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Workflow MeanROI View (Offline)

Adjusting ROIs for Time Points

If objects move laterally in the course of the time series, you can adjust the ROIs at each Time Point in order to follow the objects.

  1. You have defined at least one ROI.
  2. You are in the MeanROI view.
  1. Open the Dimensions tab in the general view options.
  2. Use the Time slider to scroll through the time points. Stop at the first time point at which you want to adjust a ROI.
  3. Open the ROI Tracing tab and activate the Enable ROI Tracing checkbox. To edit the position of a single Key frame change the Key Frame Edit Mode to single mode . Note that you can only select the key frame edit mode if the frame number is set to a value >1.
  4. Adjust the position of the ROI using drag & drop. To do this, select the ROI in the image area by pressing the left mouse button. Then move the ROI to the new position and release the mouse button. Note that rectangular or contour ROIs can also be rotated.
  5. If necessary, you can change the shape of a ROI, by right-clicking on a ROI and selecting Edit Points (e.g. for polygon contours). It is also possible to rotate a ROI if necessary.
    Note that if the area of the ROI changes, the mean intensity value will change. For ratio values in which thresholding is applied, only "valid" pixels will be respected (see Basics of Calculation of Intensity and Ratio Values). Ratios can only be performed if you have the module Physiology (Dynamics).
  6. Adjust the shape/ rotation of the ROI, by drag & drop the contour points/ via the rotation handle.
  7. Changes to the position and shape of the ROIs are adopted for all subsequent time points.
  8. Repeat the previous steps for all other time points for which you want to adjust an ROI.
    For a selected ROI you can see a list of the time points in which its position/shape was modified. As the distance (in frames) between each key frame can vary, a linear interpolation is used to smoothly progress the ROI through the time points. Alternatively, deactivate this (constant) or set it to a spline method that may better describe the progression of the object you are tracing.
  1. You have successfully adjusted the measurement regions to the course of the experiment.

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.

  1. You are in the MeanROI view or MeanROI Setup.
  2. You have defined at least one ROI.
  1. Select the Layout tab in the view options.
  2. To adjust the layout of the image and diagram display, select the desired display mode under Image and Chart.
  3. 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.
  4. 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.
  5. 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.
  6. For Offline Analysis only: Select a suitable layout for the image, chart and table display.
  7. If you want to adjust the axis scaling (range), go to the Charts tab in the view options.
  8. To define the minimum and maximum values of the axes manually, click on the Fixed button under X-/Y-Axis.
  9. The Min and Max input fields for the axis are activated.
  10. Enter the desired values first into the Max and then the Min input fields.
  11. The minimum and maximum axis values of the diagrams are adjusted. Note that the Y-axis scaling can be adjusted individually for each chart.
  12. To change the unit of the X axis, click on the Fixed button under X Units.
  13. The dropdown menu for the units is activated. You can now select the desired unit.
  14. 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.
  1. You have successfully adjusted the display of the intensity values.

Exporting a Data Table

  1. You are in the MeanROI view.
  2. You have defined at least one ROI.
  1. Select the Export tab in the View Options.
  2. In the Data Table section click on the Save As (*.csv) button.
  3. The Save As dialog opens.
  4. Enter a suitable file name, navigate to the desired folder and click on Save.
  5. All the measurement data are saved as comma-separated values in a csv file. This contains the time information, marker events, the geometric area of the ROIs, the area of ROI adjusted by threshold (only with Physiology (Dynamics) module), the mean intensity of the ROI, the mean intensity of the ROI adjusted by threshold (only with Physiology (Dynamics) module), mean ratio value (only with Physiology (Dynamics) module), focus position, and incubation events/ values (if configured) for each channel and each ROI.

Calculating a Ratio for One Wavelength

  1. To calculate ratios (quotient of two fluorescence intensities) and display ratio images, you need the Physiology (Dynamics) module.
  2. You have a suitable image data set open.
  3. You are in the MeanROI view on the Ratio tab (view option).
  1. Activate the checkbox Enable Ratio Calculation.
  2. In the Method dropdown list, select the Single Wavelength (F/F0) entry.
  3. In the Calculation dropdown list select the channel for calculating the ratio.
  4. In the Reference image (Ft0) setup, define the frames of the time series image from which you want the reference value Ft 0 to be calculated.
  5. Click on the Update button.
  6. The ratio values are calculated. The ratio image and a diagram for the ratio values are displayed in the MeanROI view. For very large images (pixels and time points) it might be necessary to use the Cache Ratio Image function on the Ratio tab, as this will eliminate flickering when playing through the images at speed.
  1. You have successfully calculated a ratio for a single wavelength dye such as Fluo-4.

Calculating a Ratio for Two Wavelengths

  1. To calculate ratios (quotient of two fluorescence intensities) and display ratio images, you need the Physiology (Dynamics) module.
  2. You have a suitable image date set open.
  3. You are in the MeanROI View on the Ratio tab (view option).
  1. In the Method dropdown list select the Dual Wavelength or any other of the Ratio Type formulas present. entry.
  2. In the Calculation dropdown lists select the channels for calculating the ratio.
  3. The ratio values are calculated automatically.
  4. Click on the Cache ratio image button to cache all the ratio images of the current time series with the given ratio calculation parameters.
  5. The ratio values are calculated. The ratio image and a ratio diagram are displayed on the MeanROI view. For very large images (pixels and time points) it might be necessary to use the Cache Ratio Image function on the Ratio tab, as this will eliminate flickering when playing through the images at speed.
  1. You have successfully calculated a ratio for a dual wavelength dye such as Fura-2.

Interaction with a Table in MeanROI View

In the MeanROI view (not the MeanROI setup) you can interact with a table in the following ways:

  • Scroll up or down or left to right. Time values are given at the far left and at the far right any markers are shown at the time point they were created. In between you find the values for each ROI in the first channel, then the second and so on, then the ratio values.
  • If you click on the column header you select the entire column and in your charts, you will see the trace corresponding to this ROI is highlighted by a thicker line. Multiple columns can be selected by pressing and holding shift key.
  • If you click any given value in a column, not only will the trace of the corresponding ROI be highlighted, the images that correspond to this time point will be displayed, and the playhead (vertical blue line) of all charts will synchronise to this time point. This allows quick and easy examination of the data/ events.

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