Physical Fractionator workflow

Purpose

The Physical Fractionator uses a two-stage systematic sampling method to estimate the number of objects in a specified region of an organ. This method combines the disector method with the fractionator sampling method.

Like the Optical Fractionator, the Physical Fractionator is typically used when the population is too large to count exhaustively. Since it relies on the disector method, pairs of thin sections are required.

The disector is a 3D stereological probe with one unique and very important attribute: it samples objects with a probability that is proportional to their number, not their size. Because of this, the disector can be used to count objects without having to make any assumptions about their size, shape, or orientation. In its simplest form, the disector involves the use of two sections, hence the name disector.

The counting rule used with the disector probe is simple. Count the objects that are present on the second section of a pair of adjacent sections that are not present on the first, proceeding systematically through a series of sections that includes the region under consideration. An equally valid counting rule is to count objects that are observable in the first section and not in the second. Essentially you determine whether or not a unique point on each object, the "top," or as in the second case, the "trailing edge," lies within the volume sampled by the disector.

We recommended that you place a reference point in an easy to find and recognizable location before starting this probe workflow. When working with a live camera feed, the placement of the reference point is essential for maintaining accurate alignment between a data file and its corresponding experimental sample when collecting data over multiple sessions. If a reference point has not been placed before opening a workflow, Stereo Investigator will automatically place it in the top left corner of the Main window. This location may not be ideal for resuming data collection in subsequent sessions.

Starting the physical fractionator workflow

  1. Click the Physical Fractionator workflow button on the Probes ribbon.

    If you haven't used the workflow recently, find the Physical Fractionator workflow button in the Number drop-down menu in the All Probes section of the Probes ribbon.

  2. You are prompted to start new subject (Start a new subject) or to continue with a subject (Load subject data from existing file).

    If you select Load subject data from existing file:

    1. Click the Browse button and select the data file.

    2. The workflow directs you to step 10 - Mark cells.

Physical fractionator workflow steps

The workflow is dynamic; your choices determine which steps are included, and what information is requested and displayed. Because of this, the step numbers you see onscreen in Stereo Investigator may be different than those in the complete workflow described here.

Click any of the underlined steps to jump to the instructions:

Indicate the Region(s) of Interest

1. Set up the subject

2. Set system to low magnification

3. Trace your region(s) of interest

4. Set system to high magnification

Define Probe Configuration

5. Define the counting frame size

6. Define SRS grid layout

7. Save sampling parameters

Align and Mark the Cells

8. Acquire alignment images

9. Match tissue sections

10. Mark cells

11. View the results

Commands available in every step of the workflow:

New workflow: Click the new workflow button to start over; the settings will revert to the defaults or those specified in the previous completed workflow.
Previous step / Next Step: Click to advance in the workflow or revisit a previous step. Alternatively, you can click the steps listed at the top of the workflow to jump to that step.
Click the help link to view information in the User Guide on completing the current step in the workflow.

See also Physical Fractionator formulas