Counting rules for Optical Fractionator

Purpose

To obtain accurate results in stereology, it is vitally important that you mark objects to be counted in a defined, consistent way (note that stereologists use the generic term ‘particles’ to describe objects being counted). Choose the same unique point of each object to mark/count and use the counting frame as a guide uniformly across your experiment.

See also Counting Rules on stereology.info

Count a unique point in each object

Identify a unique point that is present in each object (e.g., cell) that you want to count.

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, 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.

Stereo Investigator uses the centroid of detected objects as the unique point. Since it is impossible to visually determine where the centroid of objects is, you are given the choice of 3 different unique points for marking cells during the Validation workflow.

Counting rules

X- and Y-orientation counting frame rules

DO NOT MARK your unique point if any area of it:

  • Touches the red exclusion line, or is outside of the counting frame.

  • Touches both a inclusion line (green) and a exclusion line (red) at the same time.

  • Comes into focus outside of the disector height.

MARK your unique point if:

  • Any area of the unique point is touching a green inclusion line AND it does not cross an exclusion line.

  • The entirety of the unique point is inside the counting frame.

Z-orientation disector rules

DO NOT MARK your unique point if any area of it:

  • Comes into focus outside of the disector height.

  • If the unique point comes into focus while in the guard zone (shown as red in the Z meter).

MARK your unique point if:

  • The unique point comes into focus within your disector height (shown as green in the Z meter) and is in accordance with the counting frame rules.