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.

Center on live image: If you're working on a live-camera image, you can check the box to center the counting frame in the on-screen image (rather than positioning the counting frame using your mouse).

Counting Frame Size

Size the counting frame to fit approximately 1–5 objects of interest.

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.

   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.

     • If you're working on a live-camera image, you can check the box to "Center on live image".

     • 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.