3. Select the stain using the Selected Stain pulldown menu (Fig 2.). The plugin provides a number of "built in" stain vectors: Hematoxylin and Eosin (H&E), Hematoxylin and DAB (H DAB), and Hematoxylin, Eosin and DAB (H&E DAB) [1] .
4. Users can also determine their own vectors to achieve an accurate stain separation. This option is available in two ways: “From ROI” or “Load From File”. If the “From ROI” option is set, users also need to choose three ROIs to compute the stain vectors. Select small ROIs areas which are intensely stained with only one of the stain, without empty background. If the staining method uses only 2 colors instead of 3, for the 3rd selection just select a small ROI from the background. The plug-in will compute the stain vectors based on three regions [1].
The computed stain vectors will be saved in default directory (the Sedeen folder in the same directory as the image): "Path to the image/sedeen/" as “StainsFile.csv” file for future use. The stain vectors can be provided by “.csv” file by selecting “Load From File” option. An example of the “.csv” file will be provided in the plug-in directory. This file needs to be copied in default directory: “Path to the image/sedeen/”.
7. The Processing ROI option allows the % staining to be calculated over a user defined ROI. (Fig 3). If an ROI is not selected then the calculation will be done over the displayed region.
8. Clicking on the Run button will execute the algorithm. The calculated % area is shown in the results panel. A color overlay shows which pixels are included in the % stain calculation. Use the Show Result checkbox to toggle the overlay on and off. The overlay is generated at the resolution of the displayed image but the calculation is carried out at full image resolution.
[1] A. C. Ruifrok and D. A. Johnston, “Quantification of histochemical staining by color deconvolution,” Anal. Quant. Cytol. Histol., vol. 23, no. 4, pp. 291–299, 2001.
Stain Analysis Plugin was developed by Michael Schumaker and Azadeh Yazanpanah, Martel lab at Sunnybrook Research Institute (SRI), University of Toronto and was partially funded by NIH grant.
Copyright (c) 2021 Sunnybrook Research Institute
Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.