Press Select and measure the area (ctrl+M)ħ. Adjust the Saturation and Brightness bars to fine tune if desired. Adjusting the threshold can be difficult to do perfectly because you will always end up selecting regions that are not stained. Choose a different Threshold color and then go back to black (if you like) to reset the image. Initially when you move the Brightness bar you might see an inverted image. Without changing the Brightness bar, decrease the Hue bar until only the IHC stainedĪrea is selected. Click Original in one of the seven boxesĦb. Record this area (copy/paste into Excel)Ħa. Now that the appropriate area is selected, measure the area (Analyze/Measure or ctrl+M)ĥd. Press Select (one of the seven boxes at the bottom of the Threshold window)ĥc. Be sure to only select cells/tissue and not any of the background which also may be staining. Adjust the Hue and Saturation to full, and adjust the Brightness bar to a point at which all cells/tissue is selected. 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.ĥa. Open Threshold (Image/Adjust/Threshold or ctrl+shift+T)Ĥ. Load the microscope picture you want to analyze (or simply paste it)ģ. So prepare your samples on the same day, use the same microscope to take pictures using the same lighting and magnification parameters, etc.Ģ. You have to be very consistent with the way that you take your microscope pictures and prepare your slides to avoid variability. 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. be used in an Identify module or thresholded using the Threshold module to produce a.
Maybe you want to show that there are increases in staining itself, in which case you simply have to measure the stained area and not the total area. This module converts color and channel-stacked images to grayscale.
#IMAGEJ COLOR THRESHOLD HOW TO#
In this example I'm showing how to quantify the % stained area so we'll need to measure the Total Area (brown+non-brown staining) and the IHC stained Area (brown staining) and relate this as a percentage. There's a number of ways to express the data. A threshold feature allows the user to increase the selected area depending on the intensity of staining, this area can then be measured. ImageJ can be used to measure differences in staining intensity.
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