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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 Charts

  1. A Dynamics Profiler document is open in the Correlation, Diffusion, or Flow view.
  1. Click 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).

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.

Correlation View

This view displays your experiment and the correlation data of the individual measurement positions. The different areas containing the image, the charts and the table can be resized by dragging the individual area boundaries with the pressed left mouse button.

1

Preview Image
Here you can see the acquired preview image with the measurement positions marked in the view.

2

Parameter Table
This table displays the data for the different measurement positions. Each position is displayed as a row. You can select one or multiple rows, which updates the information displayed in the charts. For details, see Correlation Parameter Table.

3

Charts
Here you have the result charts visualizing the measurement data for the selected spots. You can zoom into the displayed curves with the mouse wheel or by drawing a zoom rectangle with pressed left mouse button. A right click in the chart area resets the chart (zoom) to the default view.

4

View Options
Here you have the area for your standard view options as well as the specific ones provided on the Correlation Tools tab, see Correlation Tools Tab.

Correlation Parameter Table

This table displays the data for the different measurement positions. Each position is displayed as a separate row. If a value could not be calculated for a specific position, the corresponding entry in the table is empty. A click on an entry highlights and selects the entire row. You can also select multiple rows when pressing the Ctrl button while selecting the entries. The charts are then updated accordingly. The units for individual parameters are displayed as tooltips. A right click in the table opens a specific context menu, see Parameter Table Context Menu.

Parameter

Description

Ø

Activated: Uses this position for the calculation of an average for individual values. The average is displayed on the bottom of the table.


Visibility

Toggles the visibility of the respective spot.

ID

Displays the ID for the individual spot.

Color

Sets the color for the respective measurement point by clicking on the field and selecting a color form the dialog.

Name

Displays and sets the name for the individual position.

Count Rate

Displays the count rate in kHz.

CPM

Displays the CPM in kHz. The color of the value indicates the data quality, see Data Quality Criteria (CPM).

Data Quality

Displays an indication for the fit quality.

Number of Molecules

Displays the number of molecules found at the measurement position. It is calculated by 1/correlation amplitude, where the correlation amplitude is defined as value of the correlation curve at the smallest lag time.

Concentration

Only visible after using a 3D fit model.
Displays the concentration.

Surface Concentration

Only visible after using a 2D fit model.
Displays the value for the surface concentration in nmol/cm2.

Triplet State Fraction

Displays the triplet state fraction. One physical property of the utilized fluorophore. Dependent on intensity and if applicable oxygen. A triplet state of e.g. 16% would mean that 16% of all fluorophores are in dark state.

Chi2

Displays the Chi2 value, see also Fit Calculation. Chi2 is the deviation between the measured FCS curve and the fit curve. A value of 0 would mean a 100% accordance.

Diffusion Time

Displays the diffusion time.

Diffusion Coefficient

Displays the diffusion coefficient.

α

Only visible if you have used an anomalous fit model.
Displays the anomalous parameter: 1 corresponds to free diffusion, lower values indicate constrained diffusion.

Fit Model

Displays which fit model was used.

Date

Displays the date and time of the experiment.

Correlation Tools Tab

Parameter

Description

Reference Image

Only visible if you have created a second reference image after the spot experiment.
Selects which reference image is displayed int the view.

–

Pre-Experiment

Displays the reference image acquired before the experiment.

–

Post-Experiment

Displays the reference image acquired after the experiment.

Display Chart

Selects which chart is displayed above the correlation chart.

–

Count Rate

Displays the count rate chart.

–

Residuals

Displays the residuals chart. Note that the residuals chart only contains information after a fit was executed for the respective spot.

Filter

–

Detrending

Activated: Applies a detrending filter to remove the trend (e.g. due to the photo bleaching) from the experiment data. Detrending is calculated by a low-pass filter with the filter time constant provided with the input field.

–

Dust

Activated: Applies a dust filter to remove high intensity peaks (caused e.g. by aggregated objects) from the experiment data and sets the parameter with the input field. Dust is considered, if the average intensity multiplied with the parameter value is higher than the mean signal intensity.

Fit Model

Selects the fit model for the correlation curve.

–

1 Component 2D

Selects the one component 2D model.

–

2 Component 2D

Selects the two component 2D model.

–

1 Component 3D

Selects the one component 3D model.

–

2 Component 3D

Selects the two component 3D model.

–

1 Component Anomalous 2D

Selects the one component anomalous 2D model.

–

1 Component Anomalous 3D

Selects the one component anomalous 3D model.

–

Fit

Applies the fit model to the currently selected position/curve.

–

Fit All

Applies the fit model to all positions/curves.

Amplitude Normalization

–

Activate

Normalizes the amplitude to 1.

–

Deactivate

Applies no normalization.

ωr Calibration

Selects which ωr should be used for the fit of the curves.

–

Default

Uses the default setting for the ωr calibration.

–

Custom

Opens a browser to select a file with a user generated ωr calibration. This file can be created with the ωr tab.

ωr Calibration Tab

This tab enables you to create your own custom ωr calculation that you can use for fitting the correlation curves.

Parameter

Description

Dye

Selects the dye used for the ωr calibration.

Diffusion Coefficient

Displays and sets the diffusion coefficient for the dye.

Calibrate

Calibrates the ωr value and opens a file browser to save it as a calibration file.

Diffusion View

This view displays your experiment and the diffusion data of the individual measurement positions. For information about calculations, see Calculation of Concentration and Diffusion Coefficient. The different areas containing the image, the charts and the table can be resized by dragging the individual area boundaries with the pressed left mouse button.

1

Preview Image
Here you can see the acquired preview image with the measurement positions marked in the view.

2

Parameter Table
This table displays the data for the different measurement positions. Each position is displayed as a separate row. For details, see Diffusion Parameter Table.

3

Polar Heatmap
This diagram area gives you the visual information about the diffusion in your sample and which Airyscan fibers are used for the correlation, see Polar Heatmap. A right click into the area enables you to export the polar heatmap as an image.

4

Correlation Chart
Here you have the correlation chart for the currently selected spot. You can zoom into the displayed curve with the mouse wheel or by drawing a zoom rectangle with pressed left mouse button. A right click in the chart area resets the chart (zoom) to the default view.

5

View Options
Here you have the area for your standard view options as well as the specific options provided on the Diffusion Tools tab, see Diffusion Tools Tab.

Polar Heatmap

The polar heatmap illustrates directed diffusions and barriers by using color gradients referring to the number of present molecules in specific angles. On the top you have a visual representation of the Airyscan detector that indicates the fibers that are used for the correlations. These heatmaps are calculated from six pair correlation curves and the pair correlation function (pCF). The pair correlation function is a set of cross-correlations between the central fiber and all fibers located at a given distance from the center. The rainbow pattern is used to map the diagram color to the corresponding correlation amplitude. The color mapping and legend are the same for both heatmaps and the color palette can be selected in the Diffusion Tools tab. A right click on the heatmap displays a menu that allows you to save it as an image on your PC. The displayed degrees can vary based on your system configuration.

 

Diffusion Parameter Table

This table displays the data for the different measurement positions. Each position is displayed as a separate row. If a value could not be calculated for a specific position, the corresponding entry in the table is empty. A click on an entry highlights and selects the entire row and displays the corresponding data in the chart. The units for individual parameters are displayed as tooltips. A right click in the table opens a context specific menu, see Parameter Table Context Menu.

Parameter

Description

Ø

Activated: Calculates an average of the individual values. The average is displayed on the bottom of the table.


Visibility

Toggles the visibility of the respective spot.

ID

Displays the ID for the individual spot.

Color

Sets the color for the respective measurement point by clicking on the field and selecting a color form the dialog.

Name

Displays and sets the name for the individual position.

Count Rate

Displays the count rate in kHz.

CPM

Displays the CPM in kHz. The color of the value indicates the data quality, see Data Quality Criteria (CPM).

Number of Molecules

Displays the number of molecules found at the measurement position. It is calculated by 1/correlation amplitude, where the correlation amplitude is defined as value of the correlation curve at the smallest lag time.

Concentration

Only visible after using a 3D fit model.
Displays the concentration.

Surface Concentration

Only visible after using a 2D fit model.
Displays the value for the surface concentration in nmol/cm2.

Triplet State Fraction

Displays the triplet state fraction. One physical property of the utilized fluorophore. Dependent on intensity and if applicable oxygen. A triplet state of e.g. 16% would mean that 16% of all fluorophores are in dark state.

Chi2

Displays the Chi2 value, see also Fit Calculation. Chi2 is the deviation between the measured FCS curve and the fit curve. A value of 0 would mean a 100% accordance.

Diffusion Model

Displays which fit model was used.

Diffusion Time

Displays the diffusion time.

Diffusion Coefficient

Displays the diffusion coefficient.

Date

Displays the date and time of the experiment.

Diffusion Tools Tab

Parameter

Description

Reference Image

Only visible if you have created a second reference image after the spot experiment.

Selects which reference image is displayed int the view.

–

Pre-Experiment

Displays the reference image acquired before the experiment.

–

Post-Experiment

Displays the reference image acquired after the experiment.

Correlation Chart

Selects which correlation chart and which curves are displayed in the correlation chart and allows you to set the color for the curves. You can select to see the chart for Inner Elements or Outer Elements.

–

Curve Binning

Activated: Smoothes the curves by averaging for noisy data.

Amplitude Normalization

–

Activate

Normalizes the amplitude to 1.

–

Deactivate

Applies no normalization.

Polar Heatmap Palette

Displays and selects the color palette used for the visualization in the polar heatmap.

Flow View

This view displays your experiment and the flow information. The different areas containing the image, the charts and the table can be resized by dragging the individual area boundaries with the pressed left mouse button.

1

Preview Image
Here you can see the acquired preview image with the measurement positions marked in the view.

2

Parameter Table
This table displays the data for the different measurement positions. Each position is displayed as a row. For details, see Flow Parameter Table.

3

Flow Diagram
This diagram illustrates the flow based on the Airyscan detector. The arrow indicates the flow direction. For information about the flow calculation and the classification for flow speed, see Flow Calculation. The displayed degrees can vary based on your system configuration. A right click into the area enables you to export the flow diagram as an image.

4

Correlation Chart
Here you have the correlation chart for the individual spots. You can zoom into the displayed curves with the mouse wheel or by drawing a zoom rectangle with pressed left mouse button. A right click in the chart area resets the chart (zoom) to the default view.

5

View Options
Here you have the area for your standard view options as well as the specific options provided on the Flow Tools tab, see Flow Tools Tab.

Flow Parameter Table

This table displays the data for the different measurement positions. Each position is displayed as a separate row. If a value could not be calculated for a specific position, the corresponding entry in the table is empty. A click on an entry highlights and selects the entire row and displays the corresponding data in the chart. The units for individual parameters are displayed as tooltips. A right click in the table opens a context specific menu, see Parameter Table Context Menu.

Parameter

Description

Ø

Activated: Uses this position for the calculation of an average for individual values. The average is displayed on the bottom of the table.


Visibility

Toggles the visibility of the respective spot.

ID

Displays the ID for the individual spot.

Color

Sets the color for the respective measurement point by clicking on the field and selecting a color form the dialog.

Name

Displays and sets the name for the individual position.

Count Rate

Displays the count rate in kHz.

CPM

Displays the CPM in kHz. The color of the value indicates the data quality, see Data Quality Criteria (CPM).

Data Quality

Displays an indication for the fit quality.

Number of Molecules

Displays the number of molecules found at the measurement position. It is calculated by 1/correlation amplitude, where the correlation amplitude is defined as value of the correlation curve at the smallest lag time.

Flow Direction

Displays the value that indicates the flow direction.

Flow Speed

Displays the flow speed in µm/s.

Flow Speed Confidence Interval

Displays the confidence interval for the flow speed.

Fit Model

Displays which fit model was used.

Date

Displays the date and time of the experiment.

Flow Tools Tab

Parameter

Description

Reference Image

Only visible if you have created a second reference image after the spot experiment.

Selects which reference image is displayed int the view.

–

Pre-Experiment

Displays the reference image acquired before the experiment.

–

Post-Experiment

Displays the reference image acquired after the experiment.

Correlation Chart

Selects which curves are displayed in the correlation chart and allows you to set the color for the curves.

Fit Model

Selects the fit model for the correlation curve.

–

1 Component 2D

Selects the one component 2D model.

–

1 Component 3D

Selects the one component 3D model.

–

Show Fitted Curves

Activated: Displays the fitted curves in the chart.

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.

Dynamics Profiler Calculations

Calculation of Concentration and Diffusion Coefficient

The diffusion coefficient D is calculated as indicated by the following formula:

The fluorofore concentration is given as:

  • where

    is an average number of molecules and NA ​​ is the Avogadro constant
  • and

  • where S is the structural parameter.

Data Quality Criteria (CPM)

The data quality is estimated using CPM values:

Data Quality

CPM Value

Bad (red)

Smaller than 1.

Not bad (yellow)

Between 1 and 3.

Good (green)

Higher than 3.

Filters

Detrending Filter

The detrending filter is applied to remove the trend (e.g. due to the photo bleaching) from the experiment data. The detrended signal Id(t) is calculated as follows:

where the trend, Tr(t) is the gaussian smoothed signal:

with

The FilterWindowParameter [ms] is set by the user.

Dust Filter

The dust filter allows you to remove high intensity peaks (e.g. caused by aggregated objects) from the experiment data. Calculations are done on the 500x down sampled signal data. For comparison, intensity data shown in the count rate chart is down sampled with factor 2000x. The data bin is classified as dust (filtered out) if the following criteria is fulfilled:

UserParameter * I(t) > Mean(I(t))

If both the detrending and dust filter are activated, the dust filter is applied after the detrending filter.

Fit Calculation

The unweighted fit of the correlation curve is done using the Levenberg-Marquart algorithm. Reduced chi2 is calculated as follows:

where Gt is the correlation curve value, F​t the fit value, N the number of correlation/fit values and DF the number of fit parameters.

Auto Correlation Fit Models

You can select one of the following models:

One Component 2D

One Component 3D

Two Component 2D

Two Component 3D

One Component Anomalous 2D

One Component Anomalous 3D

Parameters

The parameters used in the calculations are the following:

Parameter

Description

A

The correlation amplitude.

Tt

The triplet fraction.

Td1

The translational diffusion time.

S

The structural parameter.

τt

τt = 3µs is the fixed triplet relaxation time.

cf1

The first component fraction.

Normalization of Correlation Curves

Amplitude normalization of the correlation curves allows you to compare different correlation curves and see the differences in diffusion times between them.

If the fit of the correlation curves is already done, activating the amplitude normalization is done using the amplitude fit parameter. Selected correlation curves are rescaled that each selected curve has a correlation amplitude equal to 1 (corresponding number of molecules = 1).

where A is a fitted amplitude.

In case no fit is done, correlation curves are normalized as follows:

where Amean ​​ is the mean amplitude calculated over the first 10 points of the correlation curve:

Pair Correlation of Airyscan Fibers

The pair correlation function (pCF) is a set of cross-correlations between the central fiber and all fibers located at a given distance from the center. For a better signal to noise ratio, cross-correlations are calculated in both directions and then averaged. The pCFs are used and displayed in the polar heatmaps of the Diffusion view.

The Diffusion view can display the data of two different sets of fibers. The group called Outer Elements comprises fibers with a slightly bigger distance from the center (blue element) and is illustrated with orange in the graphic below. The second group is called Inner Elements and comprises fibers with a slightly smaller distance from the center. They are illustrated with the green color in the graphic below.

Fiber Group

Fibers

Outer Elements

Comprises fibers 9, 11, 13, 15, 17 and 19.

Inner Elements

Comprises fibers 8, 10, 12, 14, 16 and 18.

ωr Calculation

The ωr is calculated with the following formula:

During the ωr calibration, τd is calculated in the one-component 3D diffusion model

and D (diffusion coefficient) needs to be provided and added in the ωr Calibration tab. The diffusion coefficients of commonly used dyes are already specified in a dropdown list. The following requirements need to be fulfilled in order to generate a suitable calibration file:

  • Spot measurement duration of at least 60 seconds
  • Time Resolution of 0.5 µs (ωr acquisition mode)
  • The used dye concentration needs to be adjusted to match the expected FCS amplitude range (0.05 – 2)
  • The emission wavelength of the selected dye should match the selected laser line

Four values are calculated and can be accessed by opening the calibration file with a text editor:

  • W0Ring3: Effective radius of the observation spot (detector elements 1 to 19 binned together)
  • W0Ring123: Average radius of the observation spot for a single detector element (average value for detector elements 1 to 19)
  • R0Ring123: Effective distance between the detector elements for flow measurements
  • StructuralParameter: Ratio between the observation spot waist in the axial and lateral direction

The generated ωr calibration file can be used in the Correlation Tools tab to replace the default ωr calibration values.

Calculation of Autocorrelation Curve

The Dynamics Profiler autocorrelation curve is calculated according to the formula:

Correlation values are calculated for the lag times τ, commonly used in 16/8 multi-tau hardware correlators. This means the lag time τ is increased linearly with the τ =PixelDwellTime for the first 16 values. Then the lag time interval for τ is doubled, and the next eight lag times are calculated. The lag time interval is doubled after each next eight time lag values.

The very first correlation value, corresponding to the one PixelDwellTime, is skipped since it is biased due to correlation of the neighboring data points after low pass filter due to digitalization.

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