To create local focus surfaces, you must distribute support points across your tile regions and assign their focus position. Tile-region-specific focus values are then interpolated to generate a focus surface that approximates the topology of the area you want to image.
Creating a Local Focus Surface
Distributing Support Points
- To create a local focus surface you need the Tiles & Positions module.
- You have set up a Tiles experiment with at least one tile region.
- You are on the Acquisition tab in the Tiles tool.
- Click Show Viewer.
- The advanced tile setup opens.
- Select a tile region for which you want to create support points. To do this, click on the corresponding tile region in the list in the Tile Regions section of the Tiles tool. Alternatively, you can select tile regions by clicking directly on the desired tile region in the Advanced Setup view. Both methods allow you to select several tile regions when pressing the Ctrl key.
- Open the Focus Surface and Support Points section in the Tiles tool.
- To add a single support point at the current stage position, click Current Position. Alternatively, you can add a single support point to the center of the currently selected tile region by clicking Center of Tile Region.
- Under Add Multiple Support Points, you have the settings to add multiple support points. Indicate the number of Columns and Rows for the distribution of the reference points. Alternatively, recommended for larger tile regions (>200 tiles), you can use the distribution method Onion Skin. Depending on the total size and shape you might need to adjust the density parameter and/ or the maximum number of support points to optimize the result. Typically, this method works best with large irregular or rounded tile regions.
- Click Distribute.
- The support points are distributed within the selected tile region(s) and shown as yellow points
in the stage view.
- The support points of the selected tile region are displayed with their coordinates in the Local (per Tile Region) list in the Focus Surface and Support Points section of the Tiles tool.
- If necessary, you can adjust the distribution of the support points manually in the Tiles - Advanced Setup. You can change the position of the support points using drag & drop.
- Repeat the steps until you have distributed reference points across all desired tile regions.
- You have successfully distributed support points across the tile regions.
Automatic Distribution
If you activate Auto-Distribute for New Tile Regions in the Focus Surface and Support Points Section of the Tiles tool, support points are automatically added and distributed for all newly created tile regions.
Support Point Distribution
Distribute the support points evenly across your tile region. The more irregular the surface of your specimen, the more reference points you should set. An even but tilted surface requires at least 4 reference points for a solid calculation, while a simple saddle surface requires at least 9 reference points. A high reference point density leads to a more precise result, although the maximum useful density is one reference point per tile. The generic method follows a simple grid pattern to place the support points. It works well on regular rectangular tile regions smaller in size. For larger (>200 tiles) tile regions the Onion Skin will likely provide better results. In some cases, trial and error might be needed to optimize the parameters.
Verifying Z-Values of Support Points
- Click on the Verify button in the Focus Surface and Support Points section of the Tiles tool.
- The Verify Tile Regions/Positions dialog opens.
- 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.
- Click on the Move To Current Point button.
- The stage automatically moves to the support point that is highlighted in the reference point list. Alternatively, you can also double-click on the support point you want to check in the list .
- In the Live mode use the Focus tool to adjust the z-value.
- Click on the Set Z and Move to Next button.
- The checked reference point is marked with a green check mark.
- The stage moves automatically to the next support point in the list.
- Repeat the last 3 steps until you have checked all the support points.
- The message All points have been verified appears.
- Close the Verify Tile Regions/Positions dialog.
- You have adjusted and verified the z-values of all support points.
Positions always have a focus, which is determined by the z-value of the position. If you use positions in addition to tile regions, you can verify the z-values of the positions with the help of a similar dialog. Open this dialog by clicking on the Verify button in the Positions section of the Tiles tool.
Selecting Interpolation Degree
- If necessary, select the interpolation level in the Interpolation Degree dropdown list in the Focus Surface and Support Points section of the Tiles tool.
- You have successfully created a local focus surface. You can now start the experiment. To ensure that the tiles are acquired along the focus surface the software automatically selects the most appropriate focus strategy. For more information on focus strategies read the chapter Working with Focus Strategies.
The minimum number of support points necessary per tile region 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 sample demands, even if you have set 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. By default, ZEN uses a second order interpolation degree that creates a parabolic focus surface and requires at least 9 support points. Typically, this will be suitable for many samples and imaging scenarios.