Tracing vessels using automatic mode (3D)

Available with the Neurolucida 360 Ultra package only. Click here for more information.

Procedure overview

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

  • For a tracing using the defaults, click the Trace button.
  • For a more accurate or faster tracing, adjust the settings first, then click the Trace button. The settings give you the opportunity to review seed placement, which is used to initiate the automatic tracing.

Notes

  • To continue incomplete automatic tracings, you can use the user-guided tracing mode.

  • 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

  • Open an image/image stack.
  • Make image adjustments as necessary.
  • If you want to associate vessels 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.
  • If you're using the 3D Volume view, select Partial projection under Image stack settings in the Change image display panel.

Enable automatic vessel tracing mode and select a tracing method

  1. Click the Vessel button to display the Trace Vessels panel and select Automatic as the Tracing Mode.

  2. Select a tracing method:

    • Directional Kernels: With this method, four directional kernels are matched to the image data. For a given point within the vessel, 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 vessel. 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 vessel. These clusters are then used to position the nodes that define the centerline of the vessel. 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 with default settings

Click the Trace button.

  • Neurolucida 360 automatically detects and traces vessels.

  • 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.

For larger diameter vessels, you may need to manually adjust the Typical process width in the User-guided tracing options. This setting is applied to automatic tracing.

Tracing vessels with adjusted settings

  1. Click Show settings.
  2. In the Display Seeds tab, click the Display Seeds button.
    • Optional: 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 vessel sizes. In this case, enter the typical width of the larger vessels.
  3. Click the Refine Seeds tab.
    1. Click the validate Seeds button. The program applies an algorithm to evaluate seed placement.
    2. Optional: To decrease the number of unwanted seeds, increase the Refine filter value.
    3. Optional: Add or remove seeds manually.
      • To add a seed, click in the image.
      • To change the color of manually added seeds, use the color picker.
      1. Select the Remove radio button.
      2. Hover the mouse over a vessel to see the circular cursor.
      3. Hold down CTRL and scroll the mouse wheel to resize the cursor.
      4. 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.
    • Gap tolerance: Use the slider to change the maximum acceptable distance between two segments to make a connection.
    • Remove traces shorter than: Enter a value to ignore objects that may be mistaken for traces in a noisy image.
    • Connect Vessel segments: Check to have the program connect vessels according to the criteria defined in the Advanced Settings.

  5. Click the Trace button to start the automatic 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.

 

Also see Tracing with the user-guided mode(3D)