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Using ImageJ to Select & Measure Areas Based on Color 

1. Set up ImageJ:a. If you don’t have the Threshold_Colour plugin installed,

you need to get it: i. Go tohttp://www.dentistry.bham.ac.uk/landinig/software/software.html, scroll down until you see“Threshold_Colour”, and download thethresholdcolour.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 newitem called Threshold Colour should be there.

b. Go to Edit  Options Colors… and set theforeground color to white, the background color toblack.

c. Go to Analyze Set Measurements… and checkboxes next to the items you want to measure. Youprobably want Area; you may want Perimeter or  AreaFraction as well. If your image is calibrated the area

will be given in whatever units the image is calibratedin. If not, the area will be given in square pixels.  AreaFraction is the percentage of the total image areacovered 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 notcalibrated, you will only see the number of pixels in eachdimension (for example, 1276 x 816 ). If the image iscalibrated, you will see the number of pixels and you will seedimensions with a unit of length (like inches, cm, microns,etc). If the dimensions of the image are obviously notcorrect, you need to set them.

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4. If necessary, set the correct pixel size by going to AnalyzeSet Scale… and typing in the correct numbers. If you checkthe 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 versionsshow up with the name Colour-based Thresholding )

6. Make sure “Pass” is selected for all channels and move thesliders 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 twosliders 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 anderror to figure this out. Check “Exclude on Edges” if youdon’t want to count anything touching an edge of the image.Check “Include holes” if you want it to fill in holes and includethat 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.