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Dynamics Profiler

These modules allow you to use FCS (Fluorescence Correlation Spectroscopy) functionality together with the Airyscan detector. The functionality has to be licensed. Without a license, you can only open an existing document in one view with limited interaction.

The module Dynamics Profiler Basic expands the capability of the Airyscan detector and utilizes the area information gathered from 32 circular arranged detection elements for fluorescence correlation spectroscopy (FCS). A wizard guides you through the acquisition of your data. Based on a reference image, you can evaluate up to 10 measurement spots consecutively and analyze them with various fit models. Additionally, you can get access to the raw data of all Airyscan detection elements for individual analysis.
The module Dynamics Profiler Advanced additionally enables you to analyze the dynamics with pair correlation (e.g. to investigate diffusion barriers), and the active flow by providing speed in µm/s and its direction.

Overview Supported Laser Lines

The Dynamics Profiler functionality works for all RGB laser lines with the following wavelengths:

Microscope

Supported Laser Lines

LSM 990

445nm, 488nm, 514nm, 543nm, 561nm, 594nm, 639nm

LSM 910

488nm, 561nm, 639nm, 640nm

405nm Laser

Note that you can also use the laser wavelength of 405nm, but only for acquiring the reference image. Performing spot measurements is not possible with 405nm. Additionally, to be able to start the acquisition wizard, the 405nm must not be the selected main track.

Setting Up and Performing a Dynamics Profiler Acquisition

Acquisition with data compression

If you want to acquire your data with lossless compression, go to Tools > Options > Acquisition > Data Compression and make sure that Zstd (lossless) is selected.

  1. You have started ZEN with the necessary licenses and set up your hardware.
  2. You have set up an acquisition experiment with one or two Airyscan SR tracks. The FCS functionality for Airyscan is only available, if you have set up a suitable experiment with supported objective and laser line(s), see also Overview Supported Laser Lines. Note that if you have added two Airyscan tracks, the one currently selected in the Imaging Setup or Channels tool is the main track for the spot measurement, the other is considered the second track.
  1. On the Acquisition tab, in the Imaging Setup tool, click Dynamics Profiler.
  2. The Align Airyscan Detector step of the wizard opens.
  3. Click Adjustment.
  4. An automatic adjustment of the Airyscan detector starts and the button changes to a Stop button.
  5. If the automatic adjustment fails, dedicated controls are displayed on the left to manually configure the detector adjustment.
  6. Use the controls on the left side to adjust your Airyscan detector.
  7. The Quality and Status of the detector is displayed and updated on the left side.
  8. When the detector adjustment is successfully completed, the Snap Reference Image step opens automatically.
  9. Use the options of the step to set up the acquisition of the reference image. You can also start a continuous acquisition by clicking Continuous and change the parameters with direct visual feedback.
  10. If you click Continuous, a continuous acquisition starts. To continue, you have to click Stop.
  11. If you have set up your experiment with two tracks and want to acquire the reference image with both, activate Second Track.
  12. The laser setting for the second Airyscan track is displayed if you need to adjust them, the Gain slider is active for both tracks.
  13. Click Snap Reference Image.
  14. Your reference image is acquired with the current settings.
  15. Click Next.
  16. The Set Up Acquisition Spots step opens.
  17. In the Spots section, click + (activated by default) and click on your spots of interest in the reference image. Note that you can add a maximum of ten spots.
  18. The spots are added to the experiment and displayed in the list on the left.
  19. If you want to evaluate an individual spot, select it in the list and click Evaluate Spot. You can also move the spot during evaluation. You can also change the z-position of the spot during evaluation. However, note that the reference image does not adjust to the new z-position. In this case we recommend creating a new reference image by clicking Snap.
  20. Set up the parameters for your time series experiment, including the measurement time. Note that the maximum for spot measurement is 300 seconds.
  21. Click Start Experiment to start the time series experiment for all spots.
  22. The scan experiment starts. The data is displayed in the table and charts in the Center Screen Area.
  23. After the experiment is finished, the wizard closes automatically. If you have activated Create a Reference Image after Spot Measurement, a second reference image (Post Experiment image) is acquired after the spot experiment and before closing the wizard.
  1. The Dynamics Profiler document is displayed in the main user interface of ZEN. You can now save it and analyze the data with the help of the three dedicated views.
  2. Dynamics Profiler documents are also supported by ZEN Connect, see ZEN Connect.
  3. If you have activated Keep Positions and Keep Acquisition Settings in the last wizard step, the spot positions and acquisition settings are taken over to the user experiment and are available for the next acquisition with the wizard.
  4. If you open the Dynamics Profiler document in the Info view, you can see the metadata of the spot experiment. If you want to see the metadata for the reference image, open it as a separate image in ZEN, see Opening the Reference Image as Separate Image.

Exporting Data From Tables

  1. A Dynamics Profiler document is open in the Correlation, Diffusion, or Flow view.
  1. Right click in the data table.
  2. A context menu is displayed.
  3. If you want to save the complete table content as a text file, select Write Text to File.
  4. A file browser opens.
  5. Select a folder, enter a name for the document and click Save.
  6. The data of the table is saved as a tab separated text file (headers in the document correspond to the column names of the table) in the selected folder.

Exporting Data From Charts

  1. A Dynamics Profiler document is open in the Correlation, Diffusion, or Flow view.
  1. Click Dark rounded square icon with white four-point sparkle at top-left and white gear on right in the top right corner of the chart.
  2. A context menu is displayed.
  3. If you want to save the complete chart content as a text file, select Write Text to File. If you want to save the chart as a graphic, select Save Graphics to.
  4. A file browser opens.
  5. Select a folder, enter a name for the document and click Save.
  6. The data of the chart is saved as a tab separated text file (headers in the document correspond to column names/axis legends) or as a .tiff image file in the selected folder.

Opening the Reference Image as Separate Image

  1. A Dynamics Profiler document is open in one of the views.
  1. If you have acquired two reference images, select if you want to see the Pre-Experiment or Post-Experiment image with the view options tab.
  2. In the data table, toggle the visibility of the spots that should be present in the image.
  3. Right click into reference image and select Create Image from View in the context menu.
  4. The reference image is opened as a new image document in ZEN with the previously selected spots as well as all the metadata (visible in the Info view).

Cutting Out a Data Region

  1. A Dynamics Profiler document is open in the Correlation view and the count rate chart is displayed.
  1. In the table, select the spot entry for which you want to add a region. If you want to draw on region for several spots, you can also select multiple spots by pressing Ctrl.
  2. The chart is updated according to your selection.
  3. Click Dark rounded square icon with white four-point sparkle at top-left and white gear on right in the top right corner of the chart.
  4. A context menu is displayed.
  5. Select Add Remove Region.
  6. The mouse curser changes to a pen.
  7. Go to the count rate chart and click into the chart to define the lower threshold for your data region.
  8. A line is added to the chart.
  9. Go to a second measurement time position and click again.
  10. You have defined a region with a lower and upper threshold for your data. It can be moved in the chart by drag and drop of the point on the horizontal line.
  11. The region can be deleted with the Dark rounded square icon with white four-point sparkle at top-left and white gear on right button, or resized by dragging the vertical lines.
  12. Repeat these steps for every spot where you want to draw a data region.
  1. The data of the region is used for the Diffusion and Flow view. The selection is saved in the metadata of the document and is restored after it is reopened.

Saving Dynamics Profiler Documents as Compressed CZI

  1. A Dynamics Profiler document is open.
  1. Click File > Save As with Options.
  2. A file browser for saving opens.
  3. Enter the File name, navigate to the folder where you want to save the document, and open the Compression dropdown.
  4. The dropdown menu displays the different compression options.
  5. Select Lossless Compressed (ZSTD).
  6. Click Save.
  7. Your Dynamics Profiler document is now saved and compressed with a lossless data compression.

Creating a Custom ωr Calibration

  1. You have created a Dynamics Profiler document where you have set the Time Resolution to ωr in the third step of the wizard.
  2. This Dynamics Profiler document is open in the Correlation view.
  3. The objective for your spot experiment must be the same as the objective you use for the ωr calibration.
  1. In the view options, open the ωr Calibration tab.
  2. From the Dye dropdown list, select which dye you want to use for the calibration.
  3. The value for Diffusion Coefficient is updated automatically.
  4. If you know the Diffusion Coefficient for your experiment, adapt the value in the input field accordingly.
  5. Click Calibrate.
  6. A file browser opens.
  7. Select the folder where you want to save the calibration file and enter a file name.
  8. Click Save.
  9. The custom ωr calibration is created and the file is saved.

Using Custom ωr Calibration

  1. A Dynamics Profiler document is open in the Correlation view.
  2. You have a calibration file with user defined ωr calibration available, see also Creating a Custom ωr Calibration.
  3. The objective for your spot experiment must be the same as the objective you have used for the ωr calibration file.
  1. In the ωr Calibration section of the Correlation Tools tab, click Custom.
  2. A file browser opens.
  3. Navigate to the folder with your calibration file, select it and click Open.
  4. The custom ωr calibration is loaded and the file name is displayed next to the button.

Parameter Table Context Menu

When you right click in the parameter table, you have the following option:

Parameter

Description

Write Text to File

Saves the data as a .txt file. You are prompted to enter a name and select a folder before saving.

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