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Acquisition Toolkits

You can expand the software functionality with the following acquisition packages:

Toolkit	Included Modules
Base Acquisition Toolkit	Multi-Channel Time Series
Motorized Focus Acquisition Toolkit	Extended Focus Processing Software Autofocus Z-Stack
Motorized Acquisition Toolkit	Extended Focus Processing Software Autofocus Tiles & Positions Z-Stack
Advanced Acquisition Toolkit	Extended Focus Processing HDR Confocal Basic Software Autofocus Tiles & Positions Z-Stack
Smart Acquisition Toolkit	Experiment Designer Experiment Feedback Guided Acquisition

Experiment Designer

This module allows you to configure inhomogeneous acquisition experiments with support for all experiment dimensions: time series, z-stacks, tile images and channels. Experiments can consist of any number of components. A component is referred to as an experiment block. Each experiment block has a distinct number, which is shown above the block. In the dedicated tool, you can set up four different types of experiment blocks and make use of a set of powerful processing functions to extract or fuse multiblock images. Experiment Designer allows the definition of a number of iteration loops, and synchronous or asynchronous control of hardware actions during the experiment.

Experiment Feedback

This module (also known as conditional or adaptive experiments) allows you to define specific rules and actions to be performed during the acquisition of an experiment. It is possible to change the course of an experiment depending on the current system status or the nature of the acquired data during acquisition. Moreover, it is possible to integrate certain tasks like data logging or starting of an external application, directly into the ZEN experiment. A typical use case is to connect the image acquisition with automated image analysis.

Feedback experiments can be set up and controlled with the Experiment Feedback Tool and the Script Editor. For an example workflow for experiment feedback, see Workflow Experiment Feedback.

Note that we do not describe experiment feedback in detail here, as you can find a detailed instruction on how to perform feedback experiments and a lot of tutorials with the latest ZEN installation, if you activate the entry OAD Samples in the ZEISS Microscopy Installer. The tutorials and documentation are then placed in a folder on your PC like C:\Oad\Experiment Feedback. Experiment Feedback uses IronPython 3.4.

Key Features

Create smart experiments and modify the acquisition on-the-fly based on online image analysis, hardware changes or external inputs (e.g. TTL signals).
The adaptive acquisition engine allows modifying running experiments according to the rules defined inside the feedback script.
The feedback script uses ZEN commands in combination with the Python programming language.
The feedback script gives access to the current system status and results from the online image analysis during runtime of an experiment.
Data Logging or starting an external application (e.g. Python, Fiji, MATLAB, etc. ), directly from within the imaging experiment is possible.

Experiment Feedback Tool

Parameter		Description
Edit Feedback Script...		Opens the Script Editor dialog. There you can create scripts for an Experiment Feedback.
Select script runtime conditions
-	Free Run	Upon the experiment start the acquisition and the feedback script are started but run from here in a completely unsynchronized manner. The online image analysis or the script run itself will not slow down the actual image acquisition.
-	Synchronized	This mode will lead to strictly determined order of events depending on the chosen level of synchronization. The online image analysis and/ or the feedback script will be started after current acquisition is finished. In contrast to the Free Run mode, a synchronized run can slow the whole acquisition down. The big advantage of the mode is, that the synchronized run ensures a predictable workflow.
Define Script Slot		Here you define the experiment feedback sequence by arranging the slots (represented by blue buttons). The blue slots run one after another the non-blue slots are run separately. The Acquisition slot represents the actual image acquisition. The Analysis slot represents the online image analysis. The Script Run* slot represents the execution of the experiment feedback loop script. Note that the loop script will be only executed when triggered by a used observable inside the loop script. The HD Writing slot represents the slot for writing the image data to your hard drive.
Allow additional loop script runs		Triggers the main loop if observables that are not part of the multidimensional experiment (e.g. frame index, time index, block index, etc.) change. Those observables could be time or temperature of the incubation. If the checkbox is not activated only experiment observables trigger the main loop script. This only applies when the acquisition is idle. This only applies when acquisition is idle.

Guided Acquisition

This module enables you to create an automated workflow to acquire overview images, detect relevant objects with an image analysis and re-image these positions, using another experiment. Additionally, you can set up the Guided Acquisition to automatically repeat the detailed scan or the entire workflow to observe changes over time, such as the growth of organoids or embryos.

Guided Acquisition Workflow:

Scan or inspect a large area (or over a long period of time).
Perform an analysis to detect interesting objects.
Acquire detailed images for every detected object.

A possible application is to detect rare events, for example to find transfected cells. For example, the sample contains many cells that are stained with a blue dye, but only a few are additionally expressing GFP. Guided Acquisition allows you to find these cells and run another, for example high-magnification, experiment on these positions.

After performing a (low-magnification) overview scan, the image analysis detects all cells and determines which of them are expressing GFP, i.e. show a certain intensity in the GFP channel. Then the microscope revisits all GFP-expressing cells and performs a second acquisition there, for example with higher magnification, a z-stack, etc. The analysis results are automatically saved to a folder.

Preliminary Work to Guided Acquisition

For a successful Guided Acquisition experiment, you need to prepare an overview and a detailed experiment as well as an image analysis setting. If you want to process your overview image before it is analyzed, you also need a suitable setting for each processing step or function you want to execute.

Overview scan

You have defined the experiment for the overview scan. Typically, the overview scan is using a lower magnification in combination with a tile experiment.

Processing setting(s)

You have defined a suitable setting for each processing step or function you want to execute. For more information, see General Settings.
Note that if you want to use Shading Correction with a reference image, you have to define your setting in Batch mode!

Image analysis setting

You have defined a suitable image analysis setting, see Creating a New Image Analysis Setting.

Detailed scan

You can perform for example the following experiments:

simple Z-stacks with a high-NA objective lens
Multi-Channel Z-stacks using an optical sectioning method like SD, Apotome or Airyscan
Tile experiments
Note: If you have activated Auto-Distribute for New Tile Regions for support point distribution in the Tiles tool, this is saved in the experiment and used for Guided Acquisition as well.
Time Series
Note that for detailed experiments that are defined as a time series, a time series for each detected object is performed before moving to the next detected object. In order to create a time series which acquires the first time point for all detected objects before starting the acquisition for the second time point, use the Repeat Detailed Scan option.

Parcentricity

If you use different detectors for the overview and the detailed scan, it might be necessary to correct for the shift between both detectors (to ensure parcentricity). For this you have to take an image at the exact same position with both cameras and then determine the offset between the two (to ensure parcentricity). The reference to calculate this offset is the image taken with the camera for the overview experiment. You can then enter the values for the shift in X and Y in the guided acquisition setup.

Focus strategies

You have several options, to perform a focus strategy.

Detailed scan and overview scan can be defined with their own focus strategy using Focus Surface and/or Software Autofocus.

During the Guided Acquisition experiment, you can define additional focusing steps. They are independent from the focus strategy defined on the Acquisition tab, see Focus Strategy Tool.

For more information on Guided Acquisition, see Performing a Guided Acquisition.

Using Guided Acquisition Settings

Guided Acquisition offers you the possibility to save your whole experiment setup in a settings file. This file is saved in the folder for your Guided Acquisition experiment together with all the other settings, for example the overview and detailed experiment, the image analysis setting, and the image processing settings, if a processing step was selected.

Creating a Guided Acquisition setting

On the Applications tab, open the Guided Acquisition tool.
Click and select New.
Name the setting and press Enter on your keyboard or click .

You have created a setting for Guided Acquisition.

Saving a Guided Acquisition setting

When you have set up your Guided Acquisition experiment and created a setting, you can save the set up as a setting.

Click and select Save.

Your experiment set up is now saved.

Importing and exporting a Guided Acquisition setting

On the Applications tab, open the Guided Acquisition tool.
Click and select Import or Export.
A file browser opens.
Select the file you want to import or the folder where you want to export the setting to.
Click Open/Save.

You have now imported/ exported a setting.

Deleting a Guided Acquisition setting

On the Applications tab, open the Guided Acquisition tool.
Select the setting that you want to delete in the drop-down list.
Click and select Delete.
Confirm that you want to delete the file.

The selected setting is deleted.

HDR Confocal Basic

This module enables the acquisition of image data with extended dynamic range by automatic acquisition and combination of images with different excitation intensities.

Multi-Channel

This module enables you to acquire fluorescence and transmitted light images in independent channels with a technically unlimited number of independent channels for reflected light and transmitted light techniques. It provides a fully automatic generation of the required microscope setting for a channel with the possibility of adjusting the setting manually for the channel. It supports simultaneous acquisition of two channels using two synchronized cameras and offers independent exposure times and shading corrections for each channel.

Software Autofocus

This module offers a configurable image based autofocus functionality that will search through a series of axially stepped images analyzing the “sharpness” of each. The z-value of the image returning the maximum sharpness is set as the new plane of observation.

The module requires the microscope to be fitted with a motorized Z-drive. It does not require a z-piezo actuator nor is the z-piezo used by the software autofocus (SWAF) in the current implementation. On the Acquisition or Locate tab the settings for the SWAF can be adjusted in the Software Autofocus tool. These can also be called (and tested) by clicking the Find Focus (AF) button on the main button bar on Acquisition tab. SWAF settings are stored as part of an experiment on Acquisition tab.

The configuration allows the function of the SWAF run to be matched to the conditions under which the focus should be found. A basic description of the functions adjusted by the individual controls can be found further below. However, before going into the description of each parameter, we will try to address the following questions: How does the SWAF in the software attempt to locate the “focus”? And how do the parameters settings influence its behavior in this respect?

Terminology & Abbreviations

Perhaps the best place to start is with an explanation of the terms encountered when working with focus strategies before looking at the individual strategies in detail. Many of these terms are also encountered in the Tiles & Positions and Software Autofocus module. The nomenclature takes some time familiarize with due to its subtleties. Here is a list of the more common terms:

Term/Abbreviation	Description
SWAF	Stands for Software Autofocus.
DF,DF.1 or DF.2	Stands for Definite Focus, Definite Focus.1 or Definite Focus.2
Tile	One of the individual image fields that make up a tile region i.e. a 2x2 Tile region is made up of 4 tiles arranged as a grid. The tiles have a given overlap with their neighbors (default setting 10%) allowing them to be stitched together as one image if necessary. Unless otherwise specified by a focus strategy, each tile has the same z-value as the parent Tile region. After acquisition, the individual tiles are displayed together as part of the tile region to which they belong, which in turn makes up one scene.
Tile Region	In a tile experiment a tile region refers to an ordered group of individual image fields (or tiles) that belong together and are arranged in the form of a grid (these arrangements can be based on quadrilaterals, circles, ellipses or freehand polygons) with a predefined overlap (default 10%) to facilitate stitching the images together. With the help of tile regions it is possible to acquire areas with dimensions that vastly exceed the size of an individual image field. Within an experiment a number of tile regions can be acquired at various localities/ wells/ containers on the sample. Each tile region is based on an X and Y coordinate of the stage and a Z coordinate of the focus drive and are defined using the Tiles tool. After acquisition, the individual tile regions are displayed as scenes to facilitate viewing.
Position	In a tile experiment position refer to independent, individual image fields that placed at various locations on the sample. A position corresponds to (or is in some ways equivalent to) a tile region consisting of just one tile. Each position is based on an X and Y coordinate of the stage and a Z coordinate of the focus drive. Individual positions or position arrays (grouped individual positions) are defined using the Tiles tool. After acquisition, the individual positions are displayed as scenes.
Reference Channel	The channel selected as a reference z-value for focus strategies and events in particular a SWAF. The selected reference channel can be changed in the Reference channel expander or in the Channels tool. It is also possible to define a relative axial offset to the reference channel. This can be done for one or more other channels.
Focus Surface	Refers to the interpolated surface of z-values derived from support points (discrete z-values) defined by the user (or by functions such as SWAF or DF.2) prior to the experiment (or immediately before acquisition start). A focus surface can be “local” or “global”. The local form is confined to a single Tile region and attempts to describe the sample topography covered by the tile region such that all its image fields (tiles) will be in focus. The global surface form is technically identical, but is associated with a sample carrier, and defined in the sample carrier template dialogue. Thus, tile regions or positions placed on this carrier will follow the slope or contour defined by a topography that covers part or most of the sample carrier. In both cases the surface is defined by interpolation from discrete z-values – so called “support points”. Note that a positions z-value is used as its local surface, and as such does not require a support point. Global and local surfaces cannot be mixed in a single experiment (or block).
Support Point	To create a focus surface it is necessary to define one or more support points. Support points are user defined collections of z-values that correspond to the desired plane of observation at a given XY-coordinate. They can also be defined initially by a SWAF run or DF.2 recall focus function- initially, after the experiment is started, but before the first loop of images are acquired. The number of support points, defined by the user, can be distributed automatically by an algorithm, re arranged individually by hand or placed at the current stage position. The number of support points employed determines the degree of interpolation that can be used to generate the topography of the focus surface. Typically, the interpolation criteria (minimum number of support points required to generate a certain degree) should be over filled with support points, and a lower interpolation degree selected for more robust results. By default the software employs an interpolation degree of level 2 (which can generate a parabolic saddle surface with at least 9 support points). If too few support points are used the next lower level (a “tilted plane”) will be use automatically. Higher interpolation degrees have to be manually selected, but for most use cases are typically not necessary.
Z-value	The current Z coordinate of the focus drive that is used to define a Tile region, position or support point when it is created by the user. Note that the individual tiles of a tile region all have the same initial z-value unless support points are used either in the context of a local or global focus surface, a software autofocus is used to determine them individually or a definite focus stabilization adjusts them. The z-value of a position defines its z-coordinate initially when a local focus surface is used. Positions spread on a global focus surface (carrier based) are adjusted accordingly as are the individual image fields of a tile region.
Adapt Z Values/Focus Surface	The focus strategy Use Z Values/Focus Surface defined in Tiles Setup allows the Focus surface or z-values defined in the Tiles tool to be modified by the result of a SWAF or DF stabilization based on these initial values. These functions are not available when no SWAF module is present or no DF is configured. The function has several module/hardware dependent variations:
- As Additional Action	In focus strategies that use a focus surface or z-value defined by the Tile setup (tool) it is possible to optionally execute a so called “additional action” (a stabilization event) that adapts the focus surface/ z-values. This occurs after the reference z-value has been reached as defined in the tiles set-up for each discreet z-value (i.e. each tile/position or the defining focus surface). Depending on the system configuration this can be a SWAF run or a DF stabilization. In the case of a SWAF run the initially defined reference z-value is used to center the search range defined in the SWAF settings. Thus, a SWAF run can be centered on the sample topology increasing the effectiveness and/ or speed at which a maximum is detected and subsequently used for image acquisition. In certain applications, such as Correlative array tomography (CAT) this function can be performed with DF instead. In this case a local focus surface is used to make sure that the DF stabilization stays within the catchment range of the device (only important for DF.1!). Complimentary to this is the number of support points needed to initially define the surface can be significantly reduced for a large elongated Tile region - which greatly reduces set-up time to image the extremely thin (typically 70 nm thick or less) “ribbon” of serial sections.
- Update with Single Offset	In combination with a Definite Focus or SWAF if a time series is used it is possible to make use of a focus surface or z-value defined by the Tiles setup and execute a so called “update” (a stabilization event) – this makes use of a SWAF run or a DF stabilization to update the Focus surface/z-value defined initially by the Tiles set-up. In a time series the update action is performed once each time point (or every nth) at a single discrete “wait position” (default center of 1st Tile region / position). A change in Z (thermal or residual focal drift) at the wait position - if detected - is then applied to all the focus surfaces or Z-values defined in the Tiles Setup (adapting them all by the change in Z, applied as a common offset). In some cases it is useful to be able to define a specific waiting position – for example, when a special sample carrier is used where the DF reflex signal might be disturbed by its structure/optical properties at the first tile region/position. Alternatively, if using a SWAF some kind of fiducial marker or such is available at this position that does not change (e.g. bleaching or movement) can be used.
- Update with Multiple Offset	For Definite Focus.2 only an additional function is available that allows the device to be initialized on each and every z-value prior to the experiment and hence stabilize and update these individually according to their location relative to the sample/ glass interface. This function can be used with or without a time series dimension. Thus, DF.2 enables true multi-location experiments in which the user defined z-values (including support points) are used by DF.2 to create a stabilization map that is monitored and updated throughout the experiment.
Initial Definition for Z Values/ Focus Surface	This function allows you to select how the initial z-values used in the experiment are defined. By default this is By Tiles Setup and the z-values specified there (in the Tiles tool) are used. However, it is possible to define or adjust these z-values directly before the experiment (after clicking Start Experiment) either with a SWAF run or a with a DF.2 Recall Focus (Axio Observer). For the Celldiscoverer 7 this drop down offers the additional options Find Surface or Find Surface + Additional Offset to define the initial z-values. In this case the z-values are initially defined by the z-values resulting from this “pre-run” before the imaging loop starts. This can be particularly useful when working with multi well plates or chamber slides where the sample is located at a similar position relative to the carrier surface in each well or chamber. It also allows the imaging loop of the experiment itself to be speeded up and to be run in a triggered or compromised protocol (fast acquisition) thus reducing the time to complete the imaging loop of the experiment.
Stabilization Event Repetitions and Frequency	Defines the frequency and repetition of stabilization events within a given focus strategy. For the DF and SWAF focus strategies you can determine when and where in the experiment these events are executed in synchrony to the imaging loops – a loop here means time series, or positions for example, with the event synchronized to occur immediately prior to the chosen loop. A general limitation of this implementation (to limit code complexity) is that these stabilization events can only be synchronized to iterate with a single imaging loop entity i.e. the selection is only possible in a mutually exclusive manner. These settings can be accessed only when "Show all" is activated and expert mode is selected. Initially default settings are assigned and can be restored by clicking the "Standard" button. In "Expert" mode the settings are displayed and can be, if necessary, modified. Depending on the dimensions of the experiment or focus strategy different parameters can be modified to meet the experiment needs. For the Tile Region loop you can optionally select where the event occurs within the Tile region - either in the center or at the 1st Tile of the region (typically upper left hand corner). This is of use when using SWAF events as often the upper left hand corner of a Tile region might not contain sample, thus often the SWAF run will not return a suitable maxima (new z-value). Finally, focus strategies that include Definite Focus and are used with a time series dimension may also allow stabilization during the interval of the time series i.e. asynchronous to the imaging loops of the experiment. This might be necessary if the time interval is on the order of tens of minutes, or if a large thermal drift is expected (more significant for DF.1), or if the time series has no or a very short interval (i.e. fast as possible acquisition at a single position) allowing synchronized events to be disabled completely.
Focus Surface Outlier	Under Tools > Options > Acquisition > Tiles you find the option Enable Removing of Focus Surface Outlier. By two parameters you can define how so called “outlier” values are handled prior to calculation (interpolation) of a focus surface. This is particularly helpful when the z-values that will be used for this purpose contain one or more values that differ obviously from the others (for example if a SWAF run has returned a z-value that does not lie close the sample plane of interest). If not removed such values locally distort the focus surface potentially producing “blur” in the resulting images. By default a linear fit is used to detect such outliers in combination with a statistical threshold value (sigma). Values that do not meet these criteria (i.e. are significantly outside this) are classified as outliers and are not used to calculate the focus surface that will be subsequently generated for the experiment. In extreme use cases it is possible to modify the sigma value or use a mean value instead of a linear fit for this purpose, but typically these default values never need to be changed.

When is focus the "right" focus?

In microscopy, the focus can be implied from image parameters, such as the contrast or intensity, that vary with the position of the objective’s plane of observation in the sample and the level of detail at a given plane. However, an algorithm that tries to detect (and maximize) such values will only return an axial position that corresponds to a plane of interest if these coincide (which is typically the case with (thin) samples with a singular discrete plane of detail).

This becomes increasingly difficult with higher numerical aperture (NA) lenses, thicker samples, and less pronounced levels of detail (modulated as change in contrast or intensity in the resulting image). Hence, SWAF is not to be understood as a focus finder, but can be used as a method for reliably searching over a given axial range and locating such a plane in a sample. Thus, although not all samples and imaging conditions will be appropriate, SWAF is an approach that allows a useful detection of a focus plane as a start for further imaging activities.

Software Autofocus Using LSM Tracks

Confocal Tracks are also suitable as reference Channels for the Software Autofocus. As LSM acquisition is by design slower compared to Camera acquisition, some optimizations are done in the background in order to speed up the focusing action.

The typical measure for the correct focus position in confocal images is the intensity. Hence the aim of the SWAF is here not to generate images of a certain quality, but only to evaluate relative image intensities along the z-stack. This allows us to use very coarse scanning parameters.

Generally, the SWAF for LSM uses a fixed Frame Size of 64*64 pixels in combination with the fastest possible scan speed at the currently configured zoom. To further speed up the acquisition, bidirectional scanning is used. Whatever Laser power you specify in the Channels tool window for this Track is used during the SWAF action. Of note, while you assign a reference Channel, the corresponding Track with all its channels will be active during the focusing.

Some behavior depends on the selected Search Mode Full or Smart.

In the Full search, the system will use the detector gain as configured in the Channels tool window.

In contrast, the Smart search aims to start close to the likely intensity maximum of the z-Stack. This focus position is approximated by a fast line z-stack in the center of the image frame. As the line scan generates less pixels and a higher noise level, a useful dynamic range needs to be ensured. To this end, the fast line z-stack is repeated several times with increasing PMT gain. After the line scan, regardless if an intensity peak was found or not, a frame wise autofocus will follow.

In case no peak could be identified, e.g. because of a sparsely distributed sample, the Smart search will start at the original z-position and not optimization of the starting position will take place. Essentially, the Smart search will outperform the Full search on high Search Ranges and a highly varying effective focus position.

If the focus fluctuations are predictably small, a narrow Search Range in combination with a Full search might be faster. As a final remark, Camera-based Autofocus can be time-saving, especially when the search range needs to be large and a Full Search is required. While Camera and LSM cannot be combined into one image document, the deactivated camera Track may be still be used as a reference Track.

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.

Tile Regions

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

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.

Positions (Single Positions)

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

General Preparations

You have licensed this functionality and activated it under Tools > Toolkit Manager.
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.
You are on the Acquisition tab.
You have created a new experiment, defined at least one channel and correctly set the focus and exposure time.

Activate the Tiles checkbox in the Acquisition Dimensions section to display the Tiles tool.
In the Left Tool Area the Tiles tool appears in the Multidimensional Acquisition tool group.

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

You are on the Acquisition tab in the Tiles tool.

Start the Live mode to use the stage to locate a point that you want to be at the center of your tile region.
Bring the specimen into focus using the focus drive.
Open the Tile Regions section.
The Tiles mode is activated by default.
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.
Click .
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.
To add further tile regions, move the stage to another position on the sample and repeat the previous steps.
The added tile regions (TR1, TR2, etc.) are displayed in the tile regions list.
If you scroll to the right in the table, you can read the Size of the tile regions.
Save the experiment. To do this, in the Experiment Manager click and select Save As. Enter a name for the experiment in the input field (e.g. Simple Tile Experiment).
Click Start Experiment.
The Tile Region experiment is acquired.
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.

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

You are on the Acquisition tab in the Tiles tool.

Open the Positions section.
Start the Live mode to use the stage to locate a position that you want to acquire.
The X and Y coordinates of the current position are displayed in the Current X/Y display fields.
Bring the specimen into focus using the focus drive.
Click .
The current position is added to your experiment.
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).
To add further positions, move the stage to another position on the sample and repeat the previous steps.
The added positions are shown in the list in the Single Positions section with their X, Y and Z-coordinates.
Save the experiment. To do this, in the Experiment Manager click and select Save As. Enter a name for the experiment in the input field (e.g. Simple Tile Experiment).
Click Start Experiment.
The Positions experiment is acquired.
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.

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.

Adjusting Z-Values

If you add positions or tile regions, the current Z-value is automatically adopted for the tile region or position.

Learn about how to adjust and verify the Z-values of positions in the chapter Adjusting Z-Values of Positions.
Learn about how to adjust and verify the Z-values of tile regions in the chapter Adjusting Z-Values of Tile Regions. Note that the Z-values defined here are valid for all tiles in the respective tile region.

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 will still select the appropriate focus strategy for you.

Adjusting Z-Values of Tile Regions

You have set up a Tiles experiment with at least one tile region.

To check the z-value of tile regions, open the Tile Regions section in the Tiles tool.
The z-values of the tile regions are displayed in the Z column of the list.
Double-click on the list entry of the tile region that you want to check.
The stage automatically locates the center of the tile region and the associated z-position.
Use the Live mode to check the z-value of the tile region.
To adjust the z-value, set the new z-position with the Focus tool.
In the Tile Regions list, click on Options and select Set Current Z For Selected Tile Regions. Alternatively, in the Tile Regions list, right-click the tile region entry and select Set Current Z For Selected Tile Regions.
To check further tile regions, repeat steps 2 to 4.
To check and adjust large number of tile regions, click on the Verify button.
The Verify Tile Region dialog opens. There you have an interface for the verification process of each tile region.
Click on Close after you have verified all tile regions.

You have successfully checked and adjusted the individual z-values for the tile regions.

Adjusting Z-Values of Positions

You have set up a tile experiment with at least one position.
You are on the Acquisition tab in the Tiles tool.

To check and adjust the z-value of positions, open the Positions section.
The z-values are displayed in the Z column of the Single Positions list.
Double-click on the list entry of the position that you want to check.
The stage automatically locates the position.
Use the Live mode to check the z-position of the position. To adjust the z-value, set the desired position using the focus drive.
Select a position in the Single Positions list, click and select Set Current Z For Selected Positions. Alternatively, in the Single Positions list, right-click the position entry and select Set Current Z For Selected Positions.
The current z-value is set for the respective position.
To check and adjust a large number of positions, use the Verify Positions dialog. Click Verify Positions in the Positions section.
The Verify 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 Move to Current Point. Alternatively, you can double-click on the position in the list that you want to check.
The stage moves automatically to the position in the list that is highlighted in blue.
In the Live mode use the Focus (or SW Autofocus) tool to adjust the desired z-value.
Click Set Z and Move to Next.
The position is marked with a check mark.
The stage moves automatically to the next position in the list.
Repeat the last 3 steps until you have checked all the points in the list.
The message All points have been verified appears.
Close the Verify Positions dialog.

You have successfully verified and adjusted the individual z-values for positions.

Exporting Tile Images

You have acquired or opened a tile image.

On the Processing tab, open the parameters for Image Export (Ctrl+6 or via File > Export/Import > Export).
You will see the settings of the parameters for Image Export. Make sure that the Show All mode has been activated.
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.
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%.
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.
Select the Define Subset radio button.
The settings for the available dimensions open.
Open the settings for the Tiles dimension.
Select the Existing Tiles radio button.
Click on the Apply button at the top of the Processing tab.
You have exported the individual tiles from a tile image. The files can be found in the export folder indicated.

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.

To set up tiles experiments in Advanced Setup, you need the Tiles & Positions module.
You have read the general introduction, see Tiles & Positions.
You are on the Acquisition tab in the Tiles tool.

Click Show Viewer.
The Tiles Advanced Setup view opens. For more information, see Tiles Advanced Setup.
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.

Creating Tile Regions by Contour

You have generated a preview scan that will help you to position the tile regions more easily.

In the left toolbar in the Tiles section, click on the Setup by contour button.
The Contour Toolbar is displayed as the top toolbar.
In the top toolbar, select the desired contour tool.
Use the contour tool in the stage view to draw in the tile regions you want to acquire.
Tile regions are created for each marked region. They are added to the list in the Tile Regions section of the Tiles tool.

You have successfully created tile regions in Advanced Setup.

Creating Positions by Location

In the left toolbar in the Positions section, click on the Setup by location button.
The Positions Location Toolbar is displayed as the top toolbar.
In the top toolbar, click .
In the stage view, click on the location where you want to add a position.
The added positions are displayed in the stage view and the Single Positions list in the Positions section of the Tiles tool.

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

In the Tiles tool, open the Positions section and click on Position Arrays.
In the left toolbar in the Positions section, click on the Setup by array button.
The Position Array Toolbar is displayed as the top toolbar.
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.
Mark the interesting area in the Center Screen Area with a pressed left mouse button.

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

You have selected and calibrated a sample carrier template.
You are on the Acquisition tab.

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.
In the left toolbar in the Positions section, click on the Setup by sample carrier button.
The Positions Carrier Toolbar is displayed as the top toolbar.
Select the containers in which you want to distribute positions by pressing the Ctrl key and clicking on the relevant containers.
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.
Click on the Create button .
The selected containers are each filled with a Position Array (group of positions).
In the Positions section of the Tiles tool, the Position Arrays are displayed in the Position Arrays list.

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

You are in the Advanced Setup.

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.
A file browser opens.
In the file browser, select your preview scan image (*.czi) and click on Open.

You have now imported a preview scan image into the Tiles Advanced Setup.

Importing an image already opened in ZEN

You are in the Advanced Setup.
You have an image opened in ZEN.

In the Right Tool Area, in the Images and Documents tool, select the image you want to import.
Drag and drop the image into the viewer of the Advanced Setup.

You have now imported an already opened image into the Tiles Advanced Setup.

Importing from Explorer

You are in the Advanced Setup.
You have opened the path to your image (*.czi) in the Windows File Explorer.

In the Windows File Explorer, select the image you want to import.
Drag and drop the image into the viewer of the Advanced Setup.

You have now imported an image from the Windows File Explorer directly into the Tiles Advanced Setup.

Copying a Tile Region or Position

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

You have licensed this functionality and activated it under Tools > Toolkit Manager.

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.
Right-click in the setup and select Paste. Alternatively, press Ctrl+V.

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.

You have licensed this functionality and activated it under Tools > Toolkit Manager.

Select the well from where the tile region/ position setting should be copied.
The selected well is now highlighted by a blue border.
Right click within the selected well in the Center Screen Area (outside the tile region) to open the context menu.
Select Copy Container for replication.
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.
Right click in the Center Screen Area and select the context menu entry Paste Replication to and either choose Selected Container or All Container.

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.

Local and Global Focus Surfaces

Creating a Local Focus Surface

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.

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.

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.

Verifying Z-Values of Support Points

In the Tiles tool open the Focus Surface and Support Points section.
Go to the Global (on Carrier) tab.
All the support points of the selected sample carrier template are displayed in the Support Points list.
Click on the Verify button.
The Verify Global Support Points 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 Move To Current Point.
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.
In the Live mode use the Focus (or SW Autofocus) tool to set the z-value.
Click on Set Z and Move to Next.
The support point is marked with a check mark.
The stage automatically moves 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 Global Support Points dialog.

You have adjusted and verified the Z-values of all support points.

Selecting Interpolation Degree

Select the interpolation degree in the Interpolation Degree dropdown list in the Focus Surface and Support Points section.

You have successfully created a global focus surface.
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.

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.

You have licensed this functionality and activated it under Tools > Toolkit Manager.

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.
Repeat this step to rename different tile regions or positions.
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.
Under the Tile Regions/Positions list, in the Properties section Category, click on the Options button and select New from the dropdown list.
The New Category window opens.
Enter a Name and add a Description for the selected tile regions/ positions.
Assign a Color for the new category by clicking on the color bar and choosing a preferred color.
Click on OK to create the new category.
The New Category window closes and the new category is created.
As Category choose the desired category for the selected tile regions/ positions from the dropdown list.

The chosen category is now assigned to the selected tile regions/ positions.

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.
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).
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.)
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)

You have selected several different positions or tile regions and assigned different categories.

Under Positions or Tile Regions of the Tiles tool, select a position or tile region.
Right-click on the selected position/ tile, choose Sort and select By Category

The positions/ tiles will be sorted alphabetically according to the assigned categories.

Tiles Tool

The basic Tiles tool is only visible if you have a motorized stage configured with your microscope. The Tiles Advanced Setup and many other functions are only available if you licensed this functionality and activated it in the Toolkit Manager. Additionally, you must activate the corresponding checkbox on the Acquisition tab in the Experiment Manager. This tool is part of the basic license for LSM.

In the Tiles tool you configure the acquisition of images that consist of several image fields. Therefore you define Tile Regions or Positions. In addition you can set up focus surfaces and sample carrier templates here.

The Tiles tool is located in the Left Tool Area under Multidimensional Acquisition.

Parameter	Description
Show Viewer	Only available if you have the license for the Tiles & Positions functionality. Opens the Tiles Advanced Setup view in the Center Screen Area.

Parameter

Description

Show Viewer

Only available if you have the license for the Tiles & Positions functionality.

Opens the Tiles Advanced Setup view in the Center Screen Area.

The Sample Carrier, Focus Surface and Options sections are only visible if the Show All mode is activated.
If you have no license for the Tiles & Positions module you will only find Tile Regions, Positions and Options sections here.

The different sections of the tool are described in the next chapters.

Tile Regions Section

Here you can define the desired tile regions and add it to the image.
Note: The first section with controls is only visible if you have no license for the Tiles & Positions module. With a license, these controls are selected from the Left Toolbar in the Tiles Advanced Setup.

Parameter		Description
Contour		This parameter is only visible if the Show All is activated. Here you select the shape or contour of the tile region that you are adding. Simply click on the corresponding button to select the desired contour. The selected contour is highlighted in blue color.
-	Rectangle	If selected, you can create rectangular tile regions.
-	Circle	When selected, you can create circular tile regions.
Mode
-	Tiles	If selected, you have to enter the number of tiles as a reference for the size of the tile region. Enter the number of tiles in the X/Y input fields. If you are adding a circular tile region, enter the number of tiles for the diameter in the Diameter input field.
-	Size	If selected, you have to enter the size as a reference for the size of the tile region. Enter the size of the tile region in the X/Y input fields. If you are adding a circular tile region, enter the diameter of the tile region in the Diameter input field.
-	Stake	If selected, you can define a tile region by the placement of at least two markers (user defined X/Y stage coordinates). If you want to modify the tile region (expand/reduce) you have to adjust the tile region to the desired size. To complete the tile region press Done. Circular or rectangular tile region can be created in this manner by selection of the appropriate contour.
-	Add	Adds the tile region to the image. The added tile region will also appear in the Tile Regions List and is activated for acquisition. Added tile regions are displayed in the form of red grids in the stage view of the Advanced Tiles Setup.

Tile Region List Options

Parameter		Description
Tile Regions List		Displays the added tile regions. The list contains the following columns:
-	Checkbox	Activates the relevant list entry for acquisition.
-	Name	Here you can edit the name of the tile region.
-	Category	Displays the category of the tile region. Categories can be defined in the view options of the advanced tiles setup on the properties tab.
-	Tiles	Displays the number of tiles of the tile region.
-	Z	Displays the z-position of the tile region.
-	Size	Displays the size of the tile region along its x and y axes in micrometers.
Up and Down		With the Up/Down buttons you can shift the selected list entry one position up or down in the tile regions list. This allows you to modify the acquisition order. Note that the order in the list will only be respected if the sorting of tile regions/ positions is deactivated in the Options section (Stage Travel Optimization)!
Delete		Deletes the selected list entry.
Options		If you click on the button, you see the following 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 Regions	Sets the current X/Y/Z-Position for all selected tile regions.
-	Delete	Deletes the current tile region.
-	Delete All	Deletes all tile regions.
-	Activate	Activates the current tile region for acquisition.
-	Deactivate	Deactivates the current tile region for acquisition.
-	Unlock	Unlocks the current tile region. The tile regions or positions are only locked if created in carrier mode.
-	Unlock All	Unlocks all locked tile region.
-	Sort	By Center Position (Y -> X) sorts all tile regions according to their overall Y position. By Center Position (X -> Y) sorts all tile regions according to their overall X position. By Category sorts all tile regions according to their category. Note that the order in the list will only be respected if the sorting of tile regions/positions is deactivated in the Options section (Stage Travel Optimization)!
-	Convert to Positions…	Converts a selected tile region into Positions or a Position Array.
Properties Tile Region		Only visible if you have license for the Tiles & Positions module. Displays the name of the currently selected tile region if one region is selected.
Category		Only visible if you have license for the Tiles & Positions module. Shows the currently assigned category of the selected tile region. The Default category is set for all new tile regions.
Options		Only visible if you have license for the Tiles & Positions module. Opens the options for editing and creating categories.
-	New	Opens the New Category dialog to create a new category.
-	Edit	Opens the Edit Category dialog to edit the selected category.
-	Delete	Deletes the selected category and sets the category of the tile region to Default.
X		Only visible if you have license for the Tiles & Positions module. Displays and sets the x-value of the selected tile region.
Y		Only visible if you have license for the Tiles & Positions module. Displays and sets the y-value of the selected tile region.
Z		Only visible if you have license for the Tiles & Positions module. Displays and sets the z-value of the selected tile region.
Set Current Z		Only visible if you have license for the Tiles & Positions module. Sets the Z dimension at the current Z position of the focus drive.
Width		Only visible if you have license for the Tiles & Positions module. Displays and sets the width of the selected tile region.
Height		Only visible if you have license for the Tiles & Positions module. Displays and sets the height of the selected tile region.
Verify		Only visible if you have license for the Tiles & Positions module. Opens the Verify Tile Regions dialog. There you can verify each point of the tile region according focus und position.

Positions Section

Current Position section

Displays the current stage position.
Note: The first section is only visible if you have no license for the Tiles & Positions module. With a license, this functionality is visible in the Left Toolbar of the Tiles Advanced Setup.

Parameter	Description
X Position	Displays the x coordinate of the current position.
Y Position	Displays the y coordinate of the current position.
Add	Adds the current position to the Positions List and activates it for acquisition.

Display mode section

Parameter		Description
Single Positions		Shows the Single Positions List. To learn more about single positions see glossary "Position".
Position Arrays		Shows the Position Arrays List and the Positions of selected Array List, that shows a full Single Positions List for the selected position array. To learn more about position arrays see glossary "Position".
Single Position List/Position Array List		Displays the added positions/ position arrays. The list contains the following columns and buttons.
–	Checkbox	Activates the relevant list entry for acquisition.
–	Name	Displays and edits the name of the single position.
–	Category	Displays the category of the single position. Categories can be defined in the view options of the advanced tiles setup on the properties tab.
–	Contour	Only visible if you have selected Position Arrays. Displays the contour of the position array.
–	Positions	Only visible if you have selected Position Arrays. Displays the number of positions of the position array.
–	Size	Only visible if you have selected Position Arrays. Displays the size of the position array.
–	X	Displays the x-position of the single position.
–	Y	Displays the y-position of the single position.
–	Z	Displays and edits the z-position of the single position.
–	and	With the buttons you can shift selected list entry one position up or down in the tile regions list. This allows you to modify the acquisition order. Note that the Tile Regions/Positions checkbox has to be deactivated in Tiles Options. Note that the order in the list will only be respected if the sorting of tile regions/ positions is deactivated in the Options section (Stage Travel Optimization)!
–	Delete	Deletes the selected list entry.
–	Options	Opens the Options for Editing Single Positions or Options for Editing Position Arrays respectively.
Verify		Opens the Verify Tile Regions or Verify Positions Dialog.
Properties Position		Only visible if you have license for the Tiles & Positions module. Displays the name of the currently selected position.
Category		Only visible if you have license for the Tiles & Positions module. Shows the currently assigned category of the selected tile region. The Default category is set for all new tile regions.
Options		Only visible if you have license for the Tiles & Positions module. Opens the options for editing and creating categories.
-	New...	Opens the New Category dialog to create a new category.
-	Edit…	Opens the Edit Category dialog to edit the selected category.
-	Delete	Deletes the selected category and sets the category of the tile region to Default.
X		Only visible if you have license for the Tiles & Positions module. Displays and sets the x-value of the selected tile region.
Y		Only visible if you have license for the Tiles & Positions module. Displays and sets the y-value of the selected tile region.
Z		Only visible if you have license for the Tiles & Positions module. Displays and sets the z-value of the selected tile region.
Set Current Z		Only visible if you have license for the Tiles & Positions module. Sets the Z dimension at the current Z position of the focus drive.

Options for Editing Single Positions

Parameter		Description
Set Current Z for Selected Positions		Sets the current Z-Position for all selected positions.
Set Current XYZ for Selected Position		Sets the current X-Y-Z-Position for the selected position.
Delete		Deletes the current position.
Delete All		Deletes all positions.
Activate		Activates the current position for acquisition.
Deactivate		Deactivates the current position for acquisition.
Sort		Sorts the list entries according to the chosen parameter.
-	By Center Position (Y -> X)	Sorts all positions according to their overall Y position.
-	By Center Position (X -> Y)	Sorts all positions according to their overall X position.
-	By Category	Sorts all positions according to their category.
Import stage marks as positions		Only visible if you have license for the Tiles & Positions module. Imports the marks from the Stage tool as positions.

Options for Editing Position Arrays

Parameter		Description
Set Current Z for all Positions in selected Arrays		Sets the current z-position for all positions in the selected arrays.
Delete		Deletes the current position array.
Delete All		Deletes all position arrays.
Activate		Activates the current position array for acquisition.
Deactivate		Deactivates the current position array for acquisition.
Unlock		Unlocks the current position array.
Unlock All		Unlocks all locked position arrays.
Sort		Sorts the list entries according to the chosen parameter.
-	By Center Position (Y -> X)	Sorts all positions according to their overall Y position.
-	By Center Position (X -> Y)	Sorts all positions according to their overall X position.

Sample Carrier Section

Only visible if the Show All mode is activated and only available with a license for the Tiles & Position functionality.

Parameter	Description
Sample Carrier	Displays the selected sample carrier template. If no template is selected it will display None.
Select...	Not available for the Cell discoverer. Opens a dialog to select the sample carrier template.
(Edit Support Points)	Opens the sample carrier selection/editor dialog where you can edit and add global support points to the selected sample carrier.
Delete	Deletes the selected sample carrier from the sample carrier field. The template will still be available in the Select Sample Carrier Template dialog.
Calibrate...	Only available if a suitable channel is configured for the experiment. Opens the wizard which guides you through the sample carrier calibration.
Move Focus Drive to Load Position Between Containers	Activated: Moves the focus drive to the loading position during the movement of the stage to another container of the sample carrier (e.g. a well or slide). This prevents possible damage. Note that this behavior is only applied during an experiment.

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 Toolbars

In the Tiles - Advanced Setup your main features to create regions and positions are in two toolbars on top and on the left of the viewer. The options available in the top toolbar depend on what is selected in the left toolbar.

Left Toolbar

Here you select which setup you use to set tile regions or positions.

Parameter		Description
Preview		Displays the tools to define the settings for a preview scan, see Preview Scan Toolbar. Typically a low magnification objective and a channel which protects your sample (e.g. transmitted light) is used. This gives you a low resolution overview of the sample to mark tile regions and/or positions.
Tiles		Here you can select which setup you want to use to set the tile regions. The corresponding tools are displayed in the top toolbar, see Top Toolbar.
–	Setup by contour	Displays the tools to define tile regions by means of contour, see Contour Toolbar.
–	Setup by predefined	Displays the tools to define tile regions by means of number or size, see Predefined Toolbar.
–	Setup by stakes	Displays the tools to define tile regions by specifying two or more marker positions, see also Stake Toolbar.
–	Setup by carrier	Displays the tools to define tile regions automatically by means of the fill factor of the sample carrier, see also Tiles Carrier Toolbar.
Positions		Here you can select which setup you want to be used to set the positions. The corresponding tools are displayed in the top toolbar, see Top Toolbar.
–	Setup by location	Displays the tools to define positions by means of the location, see Positions Location Toolbar. You can add various positions in the Stage View using the mouse.
–	Setup by array	Displays the tools to define positions by means of position arrays, see Position Array Toolbar. You can add various contours for position arrays in the stage view.
–	Setup by carrier	Displays the tools to define positions automatically on the relevant sample carrier, see also Positions Carrier Toolbar.

Top Toolbar

Depending on what you have selected on the left toolbar of the Tiles - Advanced Setup, different features to create tiles or positions are displayed. For information on the various top toolbars, see the list below.

Preview Scan Toolbar

Here you can define the settings for a preview scan. Typically a low magnification objective and a channel which protects your sample (e.g. transmitted light) is used. This will give you a low resolution overview of the sample to mark tile regions and/or positions.

Parameter	Description
Use Experiment Settings	Uses the settings of the experiment for the preview scan.
Channel	Only available if Use Experiment Settings is deactivated. Displays the channels for the preview scan. If you deactivate Use Experiment Settings, you can deselect individual channels here.
Camera Binning	Only available if Use Experiment Settings is deactivated. Sets the binning of the camera for the preview scan experiment only. The exposure time will be compensated automatically for the preview scan. Note that higher binning will reduce the scan time (due to shorter exposures of the camera), but reduce the image resolution.
Delete existing preview images	Activated: Deletes the already existing preview images before the preview scan is started.
Start	Starts the preview scan to acquire the overview image.

Contour Toolbar

Here you can define the tile regions by means of the contour.

Parameter	Description
Selection Mode	With this tool you can select an already created tile region by clicking on it to move or edit it.
Rectangle	With this tool you can draw a rectangle tile region.
Ellipse	With this tool you can draw an elliptical tile region.
Polygon	With this tool you can draw a polygonal tile region.
Keep Tool	Activated: Keeps the selected tool active. You can use the tool several times in succession without having to reselect it.

Predefined Toolbar

Here you can define the tile regions by means of the number or size.

Parameter	Description
Rectangle	Draws a rectangular tile region.
Circle	Draws a circular tile region
Tiles	Using this mode you have to enter the number of tiles as a reference for the size of the tile region. Enter the number of tiles in the X/Y input fields. If you are adding a circular tile region, enter the number of tiles for the diameter in the Diameter input field.
Size	Using this mode you have to enter the size as a reference for the size of the tile region. Enter the size of the tile region in the X/Y input fields. If you are adding a circular tile region, enter the diameter of the tile region in the Diameter input field.
Top left anchor position	The anchor of the defined shape is at the top left.
Center anchor position	The anchor of the defined shape is centered.
Bottom right anchor position	The anchor of the defined shape is at the bottom right.
Add Tile Region	Adds the tile region to the Tile Regions List and activates it for acquisition. Added tile regions are displayed in the form of red grids in the stage view of the Advanced Tiles Setup.
Selection	Select an element in the stage view to edit or move it.
Add Tile Region	Adds the current tile definition in the image area.
Keep Tool	Activated: Keeps the selected tool active. You can use the tool several times in succession without having to reselect it.

Stake Toolbar

Here you can define the tile regions with stake markers.

Parameter	Description
Add Marker Position	Adds a marker position to the current stage and focus position.
Remove Marker Position	Removes the last marker position.
Rectangle	Draws a rectangular tile region.
Circle	Draws a circular tile region
Polygon	Draws a polygonal tile region
Done	Saves the current tile regions setup and resets the marker positions.

Tiles Carrier Toolbar

Here you can define the tile regions automatically by means of the fill factor of the sample carrier.

A sample carrier must have been selected in the Sample Carrier section of the Tiles tool.
Manually created tile regions and positions (setup by Contour and setup by Predefined) will be deleted if you switch to the setup by Carrier. If you want to combine manual and automatic setup, first use setup by Carrier and then switch to a manual setup. Tile regions that are created automatically by setup by Carrier, are defined to a container and permanently assigned and locked by default, against manual editing. You can unlock the tile regions in the Tiles tool by selecting the desired tile region and unlocking it via the options menu in the tile regions list. You can also unlock all tile regions here if necessary.

Parameter	Description
Select Sample Carrier	Only visible if no sample carrier is currently selected. Opens the dialog to select a sample carrier template.
Create	Creates a tile region for every selected sample carrier container.
Remove	Removes the tile region in every selected sample carrier container.
Fill Factor	Here you can enter the fill factor used to fill the selected container.
Columns/Rows	Here you can add single tile regions to a container by defining the number of columns and rows of the tile. The tile region is always placed at the center of the well container.

Positions Carrier Toolbar

Here you can define the positions automatically on the relevant sample carrier.

A sample carrier must have been selected in the Sample Carrier section of the Tiles tool.
Manually created tile regions and positions (setup by Contour and setup by Predefined) will be deleted if you switch to the setup by Carrier. If you want to combine manual and automatic setup, first use setup by Carrier and then switch to a manual setup. Tile regions that are created automatically by setup by Carrier, are defined to a container and permanently assigned and locked by default, against manual editing. You can unlock the tile regions in the Tiles tool by selecting the desired tile region and unlocking it via the options menu in the tile regions list. You can also unlock all tile regions here if necessary.

Parameter		Description
Select Sample Carrier		Only visible if no sample carrier is currently selected. Opens the dialog to select a sample carrier template.
Create		Creates a position array for every selected container of the sample carrier.
Remove		Removes the position array for every selected container of the sample carrier.
Distribute Positions by
–	Number	Activates the input for Number and Bias.
–	Grid	Activates the input field for an Overlap.
Number		Only visible if Number is selected. Shows the current number of positions that are distributed to newly created position array. Change the number to increase or decrease the number of single positions obtained by a position array.
Bias		Only visible if Number is selected. Adjusts the overall position of the single positions in the position array.
-	None	The single positions will be distributed evenly within the array.
-	Center	The single positions will mainly be distributed near to the center of the position array. Less positions will be at the edges of the array.
-	Edge	The positions will be distributed to the edges of the array. Less positions will be in the center of the array.
Random		Only visible if Number is selected. Activated: The single positions will be distributed randomly within the position array. The overall bias will still be taken into account.
Columns		Only visible if Grid is selected. Sets the number of columns of the position array in each container of the sample carrier.
Rows		Only visible if Grid is selected. Sets the number of rows of the position array in each container of the sample carrier.
Overlap		Only visible if Grid is selected. Shows the current degree of overlap in % of each position in the array relative to its neighbors. Both positive and negative values are possible.

Tiles View Options

This view options are specific for the Tiles & Positions module.

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.

Time Series

This modules enables the acquisition of time series (time-lapse) images with definition of intervals between images, total acquisition duration and number of time points. Time series can be started and stopped manually, at fixed times, after a waiting period or by input (trigger) signal, to analyze images already acquired or change the experiment parameters. The experiment size is limited only by free space on the hard drive and the images are acquired at maximum possible speed.

Acquiring Time Series Images

To set up Time Series experiments, you need to have licensed this functionality and activated it under Tools > Toolkit Manager. For LSM systems, this functionality is part of the system license.
You have set up a new experiment with at least one defined channel and adjusted focus and exposure time correctly, see also Set up a new experiment and Set up multi-channel experiments.
You are on Acquisition tab.

In the Acquisition Dimensions section, activate Time Series .
The Time Series tool is displayed in the Left Tool Area.
Open the Time Series tool.
Set the Duration of your time series. You are able to select an interval (days, hours, minutes, seconds, milliseconds) or cycles (1-n), e.g. 10 cycles.
Set the Interval of your time series.
Click Start Experiment.

The time series experiment starts. You have successfully set up a time series experiment.

You can display the individual images via the Time slider on the Dimensions tab.

Acquisition Toolkits

Experiment Designer

Experiment Feedback

Key Features

Experiment Feedback Tool

Guided Acquisition

Preliminary Work to Guided Acquisition

Overview scan

Processing setting(s)

Image analysis setting

Detailed scan

Parcentricity

Focus strategies

Using Guided Acquisition Settings

Creating a Guided Acquisition setting

Saving a Guided Acquisition setting

Importing and exporting a Guided Acquisition setting

Deleting a Guided Acquisition setting

See also

HDR Confocal Basic

Multi-Channel

Software Autofocus

Terminology & Abbreviations

When is focus the "right" focus?

Software Autofocus Using LSM Tracks

Tiles & Positions

General Preparations

See also

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

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

Adjusting Z-Values

See also

Adjusting Z-Values of Tile Regions

Adjusting Z-Values of Positions

Exporting Tile Images

Tiles & Positions with Advanced Setup

Creating Tile Regions by Contour

Creating Positions by Location

Creating Positions by Array

Creating Positions by Carrier

Importing Images into the Advanced Setup

Importing preview scan images

Importing an image already opened in ZEN

Importing from Explorer

Copying a Tile Region or Position

Copying Tile Region or Position settings between wells

Local and Global Focus Surfaces

Creating a Local Focus Surface

Distributing Support Points

Verifying Z-Values of Support Points

Selecting Interpolation Degree

Creating a Global Focus Surface

Verifying Z-Values of Support Points

Selecting Interpolation Degree

Assigning Categories to Tile Regions and Positions

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

Tiles Tool

Tile Regions Section

Tile Region List Options

Positions Section

Current Position section

Display mode section

Options for Editing Single Positions

Options for Editing Position Arrays

Sample Carrier Section

See also

Tiles Advanced Setup

Tiles Toolbars

Left Toolbar

Top Toolbar

See also

Preview Scan Toolbar

See also

Contour Toolbar

See also

Predefined Toolbar

See also

Stake Toolbar

Tiles Carrier Toolbar

See also