ZEISS Knowledge Base

Automated Photomanipulation

This module is exclusively available for the Celldiscoverer 7 and allows automated photoactivation and bleaching at multiple positions. It is not applicable to Tile Regions. Using this module, the system executes the following experiment steps without user interaction:

Acquisition of a multi-position image as defined in the Tiles Tool.
Identification of the photomanipulation ROIs based on a customized image analysis that was defined beforehand in the Image Analysis Wizard.
Photomanipulation experiment as defined for Bleaching and in the Time Series Tool.

For Automated Photomanipulation, you need to license the function and activate it under Tools > Toolkit Manager. The tool for the model needs to be displayed on the Applications tab.

Using Automated Photomanipulation Settings

Automated Photomanipulation offers you the possibility to save your whole experiment setup in a settings file.

Creating an Automated Photomanipulation setting

On the Applications tab, open the Automated Photomanipulation tool.
Click on Options and select New.
Name the setting and press Enter or click on .

You have created a setting for Automated Photomanipulation.

Saving an Automated Photomanipulation setting

When you have set up your Automated Photomanipulation experiment and created a setting, you can save the setup as a setting.

Click on Options and select Save.

Your experiment setup is now saved.

Importing and exporting an Automated Photomanipulation setting

On the Applications tab, open the Automated Photomanipulation tool.
Click on Options 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 on Open and/or Save.

You have now imported/exported a setting.

Deleting an Automated Photomanipulation setting

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

The selected setting is deleted.

Performing an Automated Photomanipulation Experiment

For a successful Automated Photomanipulation experiment, you have to prepare a photomanipulation experiment at multiple positions and an image analysis setting.

You have licensed this functionality and activated it under Tools > Toolkit Manager.
You have defined and saved a suitable experiment for photomanipulation at multiple positions (including Tiles, Bleaching, and Time Series).
You have defined experiment positions in the Positions section of the Tiles tool, see Positions Section.
Important: Photomanipulation with Tile Regions is not supported!
On the Acquisition tab, you have selected if the photomanipulation should be executed at All Tile Regions per Time Point (e.g. for photoactivation) or as Full Time Series per Tile Region (e.g. for photobleaching).
You have defined photomanipulation settings in the Timed Bleaching tool, see Timed Bleaching Tool.
You have defined a suitable image analysis setting using the Image Analysis Wizard or an OAD macro that detects the regions of interest where the photomanipulation should be executed, see Creating a New Image Analysis Setting.
Important: The classes in the analysis and their corresponding channel names must exactly fit to the channel names in the experiment!

On the Applications tab, open the Automated Photomanipulation tool.
Create a setting to save your experiment setup, see Using Automated Photomanipulation settings.
For Experiment, select the experiment you want to use for photomanipulation. For more information, see Set up a new experiment.
Note: You have to acquire a snap and draw one Experiment Region. Bleaching must be activated. This enables you to define the settings for Timed Bleaching. This Experiment Region is not used for the photomanipulation experiment.
For Analysis, select a suitable setting to analyze the multi-position image.
For Class, use the drop-down list to select the appropriate class or channel to identify the photomanipulation regions of interest.
For Sorting Feature, define the acquisition order of the ROIs for photomanipulation. This step depends on the selected features in the analysis (e.g. ID, mean intensity in channel x, perimeter, etc.). Per default, the photomanipulation per position is executed in the descending order of the IDs.
For Region Type, define the ROI shape and maximum ROI number per position for photomanipulation.
For Output Folder, define the folder where you want to store the experiment data.
The complete experiment including all positions and photomanipulation events is stored as one .czi file. This folder also contains the first scanned image for ROI identification (InitialAnalysisSettingImage.czi) and the table of the ROIs (SingleObjectsTable.csv).
Click on Start.
A InitialAnalysisSettingImage.czi image is acquired to identify the ROIs for photomanipulation and saved to your folder. The ROIs are listed in the SingleObjectsTable.csv table. They are automatically imported as Experiment Regions for photomanipulation.
Then the photomanipulation experiment at multiple positions is executed.
In the Mean ROI tab of the resulting .czi file, the bleach markers are shown.
To check the ROI selection, you can analyze the InitialAnalysisSettingImage.czi with the predefined analysis.
The Start button turns into a Stop button as long as Automated Photomanipulation is running. Click on Stop to stop the running Automated Photomanipulation workflow.

You have successfully performed an Automated Photomanipulation experiment.

Automated Photomanipulation Tool

Parameter		Description
Options
–	New	Creates a new Automated Photomanipulation setting. Enter a name for the setting.
–	Rename	Renames the setting.
–	Save	Saves the current setting.
–	Save as	Saves the current setting under a new name. Enter a name for the setting.
–	Import	Imports an existing setting. The setting is shown in the setting drop-down list.
–	Export	Exports the current setting.
–	Delete	Deletes the current setting.
Experiment		Selects the experiment setup to execute the photomanipulation including Tiles, Time Series and Bleaching.
Analysis		Selects the image analysis setting used to analyze the multi-position image to define the Experiment Regions for photomanipulation.
Class		Defines the class/parameter to find the photomanipulation experiment regions. Important: The classes in the analysis and their corresponding channel names must exactly fit to the channel names in the experiment.
Sorting Feature		Defines the acquisition order of the Experiment Regions for photomanipulation depending on the selected features in the analysis (e.g. ID, mean intensity in channel x, perimeter, etc.).
Region Type		Defines the shape of the photomanipulation area: Polygon, Circle, Rectangle, Custom Circle/Rectangle (Size and Offset adjustable), Circle, and Rectangle are bounding/containing the detected photomanipulation regions.
Max. Number		Defines how many photomanipulation ROIs per position should be executed.
Offset		Only visible if Custom Circle or Custom Rectangle is selected. Defines the Experiment Regions offset of the Custom Circle/Rectangle in X and Y direction.
Size		Only visible if Custom Circle or Custom Rectangle is selected. Defines the Experiment Regions size of the Custom Circle/Rectangle in width and height.
Output Folder		Selects the folder where the experiment results are saved. A subfolder will automatically be created for each run of an Automated Photomanipulation experiment, including the pre-bleach image for Experiment Regions identification, the table of all Experiment Regions, and the experiment results.
Start		Starts the Automated Photomanipulation. The experiment can be stopped with the Stop button in the Acquisition tab. Click on Stop to stop the running Automated Photomanipulation workflow.