Image Volume Spaceballs workflow >

6. Count objects

Procedure

The workflow displays a list of the sections and contours that were drawn for each section. Follow this procedure:

  1. Click the name of a contour from the list to select it.
  2. Optional: Confirm the sampling parameters to be used with Sampling.

  3. Click the Start Counting button. The first counting site is displayed. Focus and mark objects of interest as follows:

    1. Focus down until a small circle is visible in the center of the field of view; if the grid spacing is small, multiple circles may be visible.

      1. Select a marker from the Use marker drop-down menu.

      2. Mark only the circle in the center, as the other sites will be systematically visited.

    2. Select a marker type and count transections.

      1. Select a marker from the Use marker drop-down menu.

      2. Place a marker at each location where a fiber crosses the currently visible circle (shown as green in the Z meter) if the transection is within the disector height/sphere radius .

        If this point of a fiber comes into focus while in the guard zone (shown as red in the Z meter), don't count it (Guard zones).

    3. Focus through the section, marking each intersection between a fiber and the sphere, until all intersections have been counted; the circle will appear larger in each subsequent focal plane to represent the sphere or hemisphere.

      If you are using hemispheres and the current focal plane is near the equator of a hemisphere, the marker is counted as 1/2 (see Markers counted as half counts under Sampling results).

    4. Right-click and select Next Scan Site and count in the next site.

  4. Once all the sites have been visited, click Add new section or Begin next section. The workflow redirects you to the Trace your regions of interest step for your next section.

    Follow the workflow to the Count Objects step again.

  5. Continue marking each section and adding new sections until you have completed all the sections in the specimen.

    When complete, click I've finished counting.

Edge cases

For vessels that appear on the edge of the spaceball, use the “center line” rule:

  • If the center line enters and then leaves the probe, mark it twice (at the entrance point and at the exit point).
  • If the center line weaves in and out, mark the centerline every time it enters or leaves the spaceball.
  • If the center line doesn't enter the spaceball, don’t mark it.

One specimen = one file. DO NOT save each section as a new file. By using the workflow to add new sections, as described above, until all the sections in a specimen are traced and counted, you will save all these sections in one file.