Tracing trees in smart manual mode (3D)

• Snap cursor to tree: Check the box to enable this feature, which:

  • Corrects for lack of precision with mouse clicks.

  • Enables closing a loop while tracing.

  • Lets you resume tracing from an ending by clicking the ending.

• Cycle tree colors: A new color is used every time you start a new tree.

• Pan to window after each click: Each point you click that falls outside the field of view is re-centered automatically.

• Display other trees as centerline: Displays trees that are already traced as lines (instead of showing thickness).

1. Hover over the starting point of the process and, if needed, change the size of the red cursor to match the thickness of the process by holding the Ctrl key and scrolling the mouse wheel. Unlike in 2D tracing, there is no need to focus
2. Click to place the starting point. A sphere represents the starting point.
3. Hover over the next point, adjust the cursor size if needed, and click to place the next point.

   To undo the last click, press CTRL-Z.

4. Repeat step 3 for each point.
5. Click to place the last point.
6. Right-click and select Ending to end the tree.

For example, trace branch 1 (green), then trace branch 2 (blue), and finally connect 1 and 2.

1. Under Tracing Options, click the box for Snap cursor to tree to enable the option.
2. We recommend that you also enable the Cycle tree colors and Display other trees as centerline options.
3. Trace branch 1:
   1. Hover over one end of the process and, if needed, change the size of the red cursor to match its thickness by holding the Ctrl key and scrolling the mouse wheel.
   2. Click to place the first point.
   3. Hover over the next point, adjust the cursor size if needed, and click to place the point. Repeat for each point until you reach the bifurcation.

      To undo the last click, press CTRL-Z.

   4. As you reach the bifurcation, ignore it— You will connect the branches in step 4.c —and keep placing points until the process meets the soma.
   5. Click to place the last point.
   6. Right-click and select Ending from the menu.
4. Trace branch 2 and connect to branch 1:
   1. Start from the distal end of the process and place points until you reach the bifurcation.
   2. At the bifurcation, hover over branch 1. The open circle becomes solid, indicating a potential connecting point.
   3. Click to connect branch 1 and branch 2.
      • Notice that the branches are now the same color.
      • There is no need to end branch 2. It is automatically done by connecting to branch 1.

1. Under Tracing Options, we recommend that you click the boxes to enable the Cycle tree colors and Display other trees as centerline options.
2. Trace branch 1:
   1. Hover over one end of the process and, if needed, change the size of the red cursor to match its thickness by holding the Ctrl key and scrolling the mouse wheel.
   2. Click to place the first point.
   3. Hover over the next point, adjust the cursor size, and click to place the point. Repeat for each point until you reach the bifurcation.

      To undo the last click, press CTRL-Z.

   4. At the bifurcation, right-click and select Bifurcating Node from the menu.
   5. Continue to place points until you reach the end of the process.
   6. Click to place the last point then right-click and select Ending to end branch 1. The software redirects you to the bifurcating node from which you can start tracing (when you move the mouse, you see a rubber-band line between the cursor and the node).
3. Trace branch 2:
   1. From the bifurcating node, adjust the cursor size to the process thickness and click to place the first point of branch 2.
   2. Hover over the next point, adjust the cursor size, and click to place the point. Repeat for each point until you reach the outer edge of the process.
   3. Click to place the last point then right-click and select Ending to branch 2. Branches 1 and 2 are now connected.

Resume tracing if you have missed a portion of a branch and want to include it in your analysis.

1. Under Tracing Options, click the box for Snap cursor to tree to enable the option.
2. Hover over the end of the process until you see a sphere marking the end.
3. Click the sphere to resume tracing.
4. Click to place the last point.
5. Right-click and select Ending to end the branch.