Using ImageJ to Select & Measure Areas Based on Color 1. Set up ImageJ: a.

If you dont have the Threshold_Colour plugin installed, you need to get it: i. Go to http://www.dentistry.bham.ac.uk/landinig/software/ software.html, scroll down until you see Threshold_Colour, and download the thresholdcolour.zip file. ii. Unzip the file and place the contents of the folder in your ImageJ Plugins folder. iii. Start ImageJ and go to the Plugins menu: a new item called Threshold Colour should be there. b. Go to Edit Options Colors and set the foreground color to white, the background color to black. c. Go to Analyze Set Measurements and check boxes next to the items you want to measure. You probably want Area; you may want Perimeter or Area Fraction as well. If your image is calibrated the area will be given in whatever units the image is calibrated in. If not, the area will be given in square pixels. Area Fraction is the percentage of the total image area covered by the measured object. 2. Open image it should be in 24-bit (RGB) format 3. Check the spatial calibration (pixel size) of the image by looking at the top of the image window. If the image is not calibrated, you will only see the number of pixels in each dimension (for example, 1276 x 816). If the image is calibrated, you will see the number of pixels and you will see dimensions with a unit of length (like inches, cm, microns, etc). If the dimensions of the image are obviously not correct, you need to set them.

4. If necessary, set the correct pixel size by going to Analyze Set Scale and typing in the correct numbers. If you check the box next to Global, this calibration will be used for all images you open during this ImageJ session. 5. Go to Plugins Threshold Colour (Note: older versions show up with the name Colour-based Thresholding) 6. Make sure Pass is selected for all channels and move the sliders until only the areas you want to measure are showing (areas that are excluded should be covered by black pixels). Close the plugin window 7. Click on the image, go to Image Type 8-bit (Steps 8 & 9 will not work on an RGB image) 8. Go to Image Adjust Threshold and drag the two sliders until the area you want to measure is covered in red. 10. Go to Analyze Analyze Particles: set size range to exclude tiny or large objects. You may need to use trial and error to figure this out. Check Exclude on Edges if you dont want to count anything touching an edge of the image. Check Include holes if you want it to fill in holes and include that area in the measurement. Under Show select Masks to create a new image showing what is measured. Check Display Results and Summarize. Click Ok to run.