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Tiles & Positions with Advanced Setup

Advanced Setup makes it easier for you to create tile regions and positions by displaying the distribution and dimensions of tile regions and positions in the travel range of the stage. You can generate a Preview Scan and draw in tile regions or positions precisely on the basis of this template. For the preview scan you have the option of using an objective with a lower magnification and/or a different channel (e.g. transmitted light).

To ensure that the individual z values of the tile regions and/or positions are taken into account, ZEN automatically selects the most appropriate focus strategy when the checkbox Tiles is activated. For the experiment described here no further modification needs to be made. If you want to acquire all tile regions at the same z-position, then you must select None from the dropdown list in the Focus Strategy tool. The individual z-positions are then ignored and the current z-position at the time the experiment is started is used for all tile regions. In the case that you do not have the Tiles & Positions module, ZEN still selects the appropriate focus strategy for you.

  1. To set up tiles experiments in Advanced Setup, you need the Tiles & Positions module.
  2. You have read the general introduction, see Tiles & Positions.
  3. You are on the Acquisition tab in the Tiles tool.
  1. Click Show Viewer.
  2. The Tiles Advanced Setup view opens. For more information, see Tiles Advanced Setup.
  3. The live mode is activated automatically. Deactivate the live mode if you do not need it to prevent bleaching of the sample. To do this, click on the active Stop button in the Left Tool Area. This default behavior can be changed in Tools >Options > Acquisition > Tiles & Positions.

Generating a Preview Scan

  1. You have selected an objective with a relatively low magnification in your experiment settings.
  2. You are in Advanced Setup.
  1. In the left toolbar, click on Preview Scan.
  2. The Preview Scan Toolbar is displayed as the top toolbar.
  3. In the top toolbar, deactivate Use Existing Experiment Settings and select/ unselect the channels that you want to use for the preview scan.
  4. If necessary, use the live mode to adjust the focus area and exposure following a change of objective or channel.
  5. To obtain a better overview, slightly zoom out of the Advanced Setup view.
  6. Start the Live mode to use the stage to locate approximately the center of the region for which you want to generate a preview scan.
  7. In the left toolbar in the Tiles section, click on the Setup by contour button.
  8. The Contour Toolbar is displayed as the top toolbar.
  9. In the top toolbar, select the Rectangular Contour tool.
  10. In the Stage View, use the tool to draw a rectangle that approximately encloses the region for which you want to generate a preview scan.
  11. Alternatively, in the Stage View, use the live image to navigate to the edge of the area that you want to image. We recommend, for example, the bottom left corner. Add the first marker. Now navigate to the upper edge of the object and add a second marker. Finally, if necessary, navigate to the right edge and add a third marker and click on Done. You can optimize the tile region by selecting a circular contour shape for your sample instead.
  12. A tile region is created for the marked region and displayed in the list in the Tile Regions section of the Tiles tool.
  13. With the help of the Live mode, check whether the desired image region is covered by the tile region. To do this, use the stage to locate the corners and edges of the tile region and increase or reduce the yellow selection frame as necessary.
  14. In the left toolbar, click on Preview Scan and in the top toolbar click on Start.
  15. A series of snap images is acquired to generate a preview of the marked region.
  1. You have successfully generated a preview scan.

Before you continue with the actual experiment, carry out the following steps:

  1. In the Tiles Regions section, deactivate the preview tile region (TR 1) by deactivating the checkbox of the corresponding list entry. This prevents the acquisition of the preview tile region during the actual experiment.
  2. In the Microscope Control tool in the Right Tool Area, select the objective you want to use for final acquisition.
  3. Use the Live mode to adjust the focus area and exposure accordingly.
  1. You can now continue setting up the tile experiment.

Creating Tile Regions by Contour

  1. You have generated a preview scan that will help you to position the tile regions more easily.
  1. In the left toolbar in the Tiles section, click on the Setup by contour button.
  2. The Contour Toolbar is displayed as the top toolbar.
  3. In the top toolbar, select the desired contour tool.
  4. Use the contour tool in the stage view to draw in the tile regions you want to acquire.
  5. Tile regions are created for each marked region. They are added to the list in the Tile Regions section of the Tiles tool.
  1. You have successfully created tile regions in Advanced Setup.

Creating Tile Regions by Predefined

  1. In the left toolbar in the Tiles section, click on the Setup by predefined button.
  2. The Predefined Toolbar is displayed as the top toolbar.
  3. In the top toolbar, choose how many tiles in x and y dimension you want to add.
  4. Click Add Tile Region to add the respective tile region.
  1. You have now created tile regions in the Advanced Setup.

Shortcut

You can also add a predefined tile region at the current stage position by pressing the F9 button on your keyboard. The size of this region is the last defined number of tiles in x and y, or a square of 3x3 tiles if you have never defined a region before.

Creating Tile Regions by Carrier

  1. You have selected and calibrated a sample carrier with one or more wells/ containers.
  1. In the left toolbar in the Tiles section, click on the Setup by carrier button.
  2. The Tiles Carrier Toolbar is displayed as the top toolbar.
  3. In the Carrier tab, select the individual wells for which you want to create tile regions by pressing the Ctrl key and clicking on the desired wells.
  4. The selected wells are now bordered by a blue circle.
  5. In the top toolbar, select Fill Factor and enter the desired value in the Fill Factor input field.
  6. Click .
  1. According to the selected Fill Factor, the wells will be filled with a calculated number of tiles that are located around the center. To create a given size of tile region, use the Columns/Rows function in a similar manner.

Stage movement

If you only double-click one well , the stage moves to the center of that well.

Creating Positions by Location

  1. In the left toolbar in the Positions section, click on the Setup by location button.
  2. The Positions Location Toolbar is displayed as the top toolbar.
  3. In the top toolbar, click .
  4. In the stage view, click on the location where you want to add a position.
  5. The added positions are displayed in the stage view and the Single Positions list in the Positions section of the Tiles tool.
  1. You have successfully created positions in Tiles- Advanced Setup.
    Note: ZEN will query if you want to add a position that has more than 66% overlap of the field of view in either x or y with an existing position.

Shortcut

You can also add a single position at the current stage position by pressing the F10 button on your keyboard.

Creating Positions by Array

  1. In the Tiles tool, open the Positions section and click on Position Arrays.
  2. In the left toolbar in the Positions section, click on the Setup by array button.
  3. The Position Array Toolbar is displayed as the top toolbar.
  4. In the top toolbar, choose either the rectangular or circular Contour, adjust the Number of required positions and the Bias where the positions should be located.
  5. Mark the interesting area in the Center Screen Area with a pressed left mouse button.
  1. The positions will be generated automatically.

Random Distribution

If the Random checkbox is activated, the selected number of positions for the array is determined randomly within the arrays space.

Creating Positions by Carrier

  1. You have selected and calibrated a sample carrier template.
  2. You are on the Acquisition tab.
  1. To obtain a complete overview of the sample carrier, zoom out of the view using the mouse wheel. If necessary, use the panning tool (press Alt and the left mouse button) to move the stage view as needed.
  2. In the left toolbar in the Positions section, click on the Setup by sample carrier button.
  3. The Positions Carrier Toolbar is displayed as the top toolbar.
  4. Select the containers in which you want to distribute positions by pressing the Ctrl key and clicking on the relevant containers.
  5. In the top toolbar adjust the Number of required positions and the Bias where the positions should be located. Alternatively, if you want to create the positions as a Grid, adjust the Columns, Rows, and Overlap.
  6. Click on the Create button .
  7. The selected containers are each filled with a Position Array (group of positions).
  8. In the Positions section of the Tiles tool, the Position Arrays are displayed in the Position Arrays list.
  1. You have successfully used a sample carrier to create positions.

Importing Images into the Advanced Setup

In the Tiles - Advanced Setup, you have several options to open/import images. Follow the respective workflow.

Importing preview scan images

  1. You are in the Advanced Setup.
  1. In the ZEN menu bar, click on File > Tiles > Import Preview Image. Alternatively, right click in the viewer and select Import Preview Image from the context menu.
  2. A file browser opens.
  3. In the file browser, select your preview scan image (*.czi) and click on Open.
  1. You have now imported a preview scan image into the Tiles Advanced Setup.

Importing an image already opened in ZEN

  1. You are in the Advanced Setup.
  2. You have an image opened in ZEN.
  1. In the Right Tool Area, in the Images and Documents tool, select the image you want to import.
  2. Drag and drop the image into the viewer of the Advanced Setup.
  1. You have now imported an already opened image into the Tiles Advanced Setup.

Importing from Explorer

  1. You are in the Advanced Setup.
  2. You have opened the path to your image (*.czi) in the Windows File Explorer.
  1. In the Windows File Explorer, select the image you want to import.
  2. Drag and drop the image into the viewer of the Advanced Setup.
  1. You have now imported an image from the Windows File Explorer directly into the Tiles Advanced Setup.

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