"A fully integrated multiplatform image analysis utility"

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25. Processing Mouse Hindlimb SPECT/CT Images

25.1 Introduction

BioImage Suite is an integrated image analysis software suite developed at Yale University to facilitate the processing of multiple image modalities. It has been used for CT and SPECT images from cardiac animal studies, but has applications to MRI as well as human studies.

This chapter derives from the ``Mouse Suite'' tutorial, originally written by Patti Cavaliere with help from Don and Donna Dione. It guides the user through the reorientation and cropping of mouse hind limb SPECT images, and co-registration with CT images, to result in segmentation of hind limb images and statistical evaluation of the region of interest (ROI).

The procedure involves the following 5 steps.

• Step I - Flip and crop images
• Step II - Remove table from CT images
• Step III - Draw planes on CT images
• Step IV - Segment soft tissue from CT images
• Step V - Compute ROI statistics

Figure 25.1: Loading Images into the Mouse Register application

The processing is all done within the programs ``Mouse Segment'' and ''Mouse Register'' which can be found under the ''MouseSuite'' tab from the BioImage Suite main menu. To start invoke MouseRegister. Once this program opens, three windows will appear: BioImage Suite::MouseRegister, Reference Viewer, and Transform Viewer as shown in Figure 25.1.

25.2 Flip and Crop Images

25.2.1 Flip Images

Each Viewer can be designated for either CT or SPECT image processing. For this demonstration, we will use the Reference Viewer for CT, and the Transform Viewer for SPECT images. See step 6.

1. Starting with the Reference Viewer window, choose File, Switch Directory. The Select Current Directory box appears.
2. Select the directory that you want to open. Click OK.
3. Go back to the Reference Viewer window to load study images.
4. Choose File, Load. The Load Image box appears.
5. Double click the name of the study that you want to load.

   Note: Use first CT study name when choosing which CT image to load.

6. In the Transform Viewer window, go to File, Load, then choose SPECT.

   Note: Once the study is loaded, three images will appear in each of the two viewer windows. Each image will represent three orientations of the mouse designated as Coronal, Sagittal, and Axial. Hold down the left mouse button to move the color- coded-cursors. The cursor will move concurrently in both screens.

7. Make sure the CT and SPECT images are registered with each other in the x and y directions.

8. Follow steps to edit the header - this is often a problem with the specific datasets used in the preparation of this tutorial and may not apply in the general case.
   1. Go to File on the Reference Viewer window.
   2. Choose Image Header Editor.
   3. The Image Header Editor box opens.
   4. Under Voxel Dimensions, change the x-size, y-size, and z-size to 1.0, if needed.
   5. Next click Save, and then Close.
   6. If the header was changed, then repeat steps 4 and 5 to reload image.
9. Repeat steps 8 a-d to edit the header on the Transform Viewer window.

   Figure 25.2: Flipping Image Orientation (if needed)

   

10. To flip images to the correct orientation, start with the Reference Viewer(CT images) window. Choose Image Processing, then Reslice Image. The Image Processing Utility box appears - see Figure 25.2

    Click the Identity button, then hold down the left mouse button and choose Flip X. Next click Reslice! When reslicing is complete, then click Copy Results To Image. Repeat above for the Transform Viewer window(SPECT images). You are now ready to crop the images.

25.3 Cropping CT Images

Figure 25.3: Identifying the Cropping Extent.

Begin with either Reference Viewer(CT), or Transform Viewer(SPECT) window.

1. Position the cursor alongside the body so as to crop body image from background. Use care not to crop too close to body-opt for a larger image - see Figure 25.3.
2. Determine the numerical values of each coordinate visible using the scale to the right of the image. Repeat process for each side of image. Use Reference Viewer to determine x and y coordinates, and Transform Viewer to determine the z coordinates - again see Figure 25.3.

3. Record each coordinate on a piece of paper as follows:

   	LOW Value	HIGH Value
   x	( )	( )
   y	( )	( )
   z	( )	( )

4. Once coordinates are recorded, choose Image Processing on the Transform Viewer menu.
5. Next choose Reorient/Crop. A box will appear.
6. Type coordinates into table, then click Crop!. Note: New values will appear in table based on cropped size of object.
7. Click Copy Results To Image.
8. On the Transform Viewer window, Choose File, Save. A Saving Image box will appear with a prompt to rename the cropped image.
9. Rename image as CropRsliceName of study, then click the Save button.
10. Repeat steps 4 through 9 on the Reference Viewer window.

This completes cropping of CT and 1st SPECT images. Return to Transform Viewer window to begin cropping of 2nd SPECT images.

25.3.1 CROPPING 2nd SPECT IMAGES

1. Return to the Transform Viewer window. Repeat steps 3-7 of Step I, LOADING STUDY IMAGES starting on page 7, to load the 2nd SPECT image.
2. Once the study is loaded, three images will appear in the viewer window. Note: The bladder may be visible in Tc99m images in comparison to TL-201 images.

3. Choose Image Processing from the menu on the viewer window. Click Reslice Image. The Image Processing Utility box appears.

4. Click the Identity button, then hold down the left mouse button and choose Flip X. Next click Reslice!.
5. Next click Copy Results To Image.
6. Choose Reorient/Crop from the Image Processing menu.
7. Type x, y, and z coordinates into table.
8. Click Crop!, then click Copy Results To Image.
9. On the Transform Viewer window, Choose File, Save. A Saving Image box will appear with a prompt to rename the cropped image as CropResliceName of study.
10. . Minimize the Transform Viewer window and proceed to Step II REMOVING THE IMAGING TABLE FROM CT VIEWS.

25.4 Removing the Imaging Table from CT Views

Figure 25.4: Using the Segmentation/Math Morphology Tool to mask out the imaging table from the mouse CT image.

1. Return to the Reference Viewer(CT) window.
2. Choose File, Load. The Load Image box appears again.
3. Check to make sure that you are in the correct Directory, then choose the CT image that has been resliced and cropped.
4. The Reference View window will appear with (3) images.
5. Choose Segmentation on the Reference Viewer.
6. Next click Math Morphology from the dropdown menu.
7. A Segmentation Control window opens - see Figure 25.4.
8. Enter a value of 200 in the Low Threshold box. Move the sliding arrow to the far right in the High Threshold box.
9. Make sure the Display Mask is red (enabled).
10. Then click the Threshold! button. The mouse image will turn pink.
11. Position the cross-shaped cursor (+) on mouse body .
12. Click the Connectivity button. The table in the Reference Viewer window may change from pink to white.

    Note: If the table does not turn white after clicking the Connectivity button, click Erode, then re-click the Connectivity button. This Connectivity/Erode process may be repeated, up to three times. When table disappears, then press Dilate.

    NOTE: the Dilate button must be clicked the same number of times that the Erode button was clicked.

13. Uncheck the ``Display Mask'' checkbox. The mouse image will turn white again.
14. Click the Display! button. The table will disappear.
15. Go to File, Save. A Saving Image window will appear.
16. Select the name of the study, but rename it as CleanCropResliceName of study.

25.5 Draw Planes on CT Image

Figure 25.5: Bringing up the ``Virtual Butcher'' control which enables the parcellation of the CT Image into regions of interest using Cropping Planes.
Figure 25.6: The ``Planes'' - tab of the ``Virtual Butcher'' control.

1. Return to the Reference Viewer(CT) window.

2. Click 3-Slice Mode from right hand menu by holding down the left mouse button, then choose the 3D Only button when the drop-down menu appears.
3. Click the 3-slice button, then choose the Obl + Vol option.
4. Next choose Features on the Reference Viewer menu. Select Mouse Chopper from the drop down menu. A Reference Virtual Mouse Butcher dialog appears - see Figure 25.5 The ``Hind Limbs'' and ``Feet'' checkboxes should be turned on by default.
5. On the ``Virtual Mouse Butcher Dialog'' select the Planes tab - see Figure 25.6 -, then Load Planes. A Load Planes box appears with a list of studies. Choose a study directory that has been previously segmented. Load planes from that study.

   Note: A menu of eight anatomical categories will be listed under the List of Planes.

6. Next click Save Planes, then rename file for this study. Be sure to save it in the proper directory.

   Figure 25.7: Parcellating the Mouse CT Image into Regions using Cropping Planes. This figure shows the three windows: (i) the viewer, (ii) the virtual butcher control and (iii) the oblique slice control

   

   Figure 25.8: Placing the first plane to separate the hip. Once done press the ``Update Plane'' button on the butcher control to store the position and orientation of the plane.
   Figure 25.9: Placing the second plane to separate the hip.

   

   
   

   

7. Click Oblique Slice Control. An Oblique Slice Control box appears. Position this Oblique Slice Control box under the CT mouse image for better visualization of planes during this part of the segmentation process and stretch box to length of viewer window. Sliders located above the Distance will control the rotational and transitional position of the yellow transparent plane (x,y,z) - this is shown in Figure 25.7.

8. Start to adjust the first plane, Left Hipp-Horizontal, by moving the x,y,z sliders using your mouse. Position the yellow transparent plane parallel to the femur bone and above the thigh muscle - see Figure 25.8

9. Once optimal position is achieved, click Update Plane on the ReferenceVirtual Mouse Butcher window.

10. Select next anatomical category, Left Hipp-Vertical - see Figure 25.9. Align the yellow transparent plane parallel to pelvis in the space located between the head of the femur, and curve of pelvic bones. Check front and back views of the mouse by rotating the image using the left mouse button in the viewer window.

    Note: The bladder and tail may introduce unwanted isotope activity into region of interest. Bladder may be visible on frontal view of mouse. Try to align plane so as to exclude bladder and tail, if possible. If not possible, see appendix on how to remove tail from CT image.

11. Once optimal position is achieved, click Update Plane on the Reference Virtual Mouse Butcher window.

    Figure 25.10: Placing single planes at the knee (left) and the ankle (right).

    

12. Select next anatomical category, Left Knee, and align plane through point of kneecap on lateral view of mouse - see Figure 25.10 (left). Try to exclude body wall tissue before clicking Update Plane.

13. Select the next anatomical category, Left Ankle. Align the plane just above the protruding hock bone of foot - see Figure 25.10(right). Angle so as to exclude the calf muscle. Click Update Plane.

14. Repeat steps 8-13 for the Right side anatomy of mouse images. Click Update Plane after each category crop is finished.

15. Next click Save Planes, then click Save.

16. Prompt will ask if you want to overwrite. Choose OK.

17. Return to the ReferenceVirtual Mouse Butcher window.

    Figure 25.11: Generating the Mouse Map.

    

18. Choose VOI Map on the left menu by holding down the left mouse button.

19. Change the Resolution value to 1.0, then click on Generate Mouse Map - see Figure 25.11.

    Note: This step takes approximately 2-5 minutes.

20. When Generate Mouse Map is finished, a For Your Information box appears. Click OK.

21. A Reference Surfaces window will appear. Go to Edit, Paste - see Figure 25.12 (top).

    Figure 25.12: Surface Renderings of the Mouse Map.

    

22. The mouse image on the Reference Viewer window will be color coded into (7) anatomical regions(Torso, R & L foot, R & L lower leg, R & L upper leg/hip area) - see Figure 25.12.

23. Check that the Left and Right proportions are approximately equal, and that the colors do not repeat in any two regions, then click Do Not Show in the Reference Surfaces window.

    Note: The Do Not Show button alternates with the Show as Surface button, so you may need to click the Show as Surface button first.

25.6 Segment soft Tissue from CT Images

1. Return to the Reference Viewer(CT) window.
2. Click 3-D Only to change back to 3-slice Mode.
3. Choose Segmentation on the left menu of the Reference Virtual Mouse Butcher box using the left mouse button. Then choose Show Segmentation Control.
4. A Segmentation Control box appears. Choose Histogram Segmentation from the menu bar at left, if it does not come up as the default window.
5. Set the Classes field to 3. The Classes field is located at the top of the Segmentation Control box.
6. Click Segment, located toward the middle of this box.
7. Next choose Math Morphology located at the top of the menu bar at left.
8. Make sure the Display Mask is enabled (red).
9. Go back to Histogram Segmentation.
10. Set the Use Class to 2. The Use Class is located at the bottom of the Segmentation Control box.
11. Click Generate located to the right of the Use Class options.

    Note: Mouse image on Reference Viewer will turn pink again. If pink areas do not Represent ONLY bone, then go back and change Classes to 4, then click Segment again, change Use Class to 3, click Generate again.

12. Go to the Mouse VOI Map box in the Reference Virtual Mouse Butcher and click Grab.

13. After using Grab, the Muscle Min and Muscle Max should be set to ``1'' and ``1'' and the Bone Class should be set to ``2''.

    Note: These numbers will be different if the Classes and Use Class numbers were increased.

14. Click the Soft Tissue VOI button to remove the bone from image.

    Note: If the image is not satisfactory, repeat process to change Class from 4 to 5 as described in step 11.

15. When image is acceptable, go to the Reference Viewer window and choose File, Save. Choose the correct Directory, and save the new file as ModelNoboneName of study.

25.7 Compute ROI Statistics

1. Close all the Mouse Butcher windows used.
2. Open the Transform Viewer window that was minimized in Step I.
3. Choose File, Load, then select ModelNoboneNname of study.
4. Go to the Reference Viewer window.
5. Choose File, Load, then select the 1st SPECT that was cropped in Step I. You will now have (3) windows open: The Reference Viewer, Transform Viewer, and a BioImage Suite:: Mouse Register window.
6. Choose Registration, then Transformation from the left drop down menu, located in the BioImage:: Suite Mouse Register window. A Registration/Overlay Tool box appears.
7. Click Compute ROI Stats.
8. A PXTkConsole window will appear. This box will list the ROI stats computed for the SPECT image. Highlight, cut, and paste these region values into a new text document and save them as: Animal name, Isotope, Day.
9. Repeat steps 5-8 for any other SPECT images.

Note: If regions 1 or 4 values are significantly greater than the other regions, this may indicate contamination due to inclusion of the bladder or tail.

Webpage design by Marcello DiStasio.


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