Objective Range

When positions are defined near the border of the stage, areas which are found in the overview scan might not be reached with the higher magnification objective of the detailed scan.

  1. Define the overview scan only in regions which can be reached with the objective used in the detailed scan.

Auto Immersion

If you set up your Guided Acquisition experiment using an immersion objective and user input would be necessary between overview and detailed experiments, a dialog is displayed directly after clicking Start. With this dialog you can control if the immersion is created when changing the objective for the scan. The experiment runs uninterrupted afterwards.

  1. You have calibrated the XY stage.
  2. You have defined suitable experiments for overview and detailed scans, a suitable setting for each processing step or function you want to execute, and a suitable image analysis setting using the Image Analysis Wizard. For more information, see Preliminary Work to Guided Acquisition.
  1. On the Applications tab, open the Guided Acquisition tool.
  2. The parameters are displayed.
  3. Create a setting to save your experiment setup, see Using Guided Acquisition Settings.
  4. You have created a setting for your entire workflow.
  5. In the Overview Scan section, select the experiment you want to use for creating the overview image, the objective and (if available) the after-magnification lens to be used. Note that per default, objective and after-magnification are not stored as part of an experiment. If you have selected an experiment with multiple scenes or wells, you also have to select in which order the individual scenes should be acquired and processed.
  6. Your overview scan is set up.
  7. If your system is equipped with Definite Focus, the Find Surface before Overview Experiment and SW Autofocus before Overview Experiment checkboxes are visible. Activate the checkbox to perform an additional focusing step before the overview experiment.
  8. The focus action is defined.
  9. If you want to process your overview image before it is analyzed, click Add Processing Method.
  10. A processing section is added.
  11. Select a Method for processing and a corresponding Setting.
  12. The respective processing is set up.
  13. In the Image Analysis section, select a suitable setting to analyze the overview scan and detect the objects of interest.
  14. If your setting contains more than one class for analysis, the Class dropdown is displayed.
  15. Select the Class from the image analysis setting with multiple classes, which segmented regions should be imaged (e.g. with higher resolution) in the detailed experiment.
  16. If you want the detailed scans to stop after a particular amount of objects, activate Add Stop Criterion for Detailed Scans.
  17. Additional options to define an automatic stop criterion for detailed scans are displayed.
  18. Select the measurement feature that should be used to sort the objects detected by the image analysis and set the order for sorting the results. The drop down list displays all suitable measurement features as defined in selected the image analysis setting. Additionally, enter the maximum number of objects that should be scanned by the detailed experiment and select if it is the overall number of objects or the number of objects per well.
  19. You have set up a stop criterion for the detailed experiment.
  20. In the Detailed Experiment section, select the experiment for the detailed scan, the objective and, if available, the after-magnification lens to be used. Note that as default objective and after-magnification are not stored as part of an experiment.
  21. The detailed experiment is set up.
  22. If your system is equipped with Definite Focus, the checkbox Find Surface before First Detailed Experiment is available. Activate this checkbox to use the Definite Focus to find z-position of the glass surface once before the detailed scans. Activate SW Autofocus before First Detailed Experiment to perform a SW autofocus once before the detailed scans. Optionally, activate Recall Focus before each Detailed Experiment to store the difference between the glass surface and the sample and use this value for each of the detailed experiments.
  23. The focus action for the detailed experiment is defined.
  24. If you want to repeat the detailed scans or the entire Guided Acquisition workflow, activate Repeat Detailed Scan or Repeat Full Guided Acquisition Workflow.
  25. The settings for repetition are displayed.
  26. Define the settings for Cycles and Interval to set up the number of iteration and the minimum time between them. If you repeat the entire workflow, use the dropdown on the top to select which action should be performed if no objects are detected by the analysis in the first iteration.
  27. You have set up a repeated execution of the entire Guided Acquisition workflow or the detailed scan.
  28. Define the folder where you want to store the experiment data.
  29. The output folder is defined.
  30. Click Start.
  1. An overview scan is performed and a .czi image is acquired and saved to your folder.
  2. In the example above, the image in the top left shows the overview scan and three identified objects marked with an orange box. For these positions, a detailed scan is performed with a higher magnification.
  3. One Overview Scan Regions.csv and one OverviewScan Region.csv are displayed. The OverviewScan Region.csv table shows the found objects with ID, Bound Center X Stage [µm] and with Y Stage [µm], Bound Width, and Bound Height [µm], as well as Image Scene Container Name and Image Index Scene.
  4. For each detected object a detailed scan is performed. For each object, a *.czi image is acquired and stored in your folder.