Quick Guide 1: Find FRET-Pairs, Calculate Traces and Plot Histograms

1. Load data and align emission channels

Load raw image data by pressing Ctrl+L (on Windows) or go to 'File->Data->Load movie file'. Locate movie files in the dialog and press 'Open'. The movies will be loaded into the program and listed in the Files listbox.
Align the green and red ROIs (Region-Of-Interests, i.e. emission channels) in the raw image to the left. The ROIs are aligned when peaks in each color channel overlap in the overlay image to the right.
When the ROIs are aligned you can go to 'File->ROI' and set these positions as the default or apply them to other loaded files.

2. Find FRET-pairs and plot traces

Find donor (D) and acceptor (A) peaks using the peak finder panel. First, the intensity thresholds must be optimized for your setup using the two sliders: Add more or less peaks by dragging the threshold sliders until the desired number of peaks has been reached. Molecules with high intensity are added first.
FRET-pairs are automatically identified from the distance between all D and A peaks. In this example, 33 D peaks, 40 A peaks and 24 FRET-pairs were found as shown in the peakfinder panel. When you have found a proper set of thresholds you can save these thresholds as default using the save button in the peak finder panel toolbar.
Now calculate the intensity traces. Open the FRET-pair window in the Toolbar. This will open a new figure window which plots the intensity traces of individual FRET-pairs.
When the FRET-pair window is open, the intensity traces of all new FRET pairs are automatically calculated - and updated when settings are changed. Use the listbox to browse the individual molecules.

3. Plot E-histogram and export figure

To plot histograms, open the histogram window in the toolbar of the main window.
This will open a new figure window which is automatically updated based on current settings and selections.
To export the figure or data, right click inside the plot and select export option. For high-resolution figures go to 'File->Export figure'.