I will usually create a new class “Tissue” via the Annotation tab for this step. This step will likely involve some trial and error, and will be highly sample/scanner/maintenance dependent.Ībove and Below threshold: Where do you want to create your objects? If you want an object for the background, include a classification for it, otherwise leave it blank or Unclassified or as Ignore*. Threshold: There is very little I can recommend here that was not already covered in earlier steps, so I will only emphasize that you can use the number keys or Brightness&Contrast window to look for good values before you start guessing. Conversely, for the color deconvolved channels like Hematoxylin, Residual, or DAB/Eosin, the Above threshold line will be used. For tissue detection, this means using one of the first set of channels will require setting a class for the “Below threshold” class, as that will be the darker part of the image. The color deconvolved channels are in the opposite direction, with 0 being white/background, and higher values (say around 3+) being closer to black. The key to the channel value is that the Red, Green, Blue, Averaged, Min, and Max channels are all on a 0-255 scale with 0 being black and 255 being white/background. It only matters a little bit what the stain actually is, some stains like DAB absorb/scatter in all three channels and therefor will appear darker in averaged channels. Averaged channels is a measure fo overall stain intensity in that it will be low where there is a lot of staining, and high where there is background. For brightfield images I most commonly use Averaged Channels, though the stain vectors can also be useful here. Lower resolutions will run faster and can be useful for getting a feel for your chosen channel.Ĭhannel: Choose the channel to Threshold on. The 64x downsample means your image will be treated as a bunch of squares 64 pixels on a side, with the average value of all of those pixels (in red, green, and blue, for brightfield). Resolution: As long as your image has pixel size metadata, these values will start with Full for the base resolution to 64X downsample for Very Low.
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