Working with Focus Strategies

Introduction

If you want to work with focus strategies, you have to use the Focus Strategy tool. There you can select the suitable strategy and adjust the corresponding settings, e.g. defining Z-positions manually or automatically, and update these during the experiment. Note that the availability of certain focus strategies depends on your system and available components (e.g. Definite Focus.2).

General Preparations

To use focus strategies, you will need a motorized focus drive/Z-drive.
You are in the Left Tool Area on the Acquisition tab.
You have created a new experiment, defined at least one channel and adjusted the focus and exposure time.

Activate the acquisition dimensions (e. g. Tiles, Time Series) you want to use for your experiment.
Open the Focus Strategy tool.
The available focus strategies are displayed in the drop-down list.
The number of focus strategies available depends on the activated acquisition dimensions (e.g. tiles, time series), the available hardware devices (e.g. definite focus) present, and software license (e.g. Software Autofocus and Tiles & Positions module allow additional focus strategies).
Decide which strategy is best suited to your experiment. In case of a Tiles experiment, the software will automatically select the most appropriate focus strategy if you have not previously selected one. For a detailed description of all strategies, read the chapter Focus Strategy Tool.

You have successfully completed the general preparations. Now follow one of the following sets of instructions for specific focus strategies.

Using Software Autofocus

Select this focus strategy to automate the focusing of your specimen before and during acquisition with the help of the Software Autofocus. This is particularly useful for Time Series or Tiles experiments.

To use the Software Autofocus focus strategy, you will need the module.
For LSM systems, the Software Autofocus module is part of the system license.
You have completed the general preparations for using focus strategies (experiment created, at least one channel defined, acquisition dimensions activated).
You are on the Acquisition tab in the Focus Strategy tool.

Select the Software Autofocus entry from the drop-down list. Note that the Z values defined by Tiles Setup are ignored.
In the Reference Channel section, select the channel that you want to use for the focus action from the list. Expand the section if you do not see it in full. Note that the reference channel does not necessarily have to be an acquisition channel. Not all acquisition modes can be used as reference. For LSM 990 LSM Lambda tracks and Online Fingerprinting cannot be selected as reference channels. Also, Airyscan SR and MPLX tracks might fail when using such tracks as reference for reflex autofocus function.
Click on the Set as Reference Channel button.
In the Time Series Loop and/or Tiles Loop sections of the Focus Strategy tool you can define when focus actions should be performed during the course of the experiment.
Open the Software Autofocus tool.
Adjust the autofocus settings (e.g. Quality, Sampling, etc.) to your experiment conditions or use the default settings first.
Set up your tile and/or time series experiment.
To start the experiment, click on the Start Experiment button.

You have successfully used the Software Autofocus to bring images into focus automatically during the experiment.

Using Definite Focus in Time Series Experiments

Select this focus strategy to use the definite focus device to stabilize the focus in the event of temperature fluctuations during your Time Series experiments.

To use the Definite Focus focus strategy, you will need the Definite Focus hardware device.
You have completed the general preparations for using focus strategies.
You are on the Acquisition tab in the Focus Strategy tool.

Select Definite Focus as focus strategy from the dropdown list. Note that Z values defined by Tiles Setup are ignored.
In the Stabilization Event Repetitions and Frequency section select Standard mode. This mode will use our recommend default settings for stabilization. When selecting the Expert mode, you can adjust all settings according to your needs.
Set up a Time Series experiment, see Acquiring Time Series Images.
Use the Live mode to set the focus position using the focus drive.
To start the experiment, click on Start Experiment.
Definite Focus is initialized at the start of the experiment at the current focus position. The focus is then stabilized in accordance with your settings during the time series experiment. You will be reminded to set the focus accordingly prior to the experiment starting. You can do this by navigating to a suitable location (position or Tile region) and starting live or continuous. You can then continue with the experiment or cancel it.

You have successfully used the Definite Focus to stabilize the focus during a Time Series experiment.

Using Local or Global Focus Surfaces

For Local or Global Focus Surfaces you need to select the focus strategy Use Z-Values/Focus Surface defined in Tiles Setup. This strategy is selected by default, if you have licensed the Tiles & Positions module. Then you can acquire tiles images along local or global focus surfaces (tile region specific/position specific) and use the focus strategy for optimal image results.

A local/global focus surface ensures that all tiles are in focus on tilted or irregular specimens. Local focus surfaces for tile regions are interpolated on the basis of the focus positions of support points. Positions automatically have a horizontal focus area with the Z-value of the position.

The following guide explains how to use the focus strategy for local focus surfaces.

To use the Use Z-Values/Focus Surface defined in Tiles Setup focus strategy, you need a license for the Tiles & Positions module.
You have read the introduction for using focus strategies (experiment created, at least one channel defined, acquisition dimensions activated), see Introduction.
You are on the Acquisition tab in the Focus Strategy tool.

Select the Use Z-Values/Focus Surface defined in Tiles Setup entry from the drop-down list (if not selected by default).
If you want to set up the entire focus strategy, you can click Optimize this Focus Strategy to use the Focus Strategy wizard, see Focus Strategy Wizard.
In the Z-Values/Focus Surface section, select Local (per Region/Position).
Under Initial Definition for Z-Values/Focus Surface, select the By Tiles Setup entry from the drop-down list.
Alternatively, select the options By Software Autofocus or By Definite Focus: Recall Focus if you have licensed the Software Autofocus module and/or use Definite Focus.2 hardware device. This allows you to specify that the z-positions of the support points should be updated by the software autofocus and/or Definite Focus.2 immediately before acquisition.
Set up a Tiles experiment.
Create a local focus surface for the tile regions.
To start the experiment, click Start Experiment.

The tiles of tile regions and positions are acquired using the local focus surfaces.
If you are performing a Time Series experiment, we recommend to use the Adapt Z-Values/Focus Surface functions. By these you can correct the focus for small drifts that occur in long multidimensional time series experiments with living cells, for example.