyArea should now be available. Copy the following files to your Fiji plugins directory: Manual installĭownload the latest release from the repository. In the Fiji menu, go to Help -> Update… -> Manage update sites and select the ColonyArea site. InstallationĬolonyArea can be installed either through Fiji Update Sites or manually. The plugin processes each well individually and determines not the colony number, but the area of the well covered with cells, also taking the intensity into account. We have developed ColonyArea, an ImageJ-plugin that is optimized to perform standard analysis of colony formation assays conducted in 6- to 24-well dishes. These colony or focus formation assays are widely used in radiation biology and cancer biology, where they are employed to study resistance of cancer cells to radiation or the transforming potential of genes, respectively. We would really appreciate tips from more experienced users on how to improve our results.Camilo Guzmán 1,2, Manish Bagga 1,2, Amanpreet Kaur 1, Jukka Westermarck 1, and Daniel Abankwa 1ġTurku Bioscience, University of Turku, Åbo Akademi UniversityĬlonogenic assays measure the survival and growth of a single mammalian cell into a colony. It’s our first time trying to segment cells. Our best results are with automatic seeds and a radius of 25 px (that’s approx the size of a cell). For example, using the probability map from Weka Segmentation as seeds doesn’t seem to help. We have also tried playing around with the parameters for the watershed split. We tried retraining the algorithm in the Weka segmentation several times, but don’t know how to further improve the outcome. The issue is that we must keep the shape of our blobs with several cells stuck together for the watershed to be able to split them. I’m not sure Gaussian Blur is helping much since we must use very low sigma values (about 1). Erosion is definitely not helpful in our case.
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