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.

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.

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)
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.
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.