Petrimetrics

We frequently receive questions about the “correct” or “recommended” settings for grids, counting frames, and probe layouts. The short answer is that there are no “golden numbers” to use when creating your study. Remember that your goal is not to count every particle or measure every fiber—you are sampling to get an estimate. Here are some questions to ask when starting:

How precise should my estimate be? If your fiber length is uniformly long, parallel, and evenly spaced, you could go with fewer sample sites, a larger Merz radius, and a larger counting frame and you would get a relatively precise estimate. Most samples are not uniform and homogenous, however. Less homogenous samples lead to 'intersects' less predictably oriented in space; as a result, more sampling for your study is required to produce acceptable results. More sampling means more sampling sites, a smaller Merz radius, and/or larger counting frames.

Are there any guidelines to get me started? We recommend at least ten counting frames per region of interest, and aim for 200 intersections per petri dish. Remember that these are just general guidelines to get you started.

Is there a Coefficient of Error (CE) I can use? No, there is no CE for the Petrimetrics probe.

1. Load your image or work with a live image.
2. Trace your region of interest at low magnification.
3. Switch to high magnification (the power at which you will mark the intersections).
4. Use Probes>Tools>Define Counting Frame.
5. Set up the systematic random sampling with Probes>Tools>Preview SRS Layout; exit Preview SRS Layout.
6. Click Probes>All probes>Length>Petrimetrics; the program displays a warning dialog box about the Serial Section Manager.
   1. Click No since you are working on a 2D sample; the Petrimetrics dialog box opens .
   2. XY Placement of Counting Frames is based on the SRS Layout.
   3. Adjust the Merz Radius: A larger number results in larger curvilinear lines spaced farther apart; a smaller number results in smaller curvilinear lines spaced closer together, as seen here:
      

      
      Example -- Left: Merz radius = 30µm | Right: Merz radius = 10µm.

   4. Optional: Type a value for Grid Rotation select Randomize Rotation.
7. Select a marker to use for counting.
8. Place a marker wherever the test line of the Merz lines cross a fiber or blood vessel center line, or any other object for which you are estimating length.
   • When you are finished with the first site, right-click and select Next Scan Site, and place markers at the next site on all intersections of the probe with a feature.
   • Continue until all sites have been visited.
9. Click Probes>Stereology results>Probe run list for results.

See Petrimetrics