Tracing trees with the user-guided mode(3D)

1. Under Tracing Mode, select User-guided.
   • Maximum process width: As detected by the software.
   • Method: Select one of the three methods (see About the user-guided tracing methods).
2. Optional
   • Select Pan to window center after each click to avoid panning manually (to pan manually, press SHIFT and drag).
   • Uncheck Place ending using right-click: Displays the right-click menu when you right-click.
3. Hover over the area of the process where you want to place the first point. When zoomed in, you see:
   • A red cursor indicating the location of your mouse pointer.
   • A circle indicating targeting (i.e., where the software detected the process); the diameter of the circle corresponds to the detected diameter of the process at that point.
4. Click to place the first point. A sphere represents the first point.
   • To undo the first point (and delete the tree you just started), right-click once.
5. Hover over the process. A series of circles is displayed, representing the path detected by the software.
6. Keep hovering until you reach the end of the process then click to place the last point. The traced branch is displayed in 3D.

   You may need to place several points on the process to improve the accuracy of the tracing.

7. Right-click once to end the branch. If you selected Place ending using right-click, there is no right-click menu.

   When the branch is effectively ended:

   • You can see a sphere at the end of the branch or partial spheres along the branch as you hover.
   • The tree count is updated.

In this example, trace branch 1 (red) from soma to tip, then trace branch 2 (orange), and finally connect 1 and 2.

1. Under Tracing Method, select User-guided.

   Keep Cycle tree colors selected to easily visualize potential connections between branches. This option automatically selects a new color each time you start a new tree.

   Optional: Check centerline all trees when tracing to display the trees already traced as lines.

2. Trace branch 1:
   1. Start at the soma; hover over the area where you want to place the first point.
   2. Click to place the first point.
   3. Hover over the process to see the detected path.
   4. As you reach a bifurcation, ignore it— You will connect the branches in step 3 —and keep hovering until the end of the process.
   5. Click to place the last point.
   6. Right-click once to end branch 1.
3. Trace process 2 and connect to branch 1:
   1. Place the first point at the end of the process then hover over the process until you reach the bifurcation.
   2. At the bifurcation, a partial sphere appears on branch 1 (indicating a potential connecting point) and the series of circles changes color to reflect the color of branch 1.
   3. Hover over the bifurcation area until you see a partial sphere where you want to place the bifurcation point.
   4. 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.

• To abort a tree started independently of an already traced tree, right-click OR press CTRL-Z.
• To abort a tree started from another tree, right-click AND press CTRL-Z.

In this example, the orange branch was traced earlier and you realize that you missed a portion of the process. You resume tracing to include the last portion.

1. Hover over the end of the branch until you see a sphere marking the end.
2. Click the sphere to resume tracing.
3. Click to place the last point and right-click once to end the branch.

Right-click and click the check mark to end the user-guided tracing mode.

There are two aspects you can verify: directionality and node placement.

Directionality

For proper branch ordering, specify the tree origin (directionality):

1. Click the Tree or Edit button to display the Edit Trees panel.
2. Click the Points button.
3. Click the desired origin point on the tree (it must be an end point).
4. Click the Set point as origin button.

Nodes

To view and correct node placement:

1. In the classic window, turn off line thickness. Nodes are displayed as solid circles.
2. In the menu, click Trace>Edit>Select Objects to display the Edit Tool panel.
3. In the Edit Tool panel, check Individual Points.
4. Click the node you want to delete to select it.
5. Right-click and select Eliminate node.
6. Return to the 3D window to resume tracing.

The detection takes into account the outer edges of branches.

The same algorithm was used in AutoNeuron. For details on the algorithm, see Rapid automated three-dimensional tracing of neurons from confocal image stacks (Al-Kofahi, Lasek, Szarowski, Pace, Nagy, Turner, and Roysam, 2002).

The detection is implemented from a branch centerline to its outer edges (manuscript in preparation).

This algorithm generates clusters of voxels iteratively along the dendrite/axon. These clusters are then used to position the nodes that define the centerline of the dendrite/axon.

The same algorithm is used in NeuronStudio (developed by the Mount Sinai School of Medicine). For details on the algorithm, see Three-Dimensional Neuron Tracing by Voxel Scooping (Rodriguez, Ehlenberger, Hof, & Wearne, 2009).