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Find donor- and acceptor peaks and identify FRET-pairs

About the peak finder

The peak finder finds peaks in the image averaged over a certain frame interval, defined by the frame slider above the overlay image. Using an average of many frames is a quick way to reduce noise in the image used for peak detection.

Optimize image used for peak detection

Use the frame slider above the overlay image to define the frame interval of the average image. The frame interval used for the average image is shown just above the slider. Widening the interval reduces noise but may also decrease peak intensities. The optimal image is characterized by a peak intensity signal-to-noise ratio as high as possible. 

Use the frame slider to define the image used for peak detection.

Determine thresholds for the peak finder

In the peak finder panel, use the two sliders to optimize the intensity thresholds of the peakfinder. Move the peak sliders to include more or less peaks to the image until all desired peaks are found. The D-slider adjusts the number of donor peaks in the green emission channel. The A-slider adjusts the number of acceptor peaks in the red emission channel. Note how intensity thresholds in the editboxes are updated accordingly. You can choose to show only one color channel at a time using the overlay image panel button.
When both donor and acceptor peaks have been found, FRET-pairs are automatically identified based on the relative distance in between peaks in the donor and acceptor emission channels. The number of found FRET-pairs is shown in the peak finder panel and their positions are plotted in yellow in the image.
When the desired number of FRET-pairs have been identified, based on visual inspection of the overlay image, save these threshold settings as default by pressing the save icon in the peak finder panel toolbar. This will keep these settings the next time iSMS is opened.

Run peak finder with pre-defined thresholds

When the proper intensity thresholds have been found and saved as default as described above, find FRET-pairs in consecutive files by pressing the 'Run' button in the peak finder panel. This identifies all D and A peaks according to the specified thresholds and identifies FRET-pairs based on the distance threshold in between the found peaks.

Settings for the FRET-pair finder

Open the peak finder settings in the peak finder panel toolbar or navigate to 'Settings->Peak finder settings'.
The peak-finder settings.

The peak-finder settings are described below:

Default peak intensity threshold                            

Intensity of the lowest-intensity peak

Threshold for max. peaks

Relative threshold value defining the upper limit of the peak slider.

Max. number of peaks The maximum number of peaks allowed.
Use sub-pixel resolution When checked, the peak finder adjusts peak coordinates to within less than 1 pixel accuracy. This is a disadvantage for dense samples where neighbouring peaks distort the sub-pixel localization algorithm.
Max. D-A peak separation This threshold defines how far apart D and A peaks are allowed to be before being defined as a FRET-pair.
Use background subtraction When ticked, the determined peak intensities, on which the thresholds act, are background-subtracted using a local background ring. This is an advantage for narrow TIRF laser spots, but can be a disadvantage for dense samples.
Peak finder choice

The peak finder can either find peaks in the shown image or by scanning the entire movie. When scanning the entire movie the algorithm combines all FRET-pairs found through the movie. Scanning the movie always includes more false positives but may also include pairs not found by the single-image algorithmn.
Put a D at every A

This defines a FRET-pair at every A peak position. This is an advantage for high FRET samples where the D signal is very low. However, this also includes more false positives.
Put an A at every D                                                                                                                                                                              This defines a FRET-pair at every D peak position. This is an advantage for low FRET where the A signal is low. However, this also includes more false positives.

Hide peaks in overlay image

Use the toggle buttons in the toolbar to hide/show the peaks and FRET-pairs in the overlay image.