Terminology & Abbreviations

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.