First you should install CaSiAn. Running CaSiAn requires installing Java Runtime Environment (JRE) version 1.8.0. We have tested CaSiAn on Mac, Windows XP, 7 and Server 64- bit version.

After copying the files, double click on the CaSA.jar file in mac or CaSA.exe file in windows which will serve you the the first form of the tool. If double click command did not open the software, you can open the software by command line. First, go to the directory which CaSA.jar file exists and then type the command: java -jar CaSA.jar

The Main Window

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For opening a series of signals belong to one experiment you should follow these instructions:

  1. Browse the folder which contains all files of your signals. Please note that CaSiAn can open just files the .xls, .txt, .csv, .dat extension. The text files should be delimited by ‘,’ or tab separator.
  2. Enter the part of the file name which is common between all files that you intend to open.
  3. Enter the common string among the column header names that contain the intensities of calcium signals.
  4. Enter the number of column that contains the time of calcium signals.
  5. If the time column contains the frame number of the observed intensities instead of the real acquisition times, user can enter the time difference between two consecutive images in the specified input field of CaSiAn. Then CaSiAn computes the time of each sample by multiplying the frame number and the time difference between two consecutive images.
  6. Identify the names of treatments during the imaging, if there is any one.
  7. Load signals in the CaSiAn by clicking on the “open experiment” button.

The Edit Panel

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The length of a signal can be edited by the Edit panel. In this panel you identify the beginning and the ending of the signal and then by pressing the Edit button, signal will be edited. The Reset button, resets the signal to the initial signal and ignore all changes. By checking “apply to all” all changes will be applied on the all signals.

The Peak Detection Panel

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Using this panel, you can find peaks and nadirs of each signal. The user must identify how much percent of the maximum amplitude of the signal should be considered as the threshold value. The data points more than threshold value are indicated as the signal peaks. User can find peaks and nadirs of all signals by checking “apply to all” check box. After fining peaks and nadir of a signal, the average of the spike widths, the standard deviation of the spike widths, the average of amplitudes and the standard deviation of amplitudes of the signal are computed and shown in the following panel.

The Signal Plot and Interspike Interval over Time

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By loading signals, all signals are plotted in the tools and user can go through the signals by next, previous, first and last buttons. After finding peaks and nadirs of each signal, CaSiAn plots the Interspike intervals (ISIs) over time of each peak. This plots indicates the distribution of ISIs during the time.

Standard deviation of ISIs (SD) over average of ISIs (Tav)

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After detecting peaks of signals, CaSiAn is able to plot the standard deviation of ISIs over Tav. This plot shows the linear relation between SD and Tav. CaSiAn fits a linear line to the data points and shows the slope, intercept, root mean square error of the fit and also the population average of Tav. User is able to filter the data points on the base of number of peaks in each signal. The signals which have less than defined number of peaks will be removed from the data points by clicking on the “Filter signals” button.

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