Connectivity Assay >

10. Mark tissue

In this step, you can compare the reference images to the lookup images and mark particles that appear in the reference image but not in the lookup image.

Procedure

  1. Select the contour and probe run.
  2. Select continuing the probe run from the Mark by drop-down menu.

    To audit a .dat file that is already marked at the beginning of this workflow, select Mark by: Duplicating probe run for auditing from the Mark By drop-down menu.

    • The program revisits all the images, but doesn't display the markers.
    • Sampling displays the set of parameters in use (parameter sets can be saved at step 1).

  3. Click the Start counting button to start marking.

  4. Follow the instructions for your image source (selected at the start of the workflow).

    Under Show Advanced Features, use Categorize to keep track of the probe runs in the Results when you view them on the probe run list.

    1. Drag the live image of the reference section to align it with the lookup image.
    2. Refine the alignment by using the commands under Image Controls (transparency, move image buttons).

      Use the Reset button to undo and the Use Last Alignment button to apply the alignment from the previous site on that section.

    3. Once the reference image is aligned with the live lookup image, click the Begin Placing Markers button.
      1. Select a marker type.
      2. The counting frame is displayed. Place markers.

        3. If, at any time during the marking process, you are not satisfied with your alignment, click Align Reference Image with Live Image to make adjustments.

    If you see a cross section of a particle in the reference image, but not in the lookup image, then the leading edge of the particle is present between the two images and the cell should be marked.
    If you see a cross section of a particle in both the reference and the lookup image, then the leading edge of the particle is not present and the cell should not be counted.

    1. Use the button or press F2 to move to the next site. The counting frame is displayed.
    2. Click Begin placing markers and place markers.
    3. Repeat steps 3-4 for all the sites.
    4. Save the data file.
    5. If this is not the last section pair, click Add new section pair.
      • You will be directed to step 1 in the workflow.
      • Proceed as for the last section. Notice that steps 5-8 have been collapsed. This is because we cannot change these parameters from section-pair to section-pair within the same animal.
    6. When you have finished the last section-pair for this region in this subject, click I've finished marking. You are directed to step 11: View the results.

    At high power, align the reference image with the lookup image at your current site. Then mark the cells that appear in the reference image but not in the lookup image.

    1. Drag the reference section to align it with the lookup image.
    2. Refine the alignment by using the commands under Image Controls (transparency, move image buttons, rotate buttons).
      • Use the Reset button to undo and the Use Last Alignment button to apply the alignment from the previous site on that section.
    3. Once the reference image is aligned with the lookup image, click the Begin Placing Markers button.
      1. Select a marker type.
      2. The counting frame is displayed. Place markers in the site.

        3. If, at any time during the marking process, you are not satisfied with your alignment, click Align Reference Image with Live Image to make adjustments.

    If you see a cross section of a particle in the reference image, but not in the lookup image, it means that the leading edge of the particle is present between the two images and the cell should be marked.
    If you see a cross section of a particle in both the reference and the look-up image, it means the leading edge of the particle is not present and the cell should not be counted. If it is you would overestimate the number.

    1. Use the button or press F2 to move to the next site.
    2. Click Begin placing markers and place markers. The counting frame is displayed.
    3. Repeat steps 3-4 for all the sites.
    4. Save the data file.
    5. If this is not the last section pair, click Add new section pair.
      • You will be directed to step 1 in the workflow.
      • Proceed as for the last section. Notice that steps 5-8 have been collapsed. This is because we cannot change these parameters from section-pair to section-pair within the same animal.
    6. When you have finished the last section-pair for this region in this subject, click I've finished marking. You are directed to step 11: View the results.