A subjective scoring plugin for FIJI

Purpose

This plugin is designed to help score complex colony morphology (CCM) in yeast. Yeast are assayed for complex colony morphology by pinning them onto agar trays in a 96-well plate format. The trays are then scanned and the individual wells are scored based on a subjective metric of colony complexity. For a description of this method of scoring CCM, see Granek et al. 2010 and Granek et al. 2013.

This plugin implements a simple interface that: 1) crops sub-images from a larger image based on a grid; 2) displays the sub-images in a random order; 3) gets and records a user-entered score; and 4) generates a simple HTML report that matches the images to the score to allow a user to check their scoring. The plugin requires the ImageJ plugin Microarray Profile to generate a grid of coordinates for the images that are cropped and displayed.

Install the plugins

• Download the “Microarray Profile” plugin from here.
• Clone the fiji-ccm-scoring repository or download the source
• Open Fiji
• Go to Plugins -> Install and choose the Microarray Profile .jar file.
• Go to Plugins -> Install and choose the ccm-scoring_.py in this folder.
• Go to Plugins -> Scripting -> Refresh Jython Scripts
• There should now be plugins called ccm-scoring and Microarray Profile in the Plugins menu.

Name your files appropriately

• Each image you’re going to score should be named something memorable with no underscores and no special characters (e.g. spaces, dollar signs, etc.)
• Each image should be a tif file and should end with the extension .tif (not .tiff)

Create a grid

• Open the image to be scored in Fiji
• Go to Plugins -> Microarray Profile
• For 96 well plates, you can load the grid example2_plate1 in the repository to display a grid. Alternatively, you can make your own using the Microarray Profile interface.
• These are the instructions from the Microarray Profile website for aligning a grid:

1. Make sure the number of rows and columns of the cicle grid matches the image. Use Reset Grid if necessary (inputs 4,7,20 for Dot Blot).
2. Alt-drag a corner ROI so that it aligns with the corresponding corner spot in the image.
3. Shift-drag an adjacent corner so that it is postioned properly. Now the whole edge connecting the two corners should be lined up with the spots.
4. Shift-drag a spot in the interior of the opposite edge until that edge is lined up with the spots. Unless the pattern of spots is distorted, all of the ROIs should now be aligned.

• An image of an aligned grid is here. Note that each colony is completely contained in each circle. It’s OK that the circles overlap with each other.
• Once your grid has been aligned, hit the Save Grid button in the Microarray Profile interface.
• IMPORTANT Save the grid in exactly this format: “plateName_gridName”. Where “plateName” is exactly the same name as the image that the grid was aligned on but without the .tif extension. “gridName” can be any name that doesn’t contain underscores. For example, if your image was called example.tif, you could save your grid to be called example_plate1. ALSO the grid file must be saved in the same folder as the image file the grid was aligned on.
• Once you save your grid hit the Quit button in the Microarray Profile interface

Score the images

• Go to Plugins -> ccm-scoring in Fiji.
• You will be prompted to navigate to a file. You can choose as many grid files as you like. However, the full image that the grid was defined on will be opened, so if you try to open up too many you may run out of memory.
• You will be prompted for a score file name. Type in whatever you like (let’s say “example-scores.csv”). If you type in the name of a previous score file, you will append data onto it. Note: to append data onto a previous score file, you should first select the same grids that were being used previously. Otherwise, the program might crash.
• A random cell from a random plate will be displayed.
• Adjust the contrast so that the background is black and there are no saturated pixels by typing in numbers into the Min and Max fields.
• Type the score into the score box and hit enter to get the next image.
• When each image is opened, a JPEG image is stored in the same folder as the first grid file and will be called something like “example-scores_cropped” the contrast settings that a current will be applied to the image that is saved. (But, see the bug below)
• When you type in a score, it is saved in the same folder as the first grid file and will be named by your scores.
• If you want to go back to previous images, hit the “Previous Image” button. The scores you entered will be displayed along with the image they go with. To navigate forward again, select the scoring box and hit ENTER. If you change a score, it’s saved in the csv file of the scores.
• When you get to the end of the images, a dialog box will pop up telling you there’s no more images. To exit, close the “CCM scoring” window.
• When you close the window, the script will generate an HTML file that displays the thumbnails and the scores that you gave to them. The report is called something like “example-scores.html” A second file is generated called something like “example-scores-with-plate-positions.html”. This has the names of the plates and the positions so that you can correct your scores.

TODOs

• Flexibility for tif naming
• Share source images to save memory

Bugs

• Currently, when you change the min and the max, the image updates properly, but the current thumbnail is not saved with the new min/max