Tracing trees with the smart manual mode(3D)

1. Optional: Under Tracing Options, check Snap cursor to tree.
   • Useful to easily connect branches while tracing (See Tracing a bifurcating tree—Method 1).
   • Two cursors are visible: the red cursor (crosshair in a circle) indicates the location of your mouse pointer; the circle indicates targeting (i.e., where the software is detecting the process).
2. Optional: Under Tracing Options, check Pan to window center to avoid manual panning when zoomed in.

   To pan manually, press SHIFT and drag.

3. Zoom in by scrolling the mouse wheel until the magnification is suitable for manual tracing.
4. Hover over the starting point of the process and record the thickness. Unlike 2D tracing, there is no need to focus.

    To adjust thickness, hold the CTRL key and scroll the mouse wheel until the circle of the red cursor matches the apparent process diameter.

5. Click to place the starting point. A sphere represents the starting point.
6. Hover over the next point, adjust the thickness, and click to place the next point.

   To undo the last click, press CTRL-Z.

7. Repeat step 6 for each point.
8. Click to place the last point.
9. Right-click and select Ending to end the tree.

In this example, trace process 1 (green), then trace process 2 (blue), and finally connect 1 and 2.

1. Under Tracing Options, Cycle tree colors and centerline all trees when tracing.
   • Cycle tree colors automatically selects a new color each time you start a new tree.
   • centerline all trees when tracing displays the trees already traced as lines.
2. Trace branch 1:
   1. Hover over the area at the end of the process where you want to place the first point.
   2. Adjust the thickness and click to place the first point.
   3. Hover over the next point, adjust the thickness, 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 3-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.
3. Trace branch 2 and connect to branch 1:
   1. Start from the outer edge of the process and place points until you reach the bifurcation.
   2. At the bifurcation, hover over branch 1. A partial sphere (purple) appears on branch 1, 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. Zoom and pan as needed.
2. Trace branch 1:
   1. Hover over the area at the end of the process where you want to place the first point.
   2. Adjust the thickness and click to place the first point.
   3. Hover over the next point, adjust the thickness, 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 program re-directs 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 node, adjust the thickness and click to place the first point of branch 2.
   2. Hover over the next point, adjust the thickness, 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. Branch 1 and 2 are now connected.

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.
4. Right-click and select Ending to end the branch.

Right-click and select User-guided tracing.