ZEISS Knowledge Base

If you own the Physiology (Dynamics) module you can use the MeanROI setup to specify user-defined measurement regions (ROIs) before the acquisition of your time lapse experiment and analyze their time‑dependent changes in intensity online during acquisition. Ratios can also be calculated and displayed online - these are the typical functions used in physiology/ calcium Fura-2 applications.

Before the experiment

A precondition for a physiology experiment is a Time Series experiment (which can include a z-stack acquisition), which is set up in the Time Series tool. Adding a time dimension to your experiment allows you to activate the optional tool Dynamics. This tool contains the button for opening MeanROI Setup. Here you can draw in ROIs and adjust the display layout of the measurement results. Note that when the setup is opened a snap is automatically acquired, on the basis of which you can configure the settings for the subsequent experiment, like for example the ratio parameters. The structure of the MeanROI setup is based on the MeanROI view, making it easier to learn. Note that online measurements on tiles and positions experiments are not possible, but you can perform measurements on tiles or position data collected over time (i.e. multi-scene time series) in MeanROI view post-acquisition.

During the experiment

After being started, physiology experiments are displayed in the online mode of the MeanROI View. This allows you to analyze and follow the experiment during acquisition. The structure and options largely correspond to the offline mode of the MeanROI View. We therefore recommend that you familiarize yourself with the MeanROI View (offline) before performing your Physiology experiment.

After the experiment

After you have performed your Physiology experiment the data are displayed in the offline mode of the MeanROI view and can be analyzed, processed and exported there. For more information, see also Workflow MeanROI View (Offline).

To perform physiology experiments, you need the Physiology (Dynamics) module.
You have created a new experiment, defined at least one channel and adjusted the focus and exposure time, see also Set up a new experiment and Set up multi-channel experiments.
You have licensed this functionality and activated it under Tools > Toolkit Manager.
You are on the Acquisition tab.

Activate Time Series in the Acquisition Dimensions section.
The Time Series tool is displayed in the Left Tool Area under Multidimensional Acquisition.
Activate Dynamics in the experiment manager.
The Dynamics tool now is displayed in the in the Left Tool Area under Applications.
Note that the tool is not available if the Tiles or Panorama dimensions are activated. Deactivate these dimensions to make the tool available.
Set up a time series experiment, see Acquiring Time Series Images.
Open the Dynamics tool.
Click MeanROI Setup.

You have completed the general prerequisites for Physiology experiments.

Setting up the Online Ratio Calculation

You have read the Workflow Physiology (Dynamics) Experiments chapter.

In the MeanROI setup, select the Online Ratio tab in the view options .
Activate the Activate Live Ratio Generation checkbox.
In the Method dropdown list select a method for the ratio calculation. If you select the Single wavelength (F/F0) method for the calculation of the online ratio, you need to define a reference image. See Calculating the Online Ratio for Single Wavelength.
If you want to use background correction for the calculation of the online ratio, activate the desired entry under Background Correction. Note that activating Ratio fruitions in Online Ratio/ Ratio tab deactivates the background correction settings on MeanROI tab, i.e. the background functions of these tabs are mutually exclusive.
To allow you to apply a constant background value (Constant entry), an input field, in which you can enter the desired value, appears in the formula for the ratio calculation.
The ROI entry can only be selected once you have defined at least two ROI in your image.
Under Calculate complete the formula for calculating the online ratio by selecting the desired entries from the dropdown lists and indicating values in the input fields.
Activate the Threshold checkbox, if you want to set a threshold in your experiment. For information on the calculation of the ratio value R in relation to thresholds, see Calculation of the Ratio Value R.
You can adjust the settings for the ratio calculation as required and the preview image on the MeanROI setup is adapted accordingly. Press the Snap button if you need to update the images.

You have successfully activated/adjusted the calculation of the online ratio.

Calculating the Online Ratio for Single Wavelength

If you set up your online ratio calculation and select the Single wavelength (F/F0) method for the calculation of the online ratio, you need to define a reference image.

You are setting up an online ratio calculation (see also Setting up the Online Ratio Calculation).
You have selected the Single wavelength (F/F0) as the calculation method.

In the Online Ratio tab, in the input field of Reference Image Set-up, enter the number of images from which the reference image should be averaged.
Click on Define.

The images are acquired, and a reference image is calculated from them. You can now continue to set up the online ratio calculation.

Setting up an Experiment in MeanROI Setup

You have read the Workflow Physiology (Dynamics) Experiments chapter.

Activate the Dynamics checkbox in the Experiment Manager.
In the Dynamics tool, click on the MeanROI Setup button.
MeanROI setup opens.
An image is acquired automatically on the basis of which you can configure your settings. You can click on Snap at any time to update the image.

Drawing in ROIs

You are in the MeanROI View or in the MeanROI Setup on Acquisition tab.

Go to the Graphics tab in the View Options.
Select a tool for drawing in ROIs, e.g. the Polygon tool.
Activate the Keep tool checkbox.
The selected tool remains active after you have drawn in an ROI. This means you can draw in several ROIs without having to re-select the tool.
Using the selected tool, in the image view draw in the objects or regions (ROIs) for which intensity measurements are required.
The ROIs are displayed in the list (Annotations/ Measurements Layer) on the Graphics tab.
Intensity measurements are performed for each ROI and displayed in the chart area to the right of the image view.

You have successfully defined measurement regions for the intensity measurement.

Measurement time

Note that the time taken to initially create measurements will vary as some date is cached to memory. Thus, when a long time series image is opened that already contains ROIs, you might have to wait briefly until ZEN completes its measurements. The duration depends on e.g. number of ROIs, number of time points, image size, number of pixels etc.

Adjusting the Display

Here you will find out how to adjust the display of the measured intensity values in charts and tables according to your wishes.

You are in the MeanROI view or MeanROI Setup.
You have defined at least one ROI.

Select the Layout tab in the view options.
To adjust the layout of the image and diagram display, select the desired display mode under Image and Chart.
If you also want your data to be displayed in table form (note for MeanROI setup the table is only displayed after the acquisition is completed), select the desired display mode under Image and Chart with Table. With the Export tab, you can export the data table directly as a comma separated values file or create a separate data table document.
Tables created in this manner contain additional information, for details see Basics of Calculation of Intensity and Ratio Values. The table in the MeanROI view only shows two values per ROI and time point, the mean intensity of pixels above the set threshold per channel and the mean ratio value derived from (common) "valid" pixels.
In the MeanROI view (not the MeanROI setup) you can interact with a table in various ways. See Interaction with a Table in MeanROI View.
You can determine the zoom (range) of the charts of all given channels (including Ratio charts) by clicking on the chart, selecting it, and scrolling with the mouse wheel.
For Offline Analysis only: Select a suitable layout for the image, chart and table display.
If you want to adjust the axis scaling (range), go to the Charts tab in the view options.
To define the minimum and maximum values of the axes manually, click on the Fixed button under X-/Y-Axis.
The Min and Max input fields for the axis are activated.
Enter the desired values first into the Max and then the Min input fields.
The minimum and maximum axis values of the diagrams are adjusted. Note that the Y-axis scaling can be adjusted individually for each chart.
To change the unit of the X axis, click on the Fixed button under X Units.
The dropdown menu for the units is activated. You can now select the desired unit.
On the Layout tab, it is also possible to determine if a given channel view and/ or chart panel should be hidden. This is useful if these do not contain information that needs to be visible, thus increasing space on the screen for the remaining items. For example, in many applications transmitted light is used to monitor the specimen, but the intensity information (chart) is not required. The controls behave in a similar manner to the channel toggles on Dimensions tab.

You have successfully adjusted the display of the intensity values.

Starting and Influencing an Experiment

You have read the Workflow Physiology (Dynamics) Experiments chapter and set up an experiment in MeanROI Setup.
You are on the Acquisition tab.

Start your Physiology experiment by clicking on the Start Experiment button.
The time series experiment is started. The MeanROI View (online) opens and displays the current images and the intensity curves for each ROI measured online. The intensity curves are displayed in the Time Line View and in the diagrams. Note that the MeanROI view will display at the third time point. This is noticeable when the interval time is longer. Thus this display delay should fall into the typically base line of this type of experiments, i.e. prior to the first stimulus of the sample.
You can pause the experiment at any time by clicking on the Pause Experiment button and continue it again by clicking on the Continue Experiment button.
The Focus can be adjusted during the experiment. To prevent images that are not sharp being acquired, pause your experiment and use the Live acquisition button to adjust the focus. Then continue the experiment. Note that using the Live view only works with experiments run in interactive mode. In triggered acquisition scenarios this is not possible.
Adjust the display of the intensity values during the experiment by changing the settings on the Layout or Charts tab. The unit of the X-axis cannot be changed during the experiment.
You can move and change ROIs during acquisition. The changes are adopted for all time points, see Drawing in and adjusting ROIs. Note that ROI tracing functions (these allow objects to be followed in XY) are only available after an acquisition.
Activate Switches in the Time Series tool during the experiment to perform the corresponding actions.
Various events, such as the activation of switches or the pausing of the experiment, are labeled in the Time Line view by markers.
On the Dimensions tab deactivate the Follow Acquisition checkbox to analyze the data acquired up to that point. To do this, select the corresponding time points using the Time slider, the diagram sliders or the Time Line view slider in the MeanROI view.
Change the size of the area marked in blue in the Time Line View to adjust the section displayed in the charts (time axis).

You have successfully started the experiment, analyzed it online and influenced it.

Adjusting ROIs during Experiments

If objects move laterally in the course of the experiment, you can adjust the ROIs at any time during the experiment in order to follow the objects.

You have defined at least one ROI.
You have started your Physiology experiment.

In the Experiment Manger click on Pause experiment button.
Adjust the position of the ROI using drag & drop. To do this, select the ROI in the image area by left-clicking and hold the mouse button down. Then move the ROI to the new position and release the mouse button.
To change the shape of an ROI left click on an ROI and drag the bounds to adjust the size.
Changes to the position and shape of the ROIs are adopted for all time points.
Repeat the previous steps for all subsequent ROIs that you wish to adjust. Note that you can select multiple ROIs and adjust all the positions simultaneously.

You have successfully adjusted the measurement regions (ROIs) to the course of the experiment.

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Workflow Physiology (Dynamics) Experiments