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Tiles & Positions

This module enables you to acquire images that are made up of a number of individual images (tiles). To do this, it is possible to define tile regions and positions.

Acquiring a Tile Image

The tiles acquisition workbenches enable you to acquire an image of a large sample area. You define the area to be acquired and the system then acquires the corresponding tiles (images of neighboring sample areas) automatically and assemble them to a large image.

  1. The Tiles (manual) or Tiles (interactive) workbench is selected.
  2. The sample is sufficiently illuminated and in focus.
  3. The microscope is equipped with a motorized stage.
  1. Set up the camera using the Camera tool.
  2. Select the objective in the Magnification tool.
  3. Define and create the region of which you wish to acquire the tile image using the Tiles Setup (manual) or Tiles Setup (interactive) tool.
  4. The Center Screen Area displays the area you wish to acquire and a preview of the tiles to be acquired, including overlap.
  5. Set up the stitching method to be applied after acquisition using the Tile Stitching tool.
  6. Add the Focus Correction tool to apply a supporting method for focusing such as Software Autofocus or Focus Support Points / Focus Surface.
  7. In the Workbench Area, click the Start button.

The software acquires the tiles and stitches (merging the individual images together) the tile image automatically.

Acquiring a Tile Image with Extended Depth of Focus

The Tiles with EDF (interactive) workbench enables you to combine a tile image with extended depth of focus (EDF) acquisition. A Z-stack image is acquired for each tile.

  1. The Tiles with EDF (interactive) workbench is selected.
  2. The sample is sufficiently illuminated and in focus.
  3. The microscope is equipped with a motorized stage.
  1. Set up the camera using the Camera tool.
  2. Select the desired objective in the Magnification tool.
  3. Define the region of which you wish to acquire the tile image using the Tiles Setup (interactive) tool.
  4. Set up the focus range and number of slices to be acquired for each tile in the Motorized Extended Depth of Focus tool.
  5. Set up the stitching method to be applied after acquisition of using the Tile Stitching tool.
  6. In the Workbench Area, click the Start button.

The software acquires the tiles with extended depth of focus and assembles the tile image automatically.

Each tile is acquired multiple times at different focus positions. Thus, combining tile acquisition with extended depth of focus increases the acquisition time considerably.

Using Focus Support Points for Tile Images

  1. You have selected a tiles workbench, e.g. Tiles (interactive).
  2. You have set up a tiles acquisition (e.g. 3x3 tiles).
  1. Click + Tool to add a tool.
  2. Double-click the Focus Correction tool.
  3. The tool will be added to the workbench.
  4. In the Focus Correction tool, select Focus Surface.
  5. Click Distribute Default Points.
  6. 5 support points are added to the tiles acquisition.
  7. Click Verify Support Points to check each point, if the focus is set correctly.
  8. The Verify Support Point dialog will open and the stage moves to the first support point.
  9. Check if the image is in focus. If not, refocus the image. We recommend to use the Software Autofocus to help you find the correct focus setting. If the Software auto focus does not work fine, you can set the focus manually.
  10. Click Set Z & Move to Next if the focus is set correctly for the current support point.
  11. The stage will move to the next support point. Repeat the verifying process until all support points are verified.
  1. The message All points have been verified appears in the dialog. Click Close to exit the dialog and start the tiles acquisition.

Preview Images Tool

Using this tool you can select an acquired or loaded image as a preview image for a tiles acquisition. Therefore you can create an image with a lower magnification and load it as preview image. The preview image can be then used for navigating to a specific area which you want to observe in detail. You now can switch to a higher magnification and acquire another tile image.

Tiles Options Tool

This tool enables you to add advanced options when acquiring tiles.

Parameter

Description

Move Focus to Load Position Between Regions/Positions

Activated: To prevent possible damage, the focus drive is moved to the load position while moving to another tile region or position.

This option is only available in the following workbenches:

  • Position List Workbench
  • Position List with EDF Workbench
  • Tiles (measurement area) Workbench

Tile overlap

Defines the overlap of the re-tiled tiles in %.

This option is only available in the following workbenches:

  • Tiles (free drawing) Workbench
  • Tiles (interactive) Workbench
  • Tiles (manual) Workbench
  • Tiles with EDF (interactive) Workbench
  • Tiles (measurement area) Workbench

Travel in Tile Regions

Defines the stage travel behavior.

–

Meander

Default value. Acquires tile regions following a meander pattern alternately from both travel directions: from left to right and then from right to left. This scan movement is fastest.

–

Comb

Acquires tile regions following a comb pattern always from one travel direction only: from left to right. This scan movement is most precise.

–

Spiral

Acquires tile regions following a spiral pattern from the center of the region to the outer bounds in a clockwise motion. This scan movement works only for regions with rectangular or elliptical contours.

Tile Regions/Positions

Activated: Adapts the stage movement automatically to the location of the individual tile regions and positions. If you add or remove tile regions or positions, the sequence of acquisition therefore also changes.

–

Sort by Y, then X

Sorts tile regions and positions by their absolute position: first X, then Y.

–

Sort by X, then Y

Sorts tile regions and positions by their absolute position: first Y, then X.

Tiles Setup (interactive) Tool

With this tool you can interactively setup a tile region simply by moving the stage. You just have to set the start and end position of the desired tile region and the tile region is generated automatically.

If the sample height varies by a large amount, we recommend to add a focus correction method. Therefore you first have to add the Focus Correction tool. There you can select a focus correction method (e.g. Software Autofocus or Focus Surface) which is applied during the acquisition of the tile image.

 

Parameter

Description

Start with Current Position

Adds the current x/y/z position as the starting point for the new tile region. The button then changes to Extend to Current Position. Move the stage to the desired end position and click on the button to setup the tile region.

If you click on Start on top of the workbench, the final tile image will be acquired.

Delete

Deletes the created tile region.

Tiles Setup (free drawing) Tool

With this tool you can setup tile regions by drawing different contours (rectangle, ellipse, polygon) to create tile regions. The tile region is generated automatically according to the size of your drawing. When you click Start on top of the workbench, the final tile image is acquired.

Note: The Tiles (free drawing) workbench and the Tiles Setup (free drawing) tool are only visible in existing jobs where they were added in previous versions of ZEN core. They are not available any more for Free Mode or for new jobs.

If the sample height varies by a large amount, we recommend to add a focus correction method. For this, add the Focus Correction tool and select a focus correction method. This is applied during the acquisition of the tile image.

Tiles Setup (manual) Tool

With this tool you can manually setup a tile region wether by entering the number of tiles (e.g. 3 x 3) or size of the tile region (e.g. 4000µm x 4000µm). The individual images (single tiles) are acquired and then stitched together to create the final tile image.

If the sample height varies by a large amount, we recommend to add a focus correction method. For this, add the Focus Correction tool and select a focus correction method. This is applied during the acquisition of the tile image.

Parameter

Description

Create tile region

Creates a preview of the tile region which you have setup. The tile region is created at the current stage position.

The yellow grid shows the tiles to be acquired, including an overlap.

The frame shows the total (sample) area of interest.

If you click on Start on top of the workbench, the final tile image will be acquired.

Tiles

If selected, you can enter the number of tiles to be acquired, e.g. 3x3 tiles. The size of the tile region is calculated automatically.

Size

If selected, you can enter the size of the tile region to be acquired in µm, e.g. 4000 x 40000 µm. The number of tiles is calculated automatically.

Tiles Setup (measurement area) Tool

Parameter

Description

Contour

Switches the option to select rectangular or circular contour shape.

Field Size

Only visible with rectangular contour shape.
Defines the sample measurement area size. You have the following options (in mm2):

  • 0.126 (only in combination with a 20x objective and 200x total magnification)
  • The measurement is conducted with a 20x objective and 200x total magnification. The resulting standard measuring field is 355 µm x 355 µm = 0.126 mm2.
  • 0.504 (only in combination with a 10x objective and 100x total magnification)
  • Default value. The measurement is conducted with a 10x objective and 100x total magnification. The resulting standard measuring field is 710 µm x 710 µm = 0.504 mm2.
  • 2.016 (only in combination with a 5x objective and 50x total magnification)
  • The measurement is conducted with a 5x objective and 50x total magnification, the resulting standard measuring field is 1420 µm x 1420 µm = 2.016 mm2.
  • Custom

If you change the field size, the area is changed in relation.

Fields

Only visible with rectangular contour shape.
Defines the number of standard measuring fields. Per default, the measurement is conducted with a 100 x total magnification, the resulting standard measuring field is 710 µm x 710 µm = 0.5 mm2.

If you change the amount of standard measuring fields, the measurement area is changed accordingly.

Circle Diameter

Only visible with circular contour shape.
Defines the diameter of the circle contour shape.

Area

Specifies the acquired measurement area in mm2.

If you change the measurement area, the amount of fields is changed accordingly.

Add Tile Region

Adds a new tile region with the specified size values. The region is added with the top left position at the current stage position. The contour geometry of the tile region is automatically adjusted to fit an integer multiple ot the current field size. Therefore, the area might be a bit larger than expected.

The region is created in a way that an integer multiple of the current field size fits into the contour geometry (in columns and rows). Example: If you enter a field count of 6, a tile region with 3 x 2 fields is created (3 columns, 2 rows). This has also the consequence that for certain field counts (all prime numbers), a region with exactly that field cannot be created. For example, if you enter a field count of 11, a tile region with 4 x 3 (= 12) fields is created. This in general means that, if you do not change the created tile regions manually, the regions are always integer multiple of the field size.

Tile Region Table

This table provides an overview of all added tile regions. The checkbox in front of each entry activates the respective tile region. The following parameters are available in the table:

Name

Displays the name of the tile region. A click on the name allows you to edit it.

Tiles

Displays the number of tiles of the region.

Z (µm)

Displays and sets the z position of the region.

Area

Displays the area of the region in mm2.

Size

Displays the size of the region.


Move Up

Moves up the entry one position in the list.


Move Down

Moves down the entry one position in the list.


Delete

Deletes the currently selected region.


Options

–

Set Current Z for Selected Tile Regions

Sets the current z-position for all selected tile regions.

–

Set Current X/Y/Z for Selected Tile Region

Sets the current x/y/z-position for all selected tile regions.

–

Delete

Deletes the currently selected tile region.

–

Delete All

Deletes all tile regions.

–

Activate

Activates the current tile region for acquisition.

–

Deactivate

Deactivates the current tile region for acquisition.

–

Sort

Enables you to sort the entries in the tables.

–

Import Tile Regions

Opens a file browser to import a list of already defined tile regions.

–

Export Tile Regions

Opens a file browser to export the list of your currently defined tile regions as a file.

Parameter

Description

Lock Contour of all Regions

Specifies whether and how the contour geometry of all tile regions is locked.

–

None

The contour of all tile regions is not locked.

–

Area

The contour area of all tile regions remains constant.

–

Size

The contour size and area of all tile regions cannot be changed.

Adjust to Integer Multiple of Field Size

Adjust the contour geometry of all tile regions to fit an integer multiple of the current field size. After that, the area might be slightly larger. If the size of the tile regions is not locked, you can modify the size of the tile regions manually by using the mouse handles in the graphical representation.

Tiles Setup (multiple regions) Tool

With this tool you can setup multiple tile regions by drawing different contours (rectangle, ellipse, polygon). The tile regions are generated automatically according to the size of your drawings.

If the sample height varies by a large amount, we recommend to add a focus correction method. For this, add the Focus Correction tool and select a focus correction method. This is applied during the acquisition of the tile image.

Parameter

Description

Contour

Selects which contour you want to use for drawing the tile region.

–


Selection Mode

Enables you to select an already created tile region to move or resize it.

–


Rectangle

Enables you to draw a rectangular tile region.

–


Ellipse

Enables you to draw an elliptical tile region.

–


Polygon

Enables you to draw a polygonal tile region.

Tile Region Table

This table provides an overview of all added tile regions. The checkbox in front of each entry activates the respective tile region. The following parameters are available in the table:

Name

Displays the name of the tile region. A click on the name allows you to edit it.

Tiles

Displays the number of tiles of the region.

Z (µm)

Displays and sets the z position of the region.

Area

Displays the area of the region in mm2.

Size

Displays the size of the region.


Move Up

Moves up the entry one position in the list.


Move Down

Moves down the entry one position in the list.


Delete

Deletes the currently selected region.


Options

–

Set Current Z for Selected Tile Regions

Sets the current z-position for all selected tile regions.

–

Set Current X/Y/Z for Selected Tile Region

Sets the current x/y/z-position for all selected tile regions.

–

Delete

Deletes the currently selected tile region.

–

Delete All

Deletes all tile regions.

–

Activate

Activates the current tile region for acquisition.

–

Deactivate

Deactivates the current tile region for acquisition.

–

Sort

Enables you to sort the entries in the tables.

–

Import Tile Regions

Opens a file browser to import a list of already defined tile regions.

–

Export Tile Regions

Opens a file browser to export the list of your currently defined tile regions as a file.

Tiles (free drawing) Workbench

This workbench enables you to create tile images on a motorized stage by setting up the tile region with freehand drawing tools.

Note: The Tiles (free drawing) workbench and the Tiles Setup (free drawing) tool are only visible in existing jobs where they were added in previous versions of ZEN core. They are not available any more for Free Mode or for new jobs.

Tiles (interactive) Workbench

This workbench enables you to acquire an image of a large sample area composed of tiles: a tile corresponds to the area the camera is able to acquire with a single acquisition.

You can specify multiple areas of interest on your sample. The software defines the total area which needs to be acquired and thus the required number of tiles. The tiles are then acquired automatically (the stage movement is controlled by the software) and merged into a single image.

Tiles (manual) Workbench

This workbench enables you to acquire an image of a large sample area composed of tiles: a tile corresponds to the area the camera is able to acquire with a single acquisition.

You specify the area of interest on your sample you wish to acquire by defining a corresponding region in the software. You can define the region by the number of tiles to be acquired or by the size in micrometers. The tiles are then acquired automatically (the stage movement is controlled by the software) and merged into a single image.

Tiles (measurement area) Workbench

This workbench enables you to acquire one or more tile images using a motorized scanning stage by adding multiple tile regions with a defined measurement area. A number of specimens can be configured for automated tiles image acquistion. It is optimized for standard based NMI Analysis.

In case of multi-specimen-setup, you can define individual values for each sample.

Tiles with EDF Workbench

This workbench enables you to acquire an image of a large sample area by specifying multiple tile regions on your sample which you wish to combine into a large image. For each tile region, a z-stack of images is acquired and merged afterwards into an extended depth of focus (EDF) image.

The stage movement and acquisition of the individual tiles is controlled automatically.

Height Stitching and Blending Tool

This tool takes a tile image with the individual tiles placed next to each other as input and returns a single large image. For the best result and if necessary the th x, y and z position of the individual tiles is adjusted.

Parameter

Description

Acceptable difference

Defines the distance value between each tile.

Lower Threshold

Defines the minimum value to which the lowest occurring pixel value is mapped.

Upper Threshold

Defines the maximum value to which the highest occurring pixel value is mapped.

Execute Blending

Activated: The residual artifacts on the edges of the tiles are reduced.

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