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Plot intensities, ratios of intensities

Calculating graphs:

Once ROIs have been defined in the Image Window, press plot or select the Tools/Plot in the Main menu, or press Ctrl-L to generate a plot of mean pixel values (intensities) in the areas covered by the ROIs. The actual (32bit floating point) image data is plotted, not the visualized image.
To access additional functionality use the FPlot function which allows:

  • DF/F0 calculation of the mean intensities (F0 is taken as the first n frames as n set in the Preferences/Misc/DF/F calculation). This is also available in the context menu of the Plot Window.
  • Resample: set resample intervals in Preferences/'Data Loading / ROIs'/Resample. This will result evenly paced data points after copying graphs to clipboard or saving to text files. Linear interpolation is used for resample. This is also available in the context menu of the Plot Window.
  • Options of calculation of variance and punctate over diffuse index in addition to mean
  • Automatic saving of plot data into tab delimited text files.

Use FRatio Plot to calculate mean intensities in two linked Image Windows ad to plot the ratio (Image A over Image B) of mean intensities. This works better on low signal to noise images as compared to calculating FRatio images, and means of ratios.

Plot windows are kept simple and autoscaled, because their sole role to export the data for further processing in a spreadsheet or statistical software.

Managing ROIs from the Plot Window:

  • The Plot Window opened last from a Image Window remains linked to the Image Window, so any change happening in the image data or in the ROIs, the plot window will be immediately updated.
  • Use the Plot Window to select ROIs by left-clicking the corresponding trace
  • Scroll traces by < > buttons
  • Press DEL to delete selected trace and corresponding ROI (from all linked Image Windows).
  • Keep left button down and drag pointer horizontally to to scroll the original Image Window to the frame corresponding to the time point of the graph
  • Use the buttons on the status bar of the Image Window to selectively see (and copy or export) the trance corresponding to:
    • All ROIs
    • The active ROI (which is highlighted over the grayed all other traces)
    • The active ROI (which is only shown in the graph).

Calculating means of sections of traces in the Plot Window:

  • Press the Means of Marks button on the Status bar of the Plot Window.
  • Point to the trace, and select by left-clicking the section to calculate mean
  • The means of the separate grayed areas will be calculated, and shown in the hint above the pointer.
  • Saving or copying the data will result the selected mean time points and mean values for each trace.
  • Use Copy Markers and Paste Markers in the context menu of the Plot Window to transfer selection (grayed areas) between different Plot Windows.
  • This operation does not affect the image data.

Exporting data:

Plot data is exported in a tabular format: first column is the timescale (it is present even if the Image Window contains only one frame), the rest of the columns contain values corresponding to the Y-axis values of individual ROIs. When the Excel Data Window is opened plot data also appears in spreadsheet format.

  • When using the Resample option the time column is rescaled, and the y-values are interpolated.
  • Use right click context menu in the Plot Window to access: Copy Plot Data copy or Copy Y Data Only copy (the latter omits the first, time column) or the  Copy Plot Data copy in the Main menu Edit to copy graph values to the clipboard.
  • Use Save Plot Data save to generate tab delimited text files. When a Plot Window is opened by the FPlot or FRatio Plot functions, automatic saving and file naming can be set up optionally based on the Multi-Dimensional Open parameters, like base name and stage number. See details in the description of these functions.
  • Graphs can be copied as RGB image by Copy Plot Image copy plot image .