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Plots generated by the program

After the detection and reviewing of the data, some basic plots can be found under the Plots menu that allow the visualization of some of the basic parameters that the program calculates. Some plotting functions can generate multiple windows that show the data per cell as well as the mean of all cells per group/condition. The following plots are currently available:

The following sections will briefly explain these plots and how they can be used to extract data for publications.

Current trace

The current trace option allows the plotting of the currently selected release site and the current event in the detail plot to a new window. This new window can be used to modify plotting parameters such as the X and Y scale and the colors. Keep in mind that all areas that are plotted have their alpha level set to 1.0 to allow exporting to a vector format such as EPS. The main interface uses alpha level < 1.0 during the plotting, so the appearance of the new plots may look different from the interface.

Current trace example
Figure 1: Example of a current trace window. Notice the color of the patches due to the lack of transparancy.

Histograms

The histogram plots can show 4 different parameters that can be relevant: the event duration, onset time , decay rate and peak/NH4 ratio. The event duration and onset time shows a histogram with binning set to the sample interval per cell in subplots. Both the decay rate and peak/NH4 ratio have a binning set to 0.1 and may contain negative values. One figure can hold a maximum of 6 cells in a 3x2 arrangement. For more than 6 cells, additional figures are created to accomodate the additional cells. Finally, a single figure is created with the average histogram over cells per group/condition. A solid line indicates the mean frequency with a dashed line indicating the standard error of the mean (SEM). Colors are set automatically per group and are consistent between all the plots.

Histogram fusion onset
Figure 2: Example of a histogram plot. The solid line is the mean per group, while the dotted line indicates the SEM. The stimulation time is indicated by the colored bars.

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Cumulative plots

This function generates two figures with averaged data showing the cumulative frequency of onset time and duration. One figure shows the data on the absolute scale, while the other figure is on the normalized scale. The first plot from the left is the onset time, followed by a zoom of the first 4 stimulation bursts. The rightmost plot shows the plot for the event duration per group/condition. The solid line indicates the mean, and the dashed lines indicate the SEM. Please note that the duration plot does not have the stimulation plotted, as this parameter is not related to the onset of stimulation directly.

Cumulative plots (normalized)
Figure 3: Example of a normalized cumulative plot. The left plot is the fusion time per group, with solid lines representing the mean and the dotted lines the SEM. The middle plot is a zoom of the first 4 bursts of stimulation taken from the left plot. The right plot shows the fusion duration per group. The vertical dashed line in the third plot indicates the current level for transient/presistent classification.

Box and whisker plots

The box plots show the distribution of the data in a box and whisker plot. Two types of boxplots are generated: one figure for each condition with the box plots for the individual cells. Finally, a figure is generated with the grouped data. Boxplots are generated with outliers, which are defined using the following steps:

  1. calculate the median (Q2) and the first (Q1) and third (Q3) quantile
  2. calculate the Interquartile range (IQR): IQR = Q3-Q1
  3. Determine the edge of the lower and upper whiskers (LW and UW, resp.): LW = Q1−1.5×IQR and UW = Q3+1.5×IQR
  4. All points smaller than LW or higher than UW are considered (potential) outliers
The boxplot for number of events uses 3×IQR for the whiskers, meaning the points labelled as outliers in those plots are considered true outliers with a large margin of confidence. Plots can be generated for onset time, event duration and peak height. The colors used in these plots are also consistent with the other plot functions. The grouped plot also has notches, which can be used to determine significance at the 5% level: when the notches of groups show no overlap, the medians are significantly different. When plotting the duration parameter, a dashed line indicates the current setting for the classification threshold for persistent events. Values below the dashed line will be classified as transient events, while all values above the line will be classified as persistent.
Boxplot of duration
Figure 4: Example of a box and whisker plot. The dashed line indicates the current setting for transient events (10s). Circles indicate raw values, while crosses indicate (potential) outliers.


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Heat maps

The program allows the plotting of a heat map for the number of events and the first onset of events over a user-selected image. Under the Plots menu, the option for Heat maps can be found, which opens the interface for selecting a background image and the ROIs used for the analysis. At the bottom of the figure the sheetname of the current cell in the interface is displayed, which is meant to aid in the selection of the correct background image. Setting the background image can be done by pushing the Load BG image button. Please note that currently only TIF files are supported, and when selecting multipage tif files, only the first frame or channel is shown. After the background image is set, the ROIs can be loaded by pushing the Load ROIs button. Currently, only ROI files generated by ImageJ are supported, and the file has to have the .zip extension (standard when saving multiple ROIs in ImageJ). After the ROIs have loaded, the regions are plotted on top of the background image and given a color coding to indicate the number or time of the events (see figure 5).

Heat map example
Figure 5: Example heat map showing the onset of first fusion event. Stimulation started at t=30s.

The drop down menu allows the user to adjust the size of the markers used in the plot. For onset time, the color scale is set from 0 up to the ammonium application. The data can be exported using the File menu of the figure window. When saving as an .eps file, the regions are exported as vectors and can be manipulated in a vector graphics program such as Adobe Illustrator or Inkscape. Please note that the background image can not be resized in the interface; this is to be sure that the placement of the ROIs is corrrect. Once the data is exported, the image and the ROIs can be adjusted freely, but with caution.

Data Viewer

The program has an option to view core values in both a table and bar plot format. This can be done by selecting Plots | Data Viewer from the menu. This will open a new interface that allows the selection of a data type to be loaded, followed by a bar plot of the data and a table with the values per cell. As shown in Figure 6, the interface will display the bar plot per condition, and the table is sorted per condition. The table can be copied to an Excel or text file by selecting the data and pressing Ctrl + C.

Data viewer interface
Figure 6: Data viewer interface

There are currently 4 different plots that can be viewed, with 2 different tables associated with them:
  1. Nr of events: the total number of events per condition
  2. Events per site: the average number of events per site (mean±SEM)
  3. Categorization (Nr): the total number of events per category
  4. Categorization (%): category percentage of the total number of events
For plots 1 and 2, the colors are identical to the color scheme used in the rest of the interface. For plots 3 and 4, the color scheme is set to use a scale between red and yellow, with the corresponding category values detailed in the figure legend. When the data viewer is active, the main interface cannot be used until the viewer is closed.

Exporting for publication

The figures can be exported by selecting the Save As... option in the file menu of the plot window. To get the best quality setting the output format to EPS will ensure that the plots can be resized properly without artefacts. Selecting the Plot Browser in the View menu will allow the modifcation of the X and Y scale, as well as colors and enabling or disabling certain plot elements. Keep in mind that the colors used in the interface for groups are consistent between different plots: when modifying the colors manually, this consistency will be lost. For more information on the Plot Browser, see the Matlab documentation.

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