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Using ITK-SNAP to Segment Out Lateral Ventricles Protocol Developed by Vy Nguyen

UC Davis Medical Center, The M.I.N.D. Institute 10/23/06

I. Introduction In the world of cognitive neuroscience, particular interest is often directed at certain neuroanatomical structures. These “regions of interest,” as they are called, can provide great insight into the cognitive functioning as well as morphological changes of the brains of a population. The problem that researchers always face, however, is that tracing these structures on medical images to acquire volumetric data or to use as masks for functional data can be time-consuming. The solution then is to use advanced software to perform automated segmentations and save hours of manual labor. This is where ITK-SNAP comes in. ITK-SNAP is a semi-automated program that uses active contour methods specified by a variety of parameters allowing the user to segment an anatomical structure on a 3D medical image. Aside from automated segmentation, ITK-SNAP also has tools that allow the user to perform manual segmentations as well. The following protocol will you give you the basic ideas behind using the program to segment out the lateral ventricles. For more information and details on how to use ITK-SNAP, visit their website at http://www.itksnap.org. As nice as it would be to use an automated program to save us hours of doing manual labor, there is always a deeply-rooted fundamental problem that comes with the automated territory: who knows if the program is doing an accurate job? It’s very possible and incredibly likely that the volumes that result from these segmentations could be completely inaccurate. This is the reason that a program such as this needs to be validated, and the best way to do this is to perform manual tracings on a number of brains and record their results. Once this is done, a reliability analysis can be performed to determine how accurate the segmentation volumes are. I’d recommend this for any structure for which you are segmenting. There is no point in using a short-cut if the results you get are inaccurate. There are ten brains for which you will perform reliability on after developing the skills necessary to use ITK-SNAP. The parameters indicated in this protocol are specific for our population of subjects (namely, the 22q children and typically-developing children from the ages of 7-14 that we study). After becoming reliable, you can go on to use the automated program to segment ventricles in our populations. This protocol will show you how to segment your lateral ventricles so that all you will have to do when it comes to manually patching them is delete unwanted voxels, rather than fill in wanted voxels (this is very time consuming, especially if they're small groups of voxels or if they’re single voxels). So let’s proceed and get some ventricles segmented!

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II. Getting Started

Above is a screenshot of the main window of ITK-SNAP. To open a brain volume, click on: File -> Load Data -> Greyscale Image.

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Browse for the image file you’d like to open; the Image file format should be automatically chosen for you after you open the image file.

Click on Next. The following window appears:

If it isn’t already select, choose “Custom” for the orientation preset, and change the X, Y and Z-axes according to the orientation of the brain volume. For radiological images (where the patient’s right side is the screen’s left side), the RAI code should be RPI; for neurological images (where the patient’s right side is the screen’s right side), the RAI Code should be LPI. In the CABIL-Lab, because all our images are in neurological format, we should set the X, Y, and Z-axes so that the RAI Code is LPI (x-axis: left to right; y-axis: posterior to anterior; z-axis: inferior to superior). Click on Next and then, after reviewing the Image Summary window, click on Finish.

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The following window will appear asking if you want to restore the settings:

This window pops up when you’re using ITK-SNAP for the first time. You can set it so that it never appears by going to the drop down menu at the bottom, and choosing never ask me this question. I’d recommend choosing that option and clicking on Restore Settings at the bottom, but only if you are the only one using the program to perform the segmentations. This way, if you ever load up a brain that you had previously worked on, it will restore the settings (such as contrast adjustments, display options, the bounding box size, etc. – all these options are explained in the following pages) so you don’t have to redo those steps. However, if other people will be accessing the program on your computer (e.g., people performing segmentations for reliability), you do not want to restore the settings, because doing so will load up your previously stored settings and enable them to skip several steps in starting a segmentation. So if that is the case, then choose never ask me this question from the drop down menu at the bottom, and click on Don’t Restore. After choosing the desired settings, the Restore Settings window will go away, and the main window will be completely visible now:

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After loading the brain, you may find that the images are hard to see because the contrast is not properly set. In order to fix this, click on Options -> Adjust Image Contrast and the following window will appear:

You can click on one of the curve control points (in this case, there are three of them) and move them until you adjust the contrast to your liking. I find that moving the middle curve control point up and towards the left will change the contrast the way I need it to.

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You can also change the number of curve control points to better the contrast, but I find that this is not necessary. Click on Close. And last but not least, we need to change the display options so that ITK-SNAP will display the brain in the main window in the proper fashion, so we actually view the brain in neurological format. Click on Options -> Display Options and click on the Layout tab:

Be sure to uncheck the box “Patient right is screen left.” This will allow you to view the brain in LPI format in the main window. Click on Apply and then Click on Close. And voila: you are now ready to begin the preprocessing steps! III. Preparing the Volume for Preprocessing On the left hand side of the main window in the area titled “IRIS Toolbox,” click on the Snake icon. The area titled “Tool Options” will change and give the option of checking the box “Resample region.” We want to resample the region, because doing so will allow us to change the voxel sizes and increase the resolution for better segmentation results. We also want to set the bounding box (the red-dashed line in each of the three windows) around the anatomical structure we want to segment. You can do this by clicking on the red-dashed line and dragging it. In this case, we want to focus on the lateral ventricles and we do this by following the steps below:

1. Use the axial view (top-left window) and scroll through the slices using the scroller on the right side of the window to find the most anterior (using the anterior horns), posterior (using the posterior horns) and lateral parts (using the temporal horns) of your ventricles.

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2. Use your sagittal view (top-right window) to determine the most superior and inferior parts of your lateral ventricles.

3. Use your coronal view (bottom-right window) to make sure that you are

satisfied with the boundaries you chose for the bounding box. Your main window should now look something like this:

Click on Segment 3D under the Tool Options area on the left-hand menu. A window with resampling options will appear:

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Resample at a rate of 2:1 across all dimensions (voxels become half their original size) and leave the resampling method as “Cubic interpolation (high quality).” Click on Ok. Once the resampling is done, you may need to adjust the contrast again. See above on how to do just that. The result will be the following window:

Save this resampled file in the desired folder as cabil-xxx-resampled.img: File -> Save Data -> Greyscale Image Region. IV. Preprocessing the Volume During this step, we will choose a threshold value so that ITK-SNAP will know to focus on the brain matter above this value. Choose Intensity regions under the “Segmentation Pipeline” on the left-hand menu and then click on Preprocess Image. The following window will appear:

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For segmenting out the lateral ventricles, we set the Threshold direction to Above and we change the Smoothness value to 10.0. It really helps to have the “Combined display” box checked. You can play with the Upper threshold value and watch the main window to see how it affects the images in their orthogonal views. The red areas are the target areas for segmentation:

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What I find really helps is changing the Label opacity value on the bottom of the left-hand menu to something low (e.g., 29) because a value of 128 will color the image so red that it becomes difficult to see what is actually being segmented. A low value will allow you to scroll through the slices to make sure your regions of interest are “colored” properly and optimally:

A great slice to choose for finding the threshold value is an axial one in which you can see a large portion of the posterior horns. Sometimes, you will see what appears to be a grey-colored structure inside the posterior horns; they are called choroid plexi and are responsible for the production of cerebrospinal fluid (CSF). Use this slice to find the minimum threshold value needed for segmentation. The “minimum” threshold value is the smallest value used to just cover your target areas. After getting the thresholded image to fill in the CSF filled ventricular areas (which is black in structural MRI T1 images), be sure to be a little generous in setting the threshold value, so that it's a bit higher than what is necessary to just cover the non-choroid plexi areas of the ventricles (i.e., above the minimum value). The best way to see this is in an axial slice in which the septum pellucidum (the area between the bodies of the lateral ventricles) is getting smaller in width and the bodies of the lateral ventricles are getting closer to each other. Being “generous” entails setting the threshold at a level in which you do get a little bit of the septum pellucidum colored in (which at this point should have a small width at its body). This way, the left and the right lateral ventricles are “connected” in the thresholded image via the body of the septum pellucidum. You should double check this thresholded value by looking at the septum pellucidum in the coronal view. Scrolling through both the axial and coronal slices will enable you to see

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that as the lateral ventricles get closer to each other, they become connected in the thresholded image by means of the septum pellucidum. You should record the threshold value for record keeping purposes. The following image will give a better idea as to which slices to choose:

I had chosen an Upper threshold value of 73.0 because it is a value higher than the minimum value needed to cover the CSF space; the minimum value for covering the CSF was about 58.5. The “generous” value of 73.0 leads to the septum pellucidum being colored in just enough to connect the two lateral ventricles together. In this specific example, this subject has a cavum septum pellucidum (CSP), which means that there is CSF in the septum pellucidum and that this CSF must be accounted for as well when setting the threshold (we will delete the CSP later from our segmented result, since we are focusing only on the lateral ventricles). But as you can see in both the axial and coronal views that the lateral ventricles are “connected” via the CSP. Click on Ok. V. 3D Bubble Placements for Segmentation Now this is the fun part. During this step, we place 3D bubbles in our thresholded region of interest and watch them expand to fill out the ventricles as best as they can. Look at the thresholded image to get an idea as to what areas can be segmented; all the blue areas are ignored, while the white areas are targeted:

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It can be difficult to determine where to place the 3D bubbles when we cannot see the actually anatomical structures. Instead, it will be much easier to work with the greyscale image, than the thresholded image. Choose Original Greyscale image by going to the menu on the left side of the main window and changing the Display Options at the bottom from Preprocessed Image to Original Greyscale. Click on Next in the Segmentation Pipeline from the menu. The following window will appear:

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Choose bubbles that will have radii as wide as the width of the specific portion of the lateral ventricle in which you want to place the bubble; setting a bubble with too large of a radius will force the bubble to spread as far as the thresholded image and will possibly include nearby areas of CSF spaces that are not part of the ventricular system For the Axial View: Fill in posterior horns in the axial view with large bubbles that will fill up the entire posterior horns by clicking on the desired location and pressing Add Bubble. You can adjust the Radius according to your liking. Fill in the anterior portions of the lateral ventricles; this is especially important at the most anterior end of the ventricles, where the anterior horns become small in size.

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Scroll through until you reach a slice in which you can see the entire bodies of the lateral ventricles (i.e., the anterior and posterior horns are connected). Set bubbles along the bodies of each lateral ventricle to cover the entire length of the ventricles. Notice how the radii of the bubbles changed according to where they were places and how wide the ventricles were at those locations. Scroll through until you reach the most posterior ends of the horns; place small bubbles along the lengths of the horns. Notice how I try to get my bubbles to border each other as much as I can. I do this to increase the likelihood that those thresholded images will be properly segmented; they also help by covering more ventricular space than if just one bubble was placed (this also speeds up the process as well). For the Sagittal View: Scroll through the slices until you are at a slice where you can see the body of the left temporal horn.

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Place small bubbles along the body of the left temporal horn; be sure they border one another. Scroll through until you reach a slice in which the temporal horn approaches its most inferior portion; place bubbles in the temporal horn along the way, including in the inferior portion of the horn. Repeat the above steps for the right temporal horn. For the Coronal View: Finally, choose slices in which you can see the lateral ventricles at their widest point, and place bubbles of appropriate radii within the ventricles. As you're scrolling through the slices, choose slices in which the posterior horns begin to appear and be sure to place small bubbles there.

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The three views should always be used as a way to check your bubble placement as well. When satisfied with the bubble placements, click on Next on the menu. We can view the bubble placements in 3D mode by going to the bottom left-hand window and clicking on the update mesh button. In this window, you can press the Option key on the Mac or the Alt key on the PC and move the mouse to move the position of the 3D mesh. Holding down the left-click button and moving the mouse will rotate the 3D mesh. Note: All the bubbles are listed under the “Active bubbles” area under the Segmentation Pipeline. They are listed according to where they were placed by their x,y, z coordinates (sagittal, coronal, and axial, respectively). You can scroll through the slices in the orthogonal views until your slice numbers match the x,y,z coordinates to find a bubble. VI. Running the Segmentation After the bubbles have been placed, the segmentation is ready to be run. But before we run the segmentation, we need to change some parameters. Click on the Set Parameters button on the left-hand menu to change the snake parameters. The following window will appear:

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Make certain that the balloon force is set to 1.0 - this is the propagation speed of the bubbles as they spread out. Next, set the curvature force to 0.40 - as the name implies, this is the speed at which the bubbles curve to fill intensity-related voxels. A low curve velocity (e.g., 0.05) is great if you want to get detailed segmentations (such as really thin and narrow areas). However, the results may not be so great as this could lead to “leaking” of the bubbles into unwanted thresholded areas. As the curve velocity goes up, the segmentation results become smoother, which is what we want. To get a better understanding as to how these parameters affect your segmentations, you can click on Help or check the box Show animated 2D segmentation preview in the bottom right-hand corner. Click Accept. On the left-hand menu, change the Step size from 1 to 10 (rather than go one iteration at a time then, it will go in increments of ten iterations). Press the Play button to begin the segmentation. Be sure to click on the Stop button when the number hits 490 - that way, it will stop exactly at the 500-iteration mark (there is a bit of a lag). You can click on update mesh in the bottom left-hand window to view the 3D segmented result. If you check the auto-update box, the 3D window will continuously update the 3D mesh with every iteration step. I’d recommend not doing this, as it will slow down your computer as well as the segmentation process. Review your segmentation result. The program may have missed some areas that you may have covered with bubble placement. It is not a perfect program, but we must not lose sight of the purpose of this protocol. The segmentation results – unless the ventricles with all their horns are visibly large – may not look “ideal” and may appear patchy in some locations. In some instances, the bubbles may have also leaked into unwanted areas, and may give us a 3D mesh with non-lateral ventricle volumes. There is no need to panic, because all this can be fixed. Remember, our goal isn’t to get the perfect 3D mesh result, but rather to get the volumetric measurement of the lateral ventricles. As you can see in this picture, we experienced the latter. This makes sense as this subject suffers from CSP and our generous threshold value lead to leakage from the bubbles we placed in the lateral ventricles into the CSP area. We will focus on fixing this in the next step.

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Click on Finish to complete the segmentation process. Save the segmentation in the desired directory: File ->Save Data->Segmentation Data. Label it as cabil-xxx-ventricles.img. VII. Patching the Ventricles We may need to fix up our segmentation results because the bubbles may have leaked into unwanted areas. This protocol has been designed to use specific parameters so that the only patching that would need to be done are ones in which unwanted areas were deleted, as opposed to “coloring in” regions to fill in wanted areas. The latter is quite time consuming, as some slices may need single voxels or small groups of voxels colored in. Deleting regions is much faster. The following instructions are for deleting the unwanted region of the septum pellucidum. These rules apply for any unwanted regions. Using the “IRIS Toolbox” on the left-hand menu, click on the Zoom button. Using the axial view, right click the mouse (or use the option key on the Mac) and move the mouse upwards to zoom in. Find the slice at which the septum pellucidum first appears colored in (i.e., segmented out). You can also switch back to the Crosshair Tool under the IRIS Toolbox if you need to move the position of your slice to get a better view of it. Click on the Polygon Tool under the IRIS Toolbox. Under the area titled “Segmentation Label” on the menu, change the Active drawing label value from "Label 1" which is red (the default value) to "Clear Label" which is black. Essentially, the "Clear Label" is used to delete portions of your segmentations. Note: If you wanted to label your segmentation as something other than “Label 1” as you should, click on the Edit labels button and relabel the Description as something like “lateral ventricles.” Relabeling becomes important when you have multiple structures you want to segment out. Make sure the Label opacity is still set to a low enough number that you will still be able to tell what voxels have been segmented out, but still be able to distinguish the various intensities of the segmented voxels (e.g., changing the value from the default value of 128 to 29 - double check this)

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The Polygon Tool will be used to draw a shape around a region of interest (in this case, the CSP). With the label being clear, we can then delete this region from our result. Using the axial slice, use the mouse to click on locations around your region of interest to create a polygon. Then hold the Alt key (or Option key on the Mac) and click the mouse again to to close the polygon. Click on accept at the bottom of the axial window to accept the polygon shape. The segmentation information in this polygon will be deleted. Go slice by slice and delete all unwanted regions. You can click on the paste button if you wanted to use the same polygon on subsequent slices. You may be wondering why we set a threshold value so high that the septum pellucidum was going to be included in the final segmentation, only to delete those voxels afterwards. Because of narrow areas of the lateral ventricles, such as the temporal horns for instance, and because the threshold value can only come so close to covering all of the areas of the desired structure (in this case, the lateral ventricles), there will be voxels that should have been colored in (i.e., segmented out), but were not. This is the reason for the generous threshold value. But because we are focusing solely on the ventricles, we can delete the septum pellucidum voxels that were segmented out. Be sure to use the other orthogonal views as a check as well as for deleting unwanted regions Using the sagittal view, scroll to the slice in which the left temporal horn begins to appear. Continue scrolling in that direction and watch the consistency with which the segmentation occurred. You may find that some voxels are not covering any CSF ventricular spaces, but are presented on the temporal lobe, for instance. These voxels can be deleted. Repeat this step for the right temporal horn. Note: If you haven’t already done so, check to see whether or not your 3D mesh has a left and a right side (i.e., the ventricles were split into the left lateral ventricle and the right lateral ventricle). We want a 3D mesh that has the two sides separate, and deleting the septum pellucidum from our segmentation result usually does this. There may also be times, however, in which the ventricles are so large (i.e., the septum pellucidum is so thin) that it becomes unclear whether or not to delete the septum pellucidum in some slices. If that is the case, leave the segmentation on that slice as is. Use the label opacity tool to your advantage to see which segmentation voxels should be deleted.

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Once completed, click on File -> Save Data -> Segmentation Data and save the segmentation data as "cabil-xxx-ventricles-patched.img". VIII. Volumetric Measures Finally, to get the volumetric measurement of the segmentation, which is what we really care for, we need to save the volume in a text file as "cabil-xxx-ventricles-patched-volume.txt" and record the volume value for the 3D segmentation. To do this, we click on File -> Save Data -> Volumes & Statistics and save the text file as “cabil-xxx-ventricles-patched-volume.txt” in the desired directory.

Be sure to record the volume value (the third column) and not the total number of voxels (if the voxels are not 1x1x1, the volume value will not be the same as the total number of voxels). Congratulations! You have now completed your training on the ITK-SNAP Protocol for the CABIL Lab. Good luck with reliability!