6. 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.

Optional: Add a second counting frame

You can use a second counting frame to mark a population of objects that require different parameters than the objects being counted in the primary counting frame. This allows you mark objects with both sets of parameters in a single probe run which can save time. To use a second counting frame, you CTRL-click left mouse button to count/mark objects. When enabled, you can count a second object in the second frame using middle mouse button CTRL-click.

The second counting frame is useful for counting objects that are different in:

Size: You can set the main frame-size to accommodate 1–5 of the larger cells and add a second, smaller counting frame appropriate for 1–5 of the smaller cells.
Population density: Use the main counting frame for the more sparsely distributed objects and set the second counting frame with an appropriate interval to count the densely distributed objects.

Add Second Counting Frame: Check the box to add a counting frame inside the primary counting frame. The following options for the second counting frame will become available:

Centered check box:
- When checked, the second counting frame is centered in the primary counting frame.
- When unchecked, the second counting frame is positioned at the top left inside the primary counting frame.
Interval: Type in the interval at which you want a second counting frame. For example, enter "1" to include a second counting frame in each primary counting frame or enter "5" to have a second counting frame every fifth primary counting frame.

Choose a radio button to indicate how you want to indicate the size of the second counting frame:

Set CF dimensions: Select this option and text boxes will appear for you to type in the X and Y dimensions of the second counting frame (in µm).
Define as a percentage of primary CF: Select this option and enter a percentage.

To associate a marker with the second counting frame, you must hold the CTRL key down on the keyboard when placing the marker, otherwise the marker will be associated with the first counting frame even if it is contained within the area of the second counting frame.