Image Volume Fractionator workflow >

4. Define the counting frame size

Procedure

Counting Frame Display

Force the counting frame to be square: Check the box to create a square counting frame as you adjust the frame with your mouse.

Snap to increments of: Check the box and enter the desired rounding increment in µm to round the size of the counting frame to a whole number; this may be easier to remember for future experiments.

Counting Frame Size

1. Choose a unique identifying point for your object of interest (e.g., cell) that comes into focus just once, such as:
   • Cell top
   • Nucleus top
   • Nucleolus (unless there are multiple nucleoli in your cells of interest)

2. Adjust the counting frame until it is approximately large enough to have, on average, 1 to 5 identifying points that you will count using one of the following methods:

   • Type a size (µm) into the boxes labeled X: and Y:

   • Use your mouse to adjust the counting frame size by dragging the corners or edges.

   We recommend sizing counting frames so that they contain 1–5 objects. This minimizes user error and fatigue; it is difficult to count too many objects per frame, especially in a visually dense image.

3. Verify the following:

   • The counting frame is roughly in the center of the screen.

     • Hover your cursor over the counting frame and drag the mouse to move the frame.

   • There is adequate space outside the counting frame to clearly distinguish objects that are on the edge of the counting frame.
   • The tails of the counting frame are visible.