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Tiles & Positions

This module enables you to acquire high-resolution images through automatic scanning of predefined tile regions and positions of a sample. The regions of tile images and individual positions can be combined freely and a motorized stage allows automatic scanning of specimens. If you are working with a manual stage, you can still manually move the stage to different positions and acquire data at each position. This process is not automated and requires to manually adjust the stage position for each new acquisition point. Such manual positioning can be less efficient and less precise. The Panorama functionality helps in a semi- automatic way to acquire overview images with a manual stage, see also Panorama. For a full automatic scanning of predefined tile regions and positions of a sample, it is necessary to have a motorized stage on the system.

Overlapping individual images can be combined into an overview image using “stitching” algorithms. The module supports the use of sample carriers, focus correction maps, stitching and shading correction, and is compatible with Software Autofocus. Some functionality is generally available, for the full set of features you need the dedicated license for the module, see Licensing and Functionalities of Tiles & Positions.

Acquiring Tiles with different z

If you want to acquire tile regions or positions with different z-positions, you need to use a suitable focus strategy, see Using focus strategies.

Automatic Focus Strategy Selection

When you activate Tiles for the first time in any experiment, the software automatically selects the suitable focus strategy Use Z Values/ Focus Surface defined in Tiles Setup. This focus strategy is only available if you have licensed the Tiles & Positions functionality and it can be optimized with the Focus Strategy Wizard in the Focus Strategy tool, see Focus Strategy Wizard and Focus Strategy Tool. In the case that you do not have licensed the Tiles & Positions functionality, ZEN still selects the appropriate focus strategy for you.

1

Tile Regions

Defined tile regions are displayed as red rectangles in the Tiles Advanced Setup.

2

Position-Array

A position array is displayed as a dashed blue frame in the Tiles Advanced Setup. Individual positions in the array are displayed as yellow crosses.

3

Positions (Single Positions)

Individual positions in the array are displayed as yellow crosses in the Tiles Advanced Setup.

Licensing and Functionalities of Tiles & Positions

Some basic functionality for tiles and position experiments is generally available in the software, but the full Tiles & Positions functionality requires a license.

Basic functionality

The functionality generally available in ZEN includes:

  • The setup of experiments with tile regions or positions.
  • The acquisition of images through automatic scanning of the predefined regions and positions.

Licensed Functionality

If you have licensed this functionality and activated it under Tools > Toolkit Manager, the following additional functionality is available:

  • The Advanced Setup functionality to set up experiments more easily, including the possibility to copy and paste tile regions and positions.
  • The use of sample carriers for your tile or position experiments, including the functionality to generate and use your own sample carrier templates.
  • The suitable focus strategy Use Z Values/ Focus Surface defined in Tiles Setup and the Focus Strategy Wizard to guide you through the setup of a focus strategy for the best focus.
  • The creation and use of local and global focus surfaces.
  • The possibility to create and use custom categories for your tiles and positions.

General Preparations

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  2. To set up Tiles experiments, you require a motorized stage. This must be configured and calibrated correctly in accordance with the camera orientation. For more information read Calibrating the Stage and Selecting the Channel.
  3. You are on the Acquisition tab.
  4. You have created a new experiment, defined at least one channel and correctly set the focus and exposure time.
  1. Activate the Tiles checkbox in the Acquisition Dimensions section to display the Tiles tool.
  2. In the Left Tool Area the Tiles tool appears in the Multidimensional Acquisition tool group.
  1. You have successfully completed the general preparations. You can now continue with the next steps of this guide.

Setting Up a Simple Tiles Experiment Without the Tiles & Positions Module

  1. You are on the Acquisition tab in the Tiles tool.
  1. Start the Live mode to use the stage to locate a point that you want to be at the center of your tile region.
  2. Bring the specimen into focus using the focus drive.
  3. Open the Tile Regions section.
  4. The Tiles mode is activated by default.
  5. Enter the number of tiles you want in the X and Y input fields, e.g. X = 3, Y = 3 equals a tiles region containing 9 tiles.
    Alternatively, you can enter the size of the tile region that you want to add. To do this, activate the Size mode.
  6. Click Blue rounded square button with white plus sign and small gray downward arrow.
  7. The tile region is added to your experiment. The current stage position and focus determines the center and the z-position of the tile region.
  8. To add further tile regions, move the stage to another position on the sample and repeat the previous steps.
  9. The added tile regions (TR1, TR2, etc.) are displayed in the tile regions list.
  10. If you scroll to the right in the table, you can read the Size of the tile regions.
  11. Save the experiment. To do this, in the Experiment Manager click Dark blue rounded square icon with a white gear and a small white sparkle to its right and select Save As. Enter a name for the experiment in the input field (e.g. Simple Tile Experiment).
  12. Click Start Experiment.
  13. The Tile Region experiment is acquired.
  14. The individual tile regions are displayed in the acquired file as scenes and can be selected using the Scene slider on the Dimensions tab. If you deactivate the Scene checkbox, all tile regions are displayed as an overview.
  1. You have successfully set up and acquired a simple Tile Region experiment.

Z values

To ensure that the individual z values of the tile regions 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 have to 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.

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.

Setting Up a Simple Positions Experiment without the Tiles & Positions Module

  1. You are on the Acquisition tab in the Tiles tool.
  1. Open the Positions section.
  2. Start the Live mode to use the stage to locate a position that you want to acquire.
  3. The X and Y coordinates of the current position are displayed in the Current X/Y display fields.
  4. Bring the specimen into focus using the focus drive.
  5. Click Blue rounded square button with white plus sign and small gray downward arrow.
  6. The current position is added to your experiment.
  7. If you are close to a position that you added previously the software will ask you if you really want to add another position at this location (the threshold for this lies within a circle whose radius is less than half the approximate width of the cameras visible field).
  8. To add further positions, move the stage to another position on the sample and repeat the previous steps.
  9. The added positions are shown in the list in the Single Positions section with their X, Y and Z-coordinates.
  10. Save the experiment. To do this, in the Experiment Manager click Dark blue rounded square icon with a white gear and a small white sparkle to its right and select Save As. Enter a name for the experiment in the input field (e.g. Simple Tile Experiment).
  11. Click Start Experiment.
  12. The Positions experiment is acquired.
  13. The individual positions are displayed in the acquired file as scenes and can be selected using the Scene slider on the Dimensions tab. If you deactivate the Scene checkbox, all positions are displayed simultaneously as an overview.
  1. You have successfully set up and acquired a Positions experiment.

Z values

To ensure that the individual Z values of the positions are taken into account the software 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 positions at the same Z-position, then you have to 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 positions.

Shortcut

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

Exporting Tile Images

  1. You have acquired or opened a tile image.
  1. On the Processing tab, open the parameters for Image Export (Ctrl+6 or via File > Export/Import > Export).
  2. You will see the settings of the parameters for Image Export. Make sure that the Show All mode has been activated.
  3. Select the file type that you want to use. We recommend the PNG format, as this is a format that offers lossless compression with an acceptable file size.

  4. Type and source of risk

    In the case of particularly large tile images, we recommend that you reduce the size of the images you want to export. To do this, set a percentage in the Resize slider by which you want the images for export to be reduced, e.g. by 25%.

  5. Activate the Apply Display Curve and Channel Color checkbox. This means that the images will be exported with the settings you have made, e.g. tonal value corrections or contrast. If you activate the Original Data checkbox, the images are exported unchanged. In this case, the settings from the display curve, e.g. tonal value corrections and contrast, are not adopted.
  6. Select the Define Subset radio button.
  7. The settings for the available dimensions open.
  8. Open the settings for the Tiles dimension.
  9. Select the Existing Tiles radio button.
  10. Click on the Apply button at the top of the Processing tab.
  11. You have exported the individual tiles from a tile image. The files can be found in the export folder indicated.

Calibrating the Stage and Selecting the Channel

On startup of a system with motorized stage and/or focus a request will appear asking if the components should be driven to the end switches and calibrated. This ensures that you begin working with absolute coordinates in this session with the microscope. If the microscope power is cycled, then this process should be repeated. This function is of particular use if you continually work with a sample carrier e.g. 96 well plate, of the same format mounted in the same manner repeatedly with a given experiment template. If you perform a carrier calibration once with a calibrated stage, then the carrier calibration is essentially always valid. This is done in the Sample Carrier section of the Tiles tool. Note these features are only available if you have a license.

The request to calibrate stage and focus on startup can be activated/deactivated under Tools > Options > Startup/Shutdown > Stage/Focus Calibration.

 

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  1. Put your Sample Carrier on the stage.
  2. Go to the Acquisition tab.
  3. Select a low magnification objective (e.g. 10x) from the Microscope tool in the Right Tool Area.
  4. Click Live and find your focus area either using transmitted or fluorescence light.
  5. In the Stage tool of the Right Tool Area, activate the Show all mode and then click Calibrate.
  6. Check if the alignment and orientation of your camera and joystick is correct by dragging the software joystick up, down, left and right and observe whether the movement of your image corresponds to movement of the circle.
    The alignment of the camera is correct if the movement of a given stage axis is congruent to the corresponding axis of the image. An offset in the alignment will be seen as a saw tooth pattern along the edge of a tiled image (e.g. 4x4 tile region).
    In addition, check whether the image movement also corresponds accordingly when you move the hardware joystick of the stage.
  7. If the orientation of the camera to the software joystick (stage tool, right tool area) is incorrect, go to the Camera tool, activate the Show all mode, and click Model Specific. The orientation of the camera (image) can be adjusted by flipping, rotating, or mirroring.
  8. Alternatively, you can and may also need to invert the x- and y-axis of your stage in the MTB in order to align the hardware joystick and the software-controlled stage movement.
  9. Ensure that all the prerequisites (e.g. channel and camera settings) for a Tiles & Positions experiment on your sample are fulfilled. If necessary, use the Smart Setup for the setup.
  10. After you have defined at least one channel (e.g. EGFP), activate the Tiles checkbox.
  11. If you wish to work with a sample carrier, complete the following steps.
  12. Open the Tiles tool and activate the Show all mode.
  13. In the Tiles tool, open the Sample Carrier section.
  14. Click Select....
  15. Select a sample carrier template and click OK.

Copying a Tile Region or Position

To copy and paste a specific Tile Region or Position, follow these instructions.

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  1. Right-click the respective tile region or position and select Copy. Alternatively, press Ctrl+C. If you want to copy multiple tile regions and positions, select multiple regions or positions by dragging a selection box across the objects or click to select multiple objects while pressing the Ctrl button.
  2. Right-click in the setup and select Paste. Alternatively, press Ctrl+V.
  1. The copied tile regions/ positions are now pasted next to the originally copied regions/ positions.

Copying Tile Region or Position settings between wells

When you want to copy and paste a tile region or position setting (e.g. a certain arrangement of tiles, positions or local support points) from one well to other wells or even to all containers of a carrier, apply the following workflow.

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  1. Select the well from where the tile region/ position setting should be copied.
  2. The selected well is now highlighted by a blue border.
  3. Right click within the selected well in the Center Screen Area (outside the tile region) to open the context menu.
  4. Select Copy Container for replication.
  5. If you want to select specific wells, use the left mouse button to select the wells into which you want to paste the copied tile region/ position setting. To select multiple wells, press Ctrl while selecting the wells.
  6. Right click in the Center Screen Area and select the context menu entry Paste Replication to and either choose Selected Container or All Container.
  1. The copied tile region/ position setting is pasted into the selected wells or all the wells of the carrier with the same relative coordinates to the center of each well.

Assigning Categories to Tile Regions and Positions

In some cases it can be helpful to not only display the well number together with the acquired images (Path: Graphics > Frequent Annotations > Carrier Container Name) but also to create certain additional annotations for different tile regions or positions, e.g. "control condition" or "experimental condition 1".
For that purpose, the software allows you to add and edit names and categories to the different tile regions/positions that have been generated.

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  1. In order to assign individual names to different individual positions and/or tile regions in a well plate experiment, in the Tiles tool click on the respective name in the Tile Regions or Positions list. You can now edit the name field and press Enter to finish.
  2. Repeat this step to rename different tile regions or positions.
  3. To assign or edit categories of your tile regions/ positions, first select all desired tile regions/ positions that should be grouped in the same category.
  4. Under the Tile Regions/Positions list, in the Properties section Category, click on the Options button Dark blue rounded square icon with a white gear and a small white sparkle to its right and select New from the dropdown list.
  5. The New Category window opens.
  6. Enter a Name and add a Description for the selected tile regions/ positions.
  7. Assign a Color for the new category by clicking on the color bar and choosing a preferred color.
  8. Click on OK to create the new category.
  9. The New Category window closes and the new category is created.
  10. As Category choose the desired category for the selected tile regions/ positions from the dropdown list.
  1. The chosen category is now assigned to the selected tile regions/ positions.

  1. To display the name of your Tile Region/ Position later in your acquired image(s), go to Graphics > Frequent Annotations > More… and select Image.Scene.Name from the metadata list.
  2. Note that a predefined category can also be applied to a differentiated selection of Tile Regions/ Positions from more than one well.
    Note also, that the assigned color is only used as a feature in the Tiles tab (Left Tool Bar Area).
  3. To display a Tile Region/ Position Category feature (Name and/or Description) in your acquired image, you go to Graphics > Frequent Annotations > More…. Type “category” in the search bar and select the desired feature to be displayed. (Although the option "Color" is given, no reasonable element will be displayed by the software.)
  4. To adjust parameters of your annotations (e.g. font size), right-click on it and go to Format > Graphical Elements.

Displaying categories in the Tiles/ Positions List (Left Tool Area)

  1. You have selected several different positions or tile regions and assigned different categories.
  1. Under Positions or Tile Regions of the Tiles tool, select a position or tile region.
  2. Right-click on the selected position/ tile, choose Sort and select By Category
  1. The positions/ tiles will be sorted alphabetically according to the assigned categories.

Tiles Advanced Setup

Here you can visualize and plan your Tiles and Positions experiment. The advanced tiles setup opens by clicking on the Show Viewer button in the Tiles tool on the Acquisition tab of the Left Tool Area. In the Center Screen Area you can see the Stage view. When the Tiles Advanced Setup opens, the stage view is zoomed to a predefined factor. You can change the Zoom in the Dimensions tab, or by scrolling the mouse wheel.
To navigate around you have the following options:

  • Move the mouse pointer of the live navigator, then press and hold the left mouse button and drag the live navigator to the desired location. Release the left mouse button to finish.
  • Place the mouse pointer at a location where you want to move the stage and double click.
  • Use the software joystick in the Stage tool of the Right Tool Area to navigate. Alternatively use the hardware joystick of your stage controller.
  • In each corner and along each edge with the arrowheads, you can click to move the view in this direction. Additional settings and tools relating to tile regions or positions can be found in the specific view options.

1

Left Toolbar
Here you select which setup you use to set tile regions or position. for more information, see Left Toolbar.

2

Top Toolbar
Depending on what you have selected on the Left Toolbar, different features to create tiles or positions are displayed here. For more information, see Top Toolbar.

3

Stage View
The Stage View shows the full travel range of the microscope stage, along with the current stage position, the graphical display of sample carriers, and your acquired mosaic images. For more information, see Stage View.

4

View Options
General and tiles specific view options are displayed here.
Note that the functionalities between activated and deactivated Show All layout might differ.

Tiles Options

The additional options for the tiles module allow to set up several options for image acquisition and additional information. The tiles options dialog can be found in the menu bar under Tools > Options > Acquisition > Tiles & Positions.
Note: For the changes to be effective you might need to close and reopen the advanced setup viewer.

Parameter

Description

Automatically Start Live Mode in the Advanced Setup View

Activated: Automatically starts the Live mode in the Center Screen Area if you klick in the Acquisition tab in the Tiles tool on the Advanced Setup button.

Uncheck this option to prevent unnecessary specimen bleaching. The default is not activated.

Automatic Snap by Clicking the Live Navigator Buttons

Activated: Acquires an image if you click on one of the frame's blue arrow icons. The Live Navigator tool moves one frame width in the relevant direction. You can create tile images of your sample easily in this way.

Enable Stage Movement with Live Navigator

In the Live navigator tool the current stage position including the live image is shown as a frame outlined in blue. To move the frame, double-click on the position to which you want to move it. Alternatively, place the mouse cursor over the blue frame, press and hold the left mouse button and drag the live navigator to the desired location.

Activated: Allows you to move the Live Navigator tool by dragging it to a new location.

Show Stage and Focus Backlash Correction Setting in the Options

Activated: In the Tiles option, the setting to switch the backlash correction on or off is shown. Per default it is hidden.

Delimiter for CSV Export/Import

Specifies the delimiter for a CSV export or import. Select Comma (default), Semicolon or Tab.

Ask Whether Support Points/Positions Should be Overwritten

When the support points and/or positions are determined by a software autofocus run the existing points can be overwritten with the new Z values.

Activated: Shows a message box asking if the points should be overwritten if there is a autofocus Z value.

Focus Surface Outlier Determination

Ignores support points that are significantly outside the interpolated focus surface.

You have the following setting options available:

-

Maximum Interpolation Degree for Outlier Detection

This value can be 0 or 1. If 1 then a linear fit is used to detect the outlier support points. This is the default. If 0 a simple average value is used to detect outliers.

-

Threshold in Terms of the Standard Deviation (Sigma)

This parameter defines a threshold value to determine which of the support points are outliers from the fitting process. This is defined by the standard deviation (sigma value) set in the spin box. Support points not meeting this criteria are subsequently ignored when the focus surface is determined.

Delay Time After Stage Movements

Defines a delay period which is used for all stage movements in a tiles and position experiment or movements controlled in the advanced tile setup. The delay helps prevent movement in samples where, for example, a large volume of liquid is present in the sample holder. It can be used with the stage speed and acceleration options to optimise experiments with this type of sample.

Binning Compensation of Exposure Time in Preview Scans

Defines the power to which the binning ratio is modified to automatically determine the exposure time value used for a preview scan were the binning setting between the experiment and preview scan differs. The default value is 2.0 i.e. quadratic. Thus, for example the exposure time would be reduced by a factor of four if the experiment binning is 1x1 and the preview scan binning is 2x2. The value can be varied between 1.0 and 2.0 in steps of 0.1.

Live Image in Sample Carrier Calibration Wizard (relevant only for systems with camera)

-

Use Imaging Device from Selected Channel with "Acquisition" Settings

Activated: Default setting for the live image that allows navigation and focus interaction during the carrier calibration wizard.

-

Use Active Camera with "Locate" Settings

This option is only relevant for systems with a wide field (camera based) detector.
Activated: Allows you to alternatively apply locate camera settings for use in the carrier calibration wizard (live image). By default the experiment settings for the currently selected channel/track will be used.

Select Template Dialog

In this dialog you can select a sample carrier or enter a dialog to create a custom carrier. A preview of the selected carrier is shown on the right side of the list.

Parameter

Description

Workgroup Templates

Displays a list of all custom sample carrier templates.

Program Templates

Displays a list of predefined sample carrier templates for several sample carriers. When working with ZEN celldiscoverer, the available templates are grouped based on their type.

White plus sign centered on dark blue rounded-square icon
Add

Opens the New Sample Carrier Template dialog to create a new template.

White trash bin on dark blue rounded-square icon
Delete

Deletes the selected sample carrier template.

Dark blue rounded square icon with a white gear and a small white sparkle to its right
Options

–

New Template...

Opens the New Sample Carrier Template dialog to create a new template.

–

Show/Edit...

Opens the selected template in the Edit Sample Carrier Template dialog and allows editing.
ZEISS templates are read only. If you want to edit a ZEISS template, you have to use the Copy and Edit... option.

–

Copy

Copies the selected template.

–

Copy and Edit...

Copies the selected template and opens it in the Edit Sample Carrier Template dialog dialog for editing.

–

Import

Imports a template.

–

Export

Exports the selected template.

–

Delete

Deletes the selected template.

–

Refresh Templates

Refreshes the list of templates after creating a new one.

OK

Opens the selected sample carrier and closes the dialog.

Cancel

Closes the dialog without opening a sample carrier.

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