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Spaceballs step 9: Count objects

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

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 counting button. The workflow displays new options for counting.
   Focus and mark...

   1. The stage moves to the first counting site and, if you selected Refocus to top of section at each grid site in step 5, the Focus Top Of Section window is displayed.
   1. Focus above the top of the tissue until it is completely out of focus.
   2. Slowly focus back down onto the tissue until something on the tissue (e.g., a cell or interstitial components) comes into focus; this is the top of the section.
   3. Click OK.
   2. The Focus Bottom Of Section window is displayed.
      1. Focus all the way down through and past the bottom of the tissue until it is completely out of focus.
      2. Slowly focus back up until something just comes into focus. This is the bottom of the tissue (use the Z-meter to help you determine the direction that you are focusing.
      3. Click OK.

         Determining what point is the top and what point is the bottom of the tissue varies from one researcher to another. This is not an issue as long as you are the only one counting a particular experiment according to consistent criteria.

   3. 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 type from the Use marker drop-down menu.
      2. Only mark the circle in the center, as the other sites will be systematically visited.
   4. Select a marker type to count transections.
   5. 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 (shown as green in the Z meter).
      • You are following the rules of the counting frame discussed in the Spaceballs options step.

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

   6. 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).

   7. Right-click and select Next Scan Site.
4. Once all the sites have been visited, click Add new section or Begin next section in the workflow below the blue arrow button. The workflow redirects you to the Set microscope to low magnification step for your next section.
   • Follow the workflow down to the Counting Objects step again.
5. Continue marking each section and adding new sections until you have completed all the sections in this particular animal.

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 very time it enters or leaves the spaceball.
• If the center line doesn't enter the spaceball, don’t mark it.

One animal = 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 an animal are traced and counted will allow these sections to be saved as one file.

View sampling results

See Additional commands to manage the probe runs for additional options, or Spaceballs Workflow

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