Parameter

Description

Find Focus

Only visible in the tool on the Workflows tab.

Starts an autofocus search to find the focus. The autofocus search is performed for the reference channel selected in the Channels tool.

Mode

Only visible if Show All is activated.

Here you can select the sharpness measurement mode.

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Auto

This is the default setting. If selected, the software makes a choice based on the configuration of the microscope. Such that for Widefield approaches or transmitted light the sharpness measure is always Contrast based.

On the other hand, for optical sectioning methods (e.g. Spinning Disk) the software will automatically select an Intensity based approach to determine the sharpness values.

If the microscope configuration cannot be detected automatically you can manually select the sharpness measurement mode.

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Contrast

If selected the contrast-based mode will be used for sharpness measurement.

This is the standard setting for Camera acquisition.

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Intensity

If selected the intensity-based mode will be used for sharpness measurement.

This is the standard setting for Confocal acquisition.

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Reflex

Only available for LSM systems with imaging tracks other than MPLX and Airyscan SR. Using those tracks might lead to overexposure of the channel and failure for the Autofocus.

If activated, the reflex of the laser on the cover glass surface is detected.

An offset is used to focus onto the sample. You need to add the offset once by clicking the Find Offset button while the sample is in focus. The method requires a refractive index mismatch between the immersion medium and the cover glass and is therefore not for oil immersion objectives.

For Reflex mode, the Search parameter should ideally be set to Smart. After the offset was defined, you can manually reduce the search range in order to facilitate shorter focusing times. The acquisition settings (e.g. laser line, PMT gain, emission filters) are configured automatically.

Quality

Only visible if Show All is activated.

This parameter determines the merit function that will be used to calculate the contrast value of the image when Contrast mode is used by the SWAF (Software Autofocus) run to measure sharpness.

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Default

If selected, a composite of weighted merit functions is used.

Use this setting if the sample cover a greater part of the camera field of view.

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Low Signal

If selected, a single merit function to determine the value is used.

Use this setting if the image is noisy or the sample covers a small area of the field of view. As might be the case if you work with a calibration slide or beads.

Search

There are two options Smart and Full. These define a different type of primary maximizer used to run the SWAF, which in turn determines a number of additional characteristics and parameters of the entire process. To learn more about this, read the FAQ entry.

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Smart

If selected, an alternative maximizer is used that can search in a bidirectional manner and will stop when a local maximum is found in the sharpness values (i.e. a significant decrease of sharpness in both z directions). Again if an error condition is detected the Smart maximizer will throw an exception.

For specific information on the Software Autofocus using LSM Tracks in this context, also refer to chapter Software Autofocus Using LSM Tracks.

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Full

If selected, the maximizer employed with this setting uses a unidirectional movement of the z-drive stepping through the entire relative or fixed search range defined in the SWAF tool (see Autofocus Search Range). The Full maximizer will return a global maximum for the autofocus run or throw an exception when an error condition is detected.

For specific information on the Software Autofocus using LSM Tracks in this context, also refer to chapter Software Autofocus Using LSM Tracks.

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Full No Checks

Searches the whole range and selects the sharpest level. Does not apply validity criteria to maximum.

Sampling

Here you can select the step size of how the search range is sampled.

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Default

Uses the default step size (dz = 1/sqrt(2) * 2 * n* lambda/NA).

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Fine

Uses a small Z-distance (0.5 * dz) between the individual focus images that are used to calculate the best focus position.

This doubles the number of z-slices for the given range.

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Medium

Uses a medium Z-distance (2 * dz) between the individual focus images that are used to calculate the best focus position.

Halves the number of z-slices for given range.

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Coarse

Uses a large Z-distance (4 * dz) between the individual focus images that are used to calculate the best focus position.

Reduces number of z-slices by a factor of four.

Autofocus Search Range

Only visible if Show All is activated.

Here you can switch between two distinct approaches for the autofocus search range:

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Relative Range

This is the default mode.

If selected, the software autofocus is calculated over a relative range.

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Fixed Range

If selected, the software autofocus is calculated over a fixed range.

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Automatic Range

Only visible if Relative Range is selected.

Activated: Calculates the range for the autofocus search automatically depending on the objective set.
Deactivated: Allows you to set the range manually with the Range input field.

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Set Last

Only visible if Fixed Range is selected.

Defines the current z-position as the end (last) point for the software autofocus. Alternatively, you can enter the desired value in the input field to the right of the button.

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Range

Displays the area which is used for the autofocus search.

If you have selected Relative Range, deactivate Automatic Range to adapt this parameter manually.

If you have selected Fixed Range, you can adapt the range via the Set Last/Set First buttons or the input fields.

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Step Size

Displays the selected Sampling distance between the individual focus images.

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Set First

Only visible if Fixed Range is selected.

Defines the current z-position as the start (first) point for the software autofocus. Alternatively, you can enter the desired value in the input field to the right of the button.

Autofocus ROI

Here you can define a spot meter or focus region such that the pixels evaluated by the SWAF are limited to a user defined region of the image.

This is particularly usefully if you use a fiducial marker such as a speck of dirt or other constant artefact that serves as a reference for the sample focus at a fixed position over time.

The autofocus ROI is displayed in the live image of the sample enabling it to be positioned and resized, as necessary. As an additional aid to help focusing you can also use the focus bar function of the live image that monitors the image contrast in the live image or spot meter ROI.

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Spot Meter/Focus Region

Activated: Only uses the values from the Spot Meter/Focus Region to calculate the focus position.
The Focus Region is displayed in the live image as a red dashed rectangle. You can adopt it by clicking on the frame and changing its size and position.

Note that this option is not available for LSM acquisition.