Tracing trees in user-guided mode(3D)

• Snap cursor to tree: [Selected by default in User-guided mode]

• 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).

• Typical process width: Optional setting to assist with differentiation of soma from processes; this can be especially useful when staining/fluorescence is uneven along a branch.

• Method: You can use any of the included tracing methods. The tracing methods are based on algorithms that work by tracing along the trees one point at a time, and by producing three measurements at each point: (X,Y,Z) coordinate, thickness, estimated position of the next point.

  Click to expand and learn about the different methods.

  • Directional Kernels: With this method, four directional kernels are matched to the image data. For a given point within the tree, the algorithm identifies the best positions and orientations for the top, bottom, left, and right kernels surrounding the point. The positions and orientations results are combined to estimate the next point to trace. Points are estimated until a set of stopping criteria is met.

    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).

  • Rayburst Crawl: This method performs rayburst sampling measurements to obtain the diameter and centroid of the cross-section of the tree. Successive measurements are used to position the nodes that define its centerline. Points are estimated until a set of stopping criteria is met.

    For details on the rayburst sampling algorithm see Rayburst sampling, an algorithm for automated three-dimensional shape analysis from laser scanning microscopy images (Rodriguez, Ehlenberger, Hof, & Wearne, 2006).

  • Voxel Scooping: This algorithm generates clusters of voxels iteratively along the tree. These clusters are then used to position the nodes that define the centerline of the tree. As in the other methods, points are estimated until a set of stopping criteria is met.

    For details on the algorithm, see Three-Dimensional Neuron Tracing by Voxel Scooping (Rodriguez, Ehlenberger, Hof, & Wearne, 2009).

  • CNN Sectioning: This algorithm builds on the Rayburst Crawl method by making use of a pre-trained Convolutional Neural Network (CNN) to dynamically segment the local image cross-section before performing each Rayburst Sampling measurement. This segmentation step makes it possible for the algorithm to correctly estimate the diameter and centroid of hollow trees.

• Place ending using right-click:

  • When the checkbox is clear, a menu with choices will be displayed when you right click.

  • When the box is checked, right clicking ends the tree and no menu is displayed. If only the first point of a tree has been entered, right clicking will undo the first point and delete the tree you just started.

1. 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.
2. Click to place the first point. A sphere represents the first point.
3. Hover over the process. A series of circles is displayed, representing the path detected by the software. Click to trace that section.
4. Keep hovering and clicking 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.

5. Right-click to end the branch.

   If you selected Place ending using right-click, the branch will be terminated when you right click; if that option is not selected, choose Ending from the 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. Trace branch 1:
   1. Start at the soma; hover over the area where you want to place the first point, then click to place it.
   2. Hover over the process to see the detected path and click to trace it. Continue hovering and clicking to trace the branch.
   3. As you reach a bifurcation, choose one path and keep clicking and hovering until the end of the process. (You will connect the branches in the next step.)
   4. Click to place the last point.
   5. Right-click to end branch 1. If you selected Place ending using right-click, the branch will be terminated when you right click; if that option is not selected, choose Ending from the right-click menu.
2. Trace process 2 and connect to branch 1:
   1. Place the first point at the distal end of the process then hover over the process until you reach the bifurcation.
   2. At the bifurcation, a solid 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. Click where you want to place the bifurcation point 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.

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

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 to end the branch.

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

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.

1. In the 2D 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.