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Creating a Global Focus Surface

To create a global focus surface, you must distribute support points across your sample carrier and define their focus position. A focus area across the sample carrier is then interpolated from the values of these reference points.

Distributing Support Points

  1. You have configured the general settings for setting up a tile experiment (experiment created, at least one channel defined, Tiles dimension activated).
  2. To create a global focus surface, you need the Tiles & Positions module.
  3. You are on the Acquisition tab in the Tiles tool.
  1. Open the Sample Carrier section.
  2. Click on the Select... button.
  3. The Select Template dialog opens.
  4. Select the sample carrier template that you want to use.
  5. Click on Options and select Copy And Edit....
  6. A copy of the existing template is generated and opened in the sample carrier editor.
  7. To distribute support points across the sample carrier template, open the Global Support Points section.
  8. Select the containers in which you want to create global support points. To do this, press the Ctrl key and click on the containers.
  9. Click on the Distribute One Support Point For Each Selected Container button.
  10. One support point is assigned to each selected container.

  11. If you use a sample carrier without containers (e.g. slide), use the Distribute button instead, to distribute support points on the basis of columns and rows.

  12. The support points are distributed automatically across the sample carrier.
  13. You can add further support points manually using the Add button .

  14. Only create support points where you can bring the sample into focus (within the containers). This is the only way that you can determine and setup the Z-values of the support points later. The assignment of container-based global support points to the center of the container is fixed i.e. they cannot be moved. If the surface of your sample carrier is tilted but even, you will need at least 4 support points for a solid calculation. The more irregular the surface, the more support points you should distribute.

  15. To close the Editor window, click on OK.
  16. To select the edited sample carrier template, click on OK.
  17. If you wish to re-edit the global support points at any time, click on the Edit icon in the sample carrier section to open the sample carrier editor again.
  18. To calibrate the sample carrier, click on the Calibrate button and follow the wizard.
  1. You have successfully distributed support points across a sample carrier template and have selected and calibrated it.

Verifying Z-Values of Support Points

  1. In the Tiles tool open the Focus Surface and Support Points section.
  2. Go to the Global (on Carrier) tab.
  3. All the support points of the selected sample carrier template are displayed in the Support Points list.
  4. Click on the Verify button.
  5. The Verify Global Support Points dialog opens.
  6. Select the Helper Method you want to use. This will support you in determining the z-values. The options are Autofocus (AF) and Definite Focus (DF). If you have neither then you can only adjust z-values manually.
  7. Click on Move To Current Point.
  8. The stage automatically moves to the support point that is highlighted in the list. Alternatively, you can also double-click on the support point in the list.
  9. In the Live mode use the Focus (or SW Autofocus) tool to set the z-value.
  10. Click on Set Z and Move to Next.
  11. The support point is marked with a check mark.
  12. The stage automatically moves to the next support point in the list.
  13. Repeat the last 3 steps until you have checked all the support points.
  14. The message All points have been verified appears.
  15. Close the Verify Global Support Points dialog.
  1. You have adjusted and verified the Z-values of all support points.

Selecting Interpolation Degree

  1. Select the interpolation degree in the Interpolation Degree dropdown list in the Focus Surface and Support Points section.
  1. You have successfully created a global focus surface.
  2. You can now set up your tile experiment using the sample carrier. Further information on this can be found under: Using Sample Carriers. To ensure the tiles are acquired along the focus surface during the experiment the software automatically selects the most appropriate focus strategy in the Focus Strategy tool. For information on focus strategies read the chapter Working with Focus Strategies.

The minimum number of support points necessary is indicated in the Interpolation Degree dropdown list for each entry. The calculation is more solid if the number of support points exceeds this minimum number. We therefore recommend that you only increase the interpolation degree as far as the surface of the carrier demands, even if you have created more support points. If the number of support points does not correspond to the minimum number for the selected interpolation degree, the interpolation degree will be reduced automatically. Interpolation degree1 – Tilted Plane (at least 4 support points) is typically sufficient to compensate for any tilting of the sample carrier.

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