Counting rules for the counting frame

See also Counting Rules on stereology.info

Background

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 feature of each object to mark/count and use the counting frame as a guide uniformly across your experiment.

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; it can be anything visible, but there should be just one per object.

When counting cells, many researchers use the top of the cell as the unique point, but the top of the nucleus or nucleolus (provided that the nucleolus is unique to the cell) can be used instead.

Use the counting frame uniformly

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 or crosses both a inclusion line (green) and a exclusion line (red)
• 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 counting frame rules

The unique point must fall within your disector height (shown as green in the Z meter). The rest of the object may be anywhere relative to the counting frame; only the unique point matters when deciding whether or not to mark a object.

• If the unique point comes into focus while in the guard zone (shown as red in the Z meter), do not count it!
  
• When the unique point comes into focus, place a marker on it in accordance with the rules of the counting frame.