1. Quanta2 Pipeline: A Training Tutorial Imaging of Dementia and Aging (IDeA) Laboratory, UC Davis School of Medicine: Neurology Amy Liu

2. Once Quanta2 is launched in a terminal, select and confirm initials. From the main window, click the “Pipelines” button. Select the “Quanta Classic Pipeline” button. On the next window, select an image to start the pipeline. Launch Quanta2

3. After Preprocessing, the dialog box shown above will appear. Note: You can save the pipeline at any stage by using the “Save ‘n’ Exit” button. Resume by reloading the pipeline when prompted. Click on the “Continue” button to launch The Cranial Vault (TCV) Tracer. Click “OK” on the next window.

4. On the right hand side, the Pipeline Progress window is shown. This will update as the pipeline wizard progresses

5. TCV TRACER This section of the tutorial will detail functionality of The Cranial Vault (TCV) Tracer and progress through several exemplary brain slices.  Examples are traced on a Fluid Attenuated Inversion Recovery (FLAIR) image, a modality for depicting hyperintense regions.  Moving dorsally to ventrally, traces will increase in complexity.  Note the various anatomical structures that should be excluded. TCV Tracer creates an.ima file.

6. TCV Tracer Functions include: “View Original” button can be used to toggle between traced and original view of the image. “Revert Slice” button reverts the completed trace image to original. This must be done each time BEFORE retracing a slice. “Flip Y” button changes the image view vertically. Here, Flip Y may be used to display the anterior of the head at the top of the window. “Full View TCV” button displays traced slices in a new window, which can be used after completing tracing on the file and acts as a check for continuity. Graphical User Interface

7. TCV Tracer Functions include: “Save TCV” button will write the traced image as an.ima file to disk. This.ima file is a binary file that will be used in all stages of the pipeline henceforth to save post- process data of each step. “Brightness” and “Contrast” adjustment bars allow for user- preferred display settings. “Reset” buttons will restore default settings. Graphical User Interface

8. TCV Tracer: Example Depending on preference, it may be easiest to begin TCV Tracing on a slice displaying representative brain matter. Thus, scroll to a slice halfway through the brain that appears similar to this image: Locating starting slice

9. TCV Tracer: Example To start TCV tracing, click “Begin Tracing” button. This will activate tracing mode: Left mouse button is used to plot individual trace points, which will be sequentially connected. Middle mouse button, or “Undo Last Point”, erases plotted trace points. Right mouse button, or “Complete Tracing”, will complete and close the first and last points of the traced region curve. “Cancel Tracing” button erases all uncompleted trace points and deactivates tracing mode. Note: scrolling between slices is NOT possible in tracing mode. Tracing Mode

10. TCV Tracer: Example Original ImageTraced Image Using the left mouse button, trace along the interior edge of the dura mater by plotting individual points that will sequentially connect. Exclude the dura mater and superior sagittal sinus. Exclude any noise or bright signal that may appear along the interior edge of the dura mater. Clicking the right mouse button will connect the first and last plotted points, completing the trace. Note the absence of the skull, dura mater, and superior sagittal sinus in the traced image. Dura Superior Sagittal Sinus Tracing Mode 1 st and last points will connect

11. TCV Tracer: Example Continue tracing successive slices, staying along the interior edge of the dura mater and removing the structures labeled with green arrows: Dura mater Superior sagittal sinus Inferior sagittal sinus Falx cerebri Tip: Scroll 1 slice up and 1 slice down from the slice to-be-traced in order to determine brain matter from noise. If the area in question appears on the slices above and below, it should be included as brain matter in the trace. Dura Superior Sagittal Sinus Inferior Sagittal Sinus Falx cerebri Excluding Structures

12. TCV Tracer: Example Continue tracing successive slices, staying along the interior edge of the dura mater and removing the structures labeled with green arrows: Dura mater Superior sagittal sinus Appears anteriorly and posteriorly in this and subsequent slices Falx cerebri Pineal gland Often appears as bright signal Trace just posterior to the Third Ventricle Dura Superior Sagittal Sinus Pineal Falx cerebri Superior Sagittal Sinus 3 rd Ventricle Excluding Structures

13. TCV Tracer: Example Continue tracing successive slices, staying along the interior edge of the dura mater and removing the structures labeled with green arrows: Dura mater Superior sagittal sinus Appears anteriorly and posteriorly in this and subsequent slices Falx cerebri Pineal gland Often appears as bright signal Trace just posterior to the Third Ventricle 3 rd Ventricle Excluding Structures

14. TCV Tracer: Example Original ImageTraced Image As slices progress ventrally, the trace contour complexity increases. Be sure to also remove: Cerebellar vermis Medially jutting dura mater Superior sagittal sinus Here, the Third Ventricle is intact (compare with next slide). Thus, do NOT remove midbrain structures. Dura SSS Vermis 3 rd Ventricle Excluding Structures

15. TCV Tracer: Example Here, the Third Ventricle is NOT intact. Thus, remove midbrain structures: Substantia nigra Red nucleus Pons Mammillary bodies Hypothalamus Cerebral peduncle Superior colliculus Also, continue to remove: Cerebellar vermis Medially jutting dura mater Superior sagittal sinus Anterior and posterior Falx cerebri Midbrain Structures Vermis Dura 3 rd Ventricle Excluding Midbrain Structures

16. TCV Tracer: Example Here, the Third Ventricle is NOT intact. Thus, remove midbrain structures: Substantia nigra Red nucleus Pons Mammillary bodies Hypothalamus Cerebral peduncle Superior colliculus Also, continue to remove: Cerebellar vermis Medially jutting dura mater Superior sagittal sinus Anterior and posterior Falx cerebri Excluding Midbrain Structures

17. TCV Tracer: Example For slices approaching the foot, 2 or more separate traces must be drawn. This allows for the exclusion of: Optic nerves Once the optic nerves have crossed, do not include any structures located anteriorly. Amygdaloclaustral area Cribiform plate Midbrain structures Cerebellum Midbrain Structures Optic Nerve Cribiform plate Amygdaloclaustral area Excluding Ventral Structures

18. TCV Tracer: Example For slices approaching the foot, 2 or more separate traces must be drawn. This allows for the exclusion of: Optic nerves Once the optic nerves have crossed, do not include any structures located anteriorly. Amygdaloclaustral area Cribiform plate Midbrain structures Cerebellum Cribiform plate Amygdaloclaustral area Excluding Ventral Structures

19. TCV Tracer: Example For slices approaching the foot, 2 or more separate traces must be drawn. This allows for the exclusion of: Optic nerves Once the optic nerves have crossed, do not include any structures located anteriorly. Amygdaloclaustral area Cribiform plate Midbrain structures Cerebellum Completing the trace displays only the structures within the trace line. Use “View Original” button to plan your next trace. Remember to scroll up and down 1 slice to determine which structures to include. Excluding Ventral Structures

20. TCV Tracer: Example For slices approaching the foot, 2 or more separate traces must be drawn. This allows for the exclusion of: Optic nerves Once the optic nerves have crossed, do not include any structures located anteriorly. Amygdaloclaustral area Cribiform plate Midbrain structures Cerebellum Cribiform plate Amygdaloclaustral area Cerebellum Excluding Ventral Structures

21. TCV Tracer: Example For slices approaching the foot, 2 or more separate traces must be drawn. This allows for the exclusion of: Optic nerves Once the optic nerves have crossed, do not include any structures located anteriorly. Amygdaloclaustral area Cribiform plate Midbrain structures Cerebellum Excluding Ventral Structures

22. TCV Tracer: Example Original ImageTraced Image Slices toward the top of the head contain more noise and thicker dura mater. It is important to scroll between slices to determine brain matter. If noise is accidentally included in the trace: Click the “Revert Slice” button to restore the original image. Then, “Begin Tracing” again. Dura Dorsal Tracing

23. After tracing, save the TCV and close the tracing application. This will launch the dialog box shown above. Click “Continue” to launch TCV Filtering. This will remove image artifacts like bias and inhomogeneity.

24. TCV Filtering There is no graphical user interface display on this step; you will only see text output in the terminal, as shown.

25. CSF-BRAIN MODELING This section of the tutorial will detail sequential modeling of brain matter and Cerebrospinal Fluid (CSF).  Quanta2 will separate brain matter voxels from CSF and background voxels to calculate Gaussian curves.  Intensity histograms of consecutive TCV-traced slices are obtained using a user-defined statistical modeling type and the presumption of underlying voxel intensities. CSF-Brain Modeling saves to the.ima file.

26. The pipeline dialog window will pop up. Click the “Continue” button and this selection window will appear for CSF-Brain Modeling. Shown above are the default settings, which can be user defined as needed. The Gaussian curves will show 2 segmentation domains (brain matter and CSF), using an estimation sensitivity of 25 and the filtered TCV for intensity correction. Click “Continue” to proceed. CSF-Brain Modeling

27. Compare the Filtered TCV Image (upper left) with the Threshold TCV Image (bottom left) to infer if the calculated threshold is acceptable: CSF is being filtered out and brain matter remains. The yellow line represents the distribution intensity of the Original TCV image. For a FLAIR image, it should have 2 major peaks. The blue curve represents CSF and should contour a major peak of the yellow line. The red curve represents brain matter and should contour the second major peak of the yellow line. The point where the brain matter (red) and CSF (blue) curves intersect is the calculated threshold.

28. CSF-Brain Modeling Use the “>>” button to advance the modeling slice. The “<<” button allows viewing of previous slices, but will discard any changes.

29. CSF-Brain Modeling Occasionally, the computed threshold is unsatisfactory due to lack of information. In that case, the user must manually set the threshold. This can be done by: Clicking “Change Threshold” button Insufficient CSF to model:

30. CSF-Brain Modeling Occasionally, the computed threshold is unsatisfactory due to lack of information. In that case, the user must manually set the threshold. This can be done by: Clicking “Change Threshold” button followed by using the cursor to click at the desired intensity location on the histogram. Ideally, pick a point where the brain matter and CSF curves would intersect. Insufficient CSF to model:

31. CSF-Brain Modeling The filtering program often presents unsatisfactory calculated thresholds on the first and last slices, especially when the CSF curve is absent or is a straight line. Be sure to manually set the threshold. On this slice, the Gaussian CSF curve was calculated incorrectly due to large fluctuations in the distribution intensities of the yellow Original TCV line. The fluctuation changes could be detected by lowering the Estimation Sensitivity (from default 25) at the start of the CSF-Brain Modeling step. Because this occurs on only a few slices, there is no need to exit this step to reset. The threshold can easily be set manually. Manually set the threshold:

32. CSF-Brain Modeling After advancing through all slices, the program will indicate “Insufficient data to model”.

33. CSF-Brain Modeling After advancing through all slices, the program will indicate “Insufficient data to model”. Click “Exit And Save” button to proceed to next stage of the pipeline.

34. WMHI MODELING This section of the tutorial will detail sequential modeling of White Matter Hyperintensities (WMHI).  Quanta2 will calculate intensity histograms of the FLAIR image to determine hyperintensities, e.g. voxels above the user-defined threshold.  The previously traced TCV image (.ima file) will be used. Also, erosion of the created CSF mask is required because FLAIRs have a bright lining, which skews threshold calculations. WMHI Modeling creates a _WM.ROI file.

35. The pipeline dialog will pop up. Click “Continue” to proceed to White Matter Modeling. A selection window resembling the above will appear. Please select the options as shown above for optimal resolution and click “Continue”. The Zmask, conversion of images using another statistical method, is not necessary with FLAIRs. White Matter Modeling

36. The red Gaussian curve generated is used to compute the white matter threshold to 3.5 (default), or user- defined, standard deviations from the mean. Compare the Thresholded Eroded TCV Image (upper left) with the White Matter Image (bottom left) to infer if the calculated threshold is acceptable: noise is being filtered out and white matter remains.

37. White Matter Modeling Although the computed threshold is correct in many cases, noise appears in undesired locations on the image. These erroneous bright signals must be manually removed by: Clicking “Edit WMHI” button. Noise

38. White Matter Modeling Although the computed threshold is correct in many cases, noise appears in undesired locations on the image. These erroneous bright signals must be manually removed by: Clicking “Edit WMHI” button. This brings up a 3-paneled window containing Scratch Pad Image, Modeled Image, and TCV Image.

39. White Matter Modeling Focusing on the Modeled and TCV Images, use the “X” cursor to locate bright signals: Within ventricles Around the outside edge of the brain Along motion lines. These are NOT WMHIs. Holding and dragging the left mouse button over the erroneous signal erases (default). Holding and dragging the right mouse button over the area restores, if you have made a mistake.

40. White Matter Modeling Focusing on the Modeled and TCV Images, use the “X” cursor to locate bright signals: Within ventricles Around the outside edge of the brain Along motion lines. These are NOT WMHIs. Holding and dragging the left mouse button over the erroneous signal erases (default). Holding and dragging the right mouse button over the area restores, if you have made a mistake. Finishing WMHI editing on the slice by clicking “Save and Close Window”, or middle mouse button. Corrected:

41. White Matter Modeling Focusing on the Modeled and TCV Images, use the “X” cursor to locate bright signals: Within ventricles Around the outside edge of the brain Along motion lines. These are NOT WMHIs. Holding and dragging the left mouse button over the erroneous signal erases (default). Holding and dragging the right mouse button over the area restores, if you have made a mistake. Finishing WMHI editing on the slice by clicking “Save and Close Window”, or middle mouse button. Repeat this procedure with every image slice. Corrected:

42. White Matter Modeling The “>>” button will advance you through image slices. The “<<” button will navigate to previous slices, but will discard any changes.

43. White Matter Modeling Due to lack of information, the threshold calculated is not accurate and the user must manually mark the threshold. Use the “Change Threshold” button Ideally, pick a point where the WMHI appears without displaying noise. followed by using the cursor to click at the desired intensity location on the histogram. The program often displays noise on the first and last slices, especially when the red Gaussian curve is not smooth or cannot be calculated from the large intensity differences. Noise

44. White Matter Modeling As an example, the top window displays a manually set threshold intensity that is too low. Tissue other than WMHIs are displaying as bright signal. There is no way to distinguish and erase erroneous WMHI. Use the “Change Threshold” button to reset a higher threshold intensity. The bottom window displays a manually set threshold intensity that is too high. While some WHMIs are displayed as bright signal, much of the tissue is not. This will cause the WM burden to appear lower than it actually is. Use the “Change Threshold” button to reset a lower threshold intensity.

45. White Matter Modeling After changing the threshold, click the “Edit WMHI” button to inspect and erase erroneous signals. Finish WMHI editing by clicking “Save and Close Window”, or middle mouse button.

46. White Matter Modeling Once the last slice is reached, the program will indicate “Last slice in image. You must go back!” or “Insufficient data to model”.

47. White Matter Modeling Once the last slice is reached, the program will indicate “Last slice in image. You must go back!” or “Insufficient data to model”. Click “Save WMHI Array” button. Then, click “Exit and Save” button. Data will be lost if BOTH of these commands are not initiated.

48. The next dialog window will ask if you wish to trace Regions Of Interest (ROIs). If so, click “Continue”. Tracing ROIs will be explained in a separate tutorial. If not, click “Skip Next Step”.

49. Par File After skipping ROI trace, the last step in the pipeline involves writing a Par File. This is a comma-separated text file that stores volume data about the analyzed brain. Click “Write Report to File” button to write all the analyzed volumes (in cubic centimeters) to a.par file. Close the dialog box to return to Quanta2 Main.

50. Par File Reader To access analyzed volume data, the.par file can be read using Parfile Reader This is located under the “Utilities” menu in Quanta2 Main. Click the “Operations” menu to “Open” and select a.par file to read. The information displayed here can be exported to a.csv file, which is compatible with most spreadsheet programs. Click the “Operations” menu to “Export as CSV”. The.csv file will be saved in the same directory as the.par file.