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

  1. You have licensed this functionality and activated it under Tools > Toolkit Manager.
  2. You have defined and saved a suitable experiment for photomanipulation at multiple positions (including Tiles, Bleaching, and Time Series).
  3. You have defined experiment positions in the Positions section of the Tiles tool, see Positions Section.
    Important: Photomanipulation with Tile Regions is not supported!
  4. 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).
  5. You have defined photomanipulation settings in the Timed Bleaching tool, see Timed Bleaching Tool.
  6. 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!
  1. On the Applications tab, open the Automated Photomanipulation tool.
  2. Create a setting to save your experiment setup, see Using Automated Photomanipulation settings.
  3. For Experiment, select the experiment you want to use for photomanipulation. For more information, see Setting 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.
  4. For Analysis, select a suitable setting to analyze the multi-position image.
  5. For Class, use the drop-down list to select the appropriate class or channel to identify the photomanipulation regions of interest.
  6. 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.
  7. For Region Type, define the ROI shape and maximum ROI number per position for photomanipulation.
  8. 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).
  9. Click on Start.
  10. 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.
  11. Then the photomanipulation experiment at multiple positions is executed.
  12. In the Mean ROI tab of the resulting .czi file, the bleach markers are shown.
  13. To check the ROI selection, you can analyze the InitialAnalysisSettingImage.czi with the predefined analysis.
  14. 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.
  1. You have successfully performed an Automated Photomanipulation experiment.