Sean Lauber:ImageJ - Threshold Analysis

ImageJ can be used to measure differences in staining intensity. A threshold feature allows the user to increase the selected area depending on the intensity of staining, this area can then be measured. In order to quantify IHC staining, we need to measure the Total Area (brown+non-brown staining) and the IHC stained Area (brown staining) and relate this as a percentage. Download ImageJ here: http://rsbweb.nih.gov/ij/ (freeware).

Realize that quantification of IHC is considered to not be possible for a variety of reasons (see "Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II"), but may still be useful. You have to be very consistent with the way that you take your microscope pictures and prepare your slides to avoid variability. So prepare your samples on the same day, use the same microscope to take pictures using the same lighting and magnification parameters, etc.

The figure that I'm referring to is at the bottom of the page (Figure A-E).

1. Open ImageJ
2. Load the microscope picture you want to analyze (or simply paste it)
3. Open Threshold (Image/Adjust/Threshold or ctrl+shift+T)
4. Set all the parameters (Hue, Saturation, Brightness) to zero, in addition I like the following parameters: Thresholding method: Default; Threshold color: Black (you might prefer white or red); Color space: HSB; Dark background: unchecked.
5. You should see what's in Figure A
6. To measure the Total Area
   1. Adjust the Hue and Saturation to full, and adjust the Brightness bar to a point at which all cells/tissue is selected. See Figure B. Be sure to only select cells/tissue and not any of the background which also may be staining. You want to exclude white space)
   2. Press Select (one of the seven boxes at the bottom of the Threshold window)
   3. Now that the appropriate area is selected, measure the area (Analyze/Measure or ctrl+M) (Figure C).
   4. Record this area (copy/paste into Excel)
7. To measure the IHC stained area:
   1. Click Original in one of the seven boxes
   2. Without changing the Brightness bar, decrease the Hue bar until only the IHC stained area is selected. See Figure D. Initially when you move the Brightness bar you might see an inverted image. I think this is a bug. Choose a different Threshold color and then go back to black (if you like) to reset the image. Adjusting the threshold can be difficult to do perfectly because you will always end up selecting regions that are not stained. Adjust the Saturation and Brightness bars to fine tune if desired. Compromise!
   3. Press Select and measure the area (ctrl+M), see Figure E.
   4. Record into Excel
8. Now calculate the %IHC stained area: (IHC stained area)/(Total area) * 100%

And repeat!