SRS image stack workflow > Acquire Configuration >

6. Counting frame settings

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.

Optional: Add a second counting frame

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.

You can count two objects per counting frame using an ordinary mouse-click for one object and middle mouse button-click to count a second object (see Count objects).

. 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.

To use a second counting frame, you CTRL-click to count/mark objects. When enabled, you can count a second object in the second frame using middle mouse button CTRL-click.

Counting Frame Display

  • Constrain the counting frame shape to a square, and/or snap according to increments of your choice.

Counting Frame Size

  • Manually enter the counting frame dimensions.

or

  • Resize the frame displayed on the screen.
    • Drag the cursor until the counting frame is the desired size.
    • Make size adjustments by dragging out any of the counting frame's corners or sides.

 

  • To center the frame, place the cursor in the center of the frame and drag it to the middle of the window.
  • To start again, click Undo My Changes.