Tracing trees in automatic mode (3D)

Procedure overview

There are two ways to implement automatic tree detection/tracing:

  • To use the default settings, click the Trace button.
  • For more control, adjust the settings first (see description below), then click the Trace button.

Notes

  • To process multiple files with the configuration used for a representative image stack, save your 3D Settings (learn more here) for later use. Use the batch trace function available in the Batch pipeline to further expedite your work.

  • When working with multiple images, make sure that the image you want to use for tracing is checked in the Image Organizer.

Before you start

Open and adjust the image/image stack

  1. Open an image/image stack.

  2. Make image adjustments as necessary.

  3. Detect somas.

  4. If you want to associate trees with a specific color channel, select a single channel using either the Channel panel on the left side of the 3D Environment or the Image Adjustment tool (on the Image and Workspace ribbons) in the 2D window.

Enable automatic tree tracing mode and select a tracing method

  2. Click Tree to display the Trace Trees panel and select Automatic as the Tracing Mode.

  3. Select a tracing method:

    • 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).

Tracing trees with default settings

Click the Trace button.

  • Neurolucida 360 automatically detects and traces trees.

  • If the results do not meet your expectations, click the Clear Tracing button to discard the tracing. You can then trace again using a different tracing method or using adjusted settings as described below.

Tracing trees with adjusted settings

  1. Click the Show settings button.

  2. In the Display Seeds tab, click the Display Seeds button.

    • To change the seed color, use the color picker next to the Hide Seeds button.

    • To adjust the number of seeds, use the Sensitivity slider and the Density buttons.

    • Typical process width: This setting helps guide the algorithm during tracing and is especially helpful when there is a large range in process sizes. In this case, enter the typical width of the larger processes.

  3. Click the Refine Seeds tab.

    1. Click the validate Seeds button. The program applies an algorithm to evaluate seed placement.

    2. To decrease the number of unwanted seeds, increase the Refine filter value .

    3. Add or remove seeds manually.

      • To change the color of manually added seeds, use the color picker.
      • To add a seed, click in the image.
      1. Select the Remove seeds within circular cursor radio button.
      2. If needed, change the size of the cursor to by holding the Ctrl key and scrolling the mouse wheel.
      3. Click in the image to remove seeds within the cursor radius OR hold down CTRL and drag to remove contiguous seeds.

  4. Click the Trace tab to modify the tracing settings.

    • Sensitivity: Use the slider or type a value to adjust sampling density. Too high a value can generate tracings based on nonspecific staining/fluorescence; too low can cause tracing to stop prematurely.
    • Gap tolerance: Use the slider to change the maximum acceptable distance between two segments to make a connection.
    • Remove traces shorter than: Check the box and enter a value to ignore objects that may be mistaken for traces in a noisy image.
    • Connect branch segments: Check to have the program connect branches according to the Branch Connections Criteria defined in the Advanced Settings (see details in Advanced settings for automatic tracing (3D)).

  5. Click the Trace button to start tracing.

  6. To change the tracing settings, click the Clear Tracing button, readjust the settings, and click Trace.

  7. (optional) Click the 3D Settings button to save your settings for later use. Learn more here.

 

See also Tracing trees in Smart manual mode(3D), Tracing trees with the user-guided mode(3D), Editing trees (3D)