TITLE: Drawing ROIs in ITK-Snap
AUTHORS: Courtney Comrie

[STEPS]
?. [ Introduction]
This protocol will show you two different methods for drawing ROIs in ITK-SNAP. The first method will be drawing ROIs manually and the second method will show the automatic ROI with the snake tool.Note: The example in this protocol uses the Fornix Fimbria in a ferret brain as the ROI.
?. [Manual ROIs]
Open up ITK-SNAP by typing:
?. [Manual ROIs]
Open NIFTI file on ITK-SNAP.
?. [Manual ROIs]
Zoom into the brain until you are satisfied with your view.To zoom in select the "Zoom/Pan Mode" button, and then the up arrow boxed in the image below.
?. [Manual ROIs]
Identify the ROI you want to segment on your brain.There are resources on the internet, and in lab to help identify the tracts. For this protocol a Brain Atlas of the Ferret was utilized.
?. [Manual ROIs]
Select the "Polygon Mode" and on your ROI trace an outline.Once you are happy with your shape select the accept button in the bottom right hand corner.
?. [Manual ROIs]
Continue the previous step through the slices till your ROI is finished.
?. [Manual ROIs]
Lastly, save your ROI as a NIFTI file, and open it in ITK-SNAP to ensure there are no mistakes.
?. [Auto ROIs]
Open up ITK-SNAP by typing:
?. [Manual ROIs]
Save the ROI by clicking on Segmentation>Save Segmentation Image.Title your ROI and make sure to delete the ".gz" at the end. You want a ".nii" file, not a ".nii.gz" file.
?. [Auto ROIs]
Open NIFTI file on ITK-SNAP.
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Select the snake tool in the top left corner.Then select the Segment 3D button.
?. [Auto ROIs]
Change the Presegmentation mode to Classification from Thresholding.
?. [Auto ROIs]
Zoom into the brain until you are satisfied with your view.To zoom in select the "Zoom/Pan Mode" button, and then the up arrow boxed in the image below.
?. [Auto ROIs]
Starting with Label 1, use either the polygon or paintbrush mode mark the tract/ROI.The labels are identified by color on the right hand side of the above image. Create another label, and mark an area that is not your ROI. Select "Train Classifier."
?. [Auto ROIs]
The goal is to completely single out your tract/ROI onto a single ROI from the surrounding grey matter (and other white matter tracts). This is achieved by adding labels to the area surrounding the ROI and updating the "Train Classifier" until the desired effect is achieved.  Typically you will end up using 3-5 labels, but that is dependent on the brain and selected ROI.
?. [Auto ROIs]
Note: Should for any reason you wish to clear the marks after training the classifier, select "Clear Examples" to delete marks made with the brush or polygon mode.
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Once you are happy with the trained classifier, select "Next" in the bottom right hand corner.
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Move the cursor to your ROI on the image and select "Add Bubble at Cursor." Ensure that your bubble is correctly sized and placed on the desired region. Select Next.
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Grow the bubble by selecting the play button.As the bubble grows, you can select update at the bottom of the left hand corner to see a 3D visualization of your ROI.
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Select Finish.
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Lastly, save the ROI by clicking on Segmentation>Save Segmentation Image. Title your ROI and make sure to delete the ".gz" at the end. You want a ".nii" file, not a ".nii.gz" file.
?. [Auto ROIs]
Above is the Snake-Tool ROI.