This guide describes how to trace neurons using semi-automated filament tracing. This uses the AutoPathalgorithm (used in the filament tracing wizard) to automatically computes all the paths from a user-defined starting point to the end of the structure. After all possible paths are calculated by the algorithm, the filaments are traced by the user by moving the mouse over the structure of interest.

1. First add a filament to the surpass scene:

2. Choose Skip automatic creation, edit manually:

3. Select the AutoPath method radio button and set the diameter of the object being traced:

4. Choose a starting point (e.g. the cell body). Ensure that the Pointer type is Select under the Camera navigation panel. Use the mouse wheel to make the cube region of interest indicator roughly the same size as the cell body. Select the cell body as the starting point by holding the shift key on the keyboard and pressing the right mouse button.

5. Imaris now does some calculations that may take a minute or so depending on the complexity of the image.

6. Move the mouse over the image to trace the dendrite of interest. Imaris will automatically trace the object based on the earlier calculations.

7. Move the mouse to the end of the object to be traced. Pressing the shift key plus clicking the left mouse button permanently draws the traced path. Use the mouse wheel to change the size of the region of interest to be roughly the same size as the object being traced. This will help in tracing the correct path.

8. Mistakes can be corrected using AutoDepth. It is also possible to use AutoDepth to trace the neuron in its entirety.  In Autodepth mode the x-y position is traced by the user while the depth is calculated by Imaris. Hold the shift button and left click to place a starting point. While holding shift down click again, this time dragging the mouse along the process you wish to trace.

9. For the most difficult tracing it is possible to use the manual mode. When using the manual tracing mode select between YZ, XZ and XY planes using the radio buttons:

 

When using manual tracing click the right mouse button cycles through the three planes. The plane position can be adjusted using the slider labelled position. The controls in manual mode are as follows:

  • Left-click plane & drag: Change plane position perpendicular to its orientation
  • Shift + left-click: Set a filament vertex
  • Shift + left-click plane & drag: Draw a filament
  • Right-click plane: Change the plane orientation (from XY to YZ to XZ)
  • Mouse wheel: Change the “pencil” diameter for drawing the filament
  • PageUp: Increase the “pencil” diameter for drawing the filament
  • PageDown: Decrease the “pencil” diameter for drawing the filament
  • Num plus: Increase the extended section (of the drawing plane)
  • Num minus: Decrease the extended section (of the drawing plane)
  • Arrow up: Change slice position up
  • Arrow down: Change slice position down
  • X: Set the plane orientation to YZ
  • Y: Set the plane orientation to XZ
  • Z: Set the plane orientation to XY