Image Volume Fractionator workflow >

7. Count objects

Procedure

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

  1. Click the name of a section to select it. All contours within the section are listed below it.

  2. Click the name of a contour to select it for counting.

  3. Click Start counting. The workflow displays new options for counting and the first counting site is displayed.

  4. Start counting in the first site.

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

    2. Optional: Check the Enable middle mouse button for placing markers box to mark two populations simultaneously with identical sampling parameters (use for population estimates of two similarly frequent populations).

      • If you check the box to Enable middle mouse button for placing markers, select a different marker from the Middle Mouse Button Marker drop-down menu.

    3. Focus through the section to find the unique point of the particle. If you focus above or below the disector (i.e., outside of the green area displayed in the Z meter), the cursor changes to indicate that you're outside of the appropriate area.

    4. When the unique point comes into focus, click to place a marker on it in accordance with the rules of the counting frame. See "Counting Rules"

    Many researchers are counting cells and use the cell top as the unique point to count, but the top of the nucleus or nucleolus (provided that the nucleolus is unique to the cell) is a good alternative. The unique point can be anything visible, but there should be just one per object. The unique point must fall within the counting frame, or touching the green inclusion line ( see "Counting Rules"), or within the disector height (shown as green in the z meter). The rest of the object you are counting may be anywhere relative to the counting frame and top and bottom of the disector. Only the location of the unique point matters when deciding whether or not to mark an object as counted.

    1. Repeat steps c-d for the other objects that can be counted in the site.

    2. Once you've marked all the objects in the site, click next to move to the next counting site.Repeat steps 3-4 for all sites in a section.

  5. Once all the sites have been visited, the probe run is displayed under Regions of Interest .

  6. Click Begin Next Section. If no contour has been traced in this section, the workflow redirects you to the Trace your regions of interest step for your next section.

  7. Follow the workflow to the Count Objects step again.

  8. Continue counting in each section until you have completed all the sections in the subject.

  9. When complete, click I've Finished Counting.