Analysis of Direct FRET data consists of determination of Donor and Acceptor crosstalk factors, calculation of Net FRET and FRET efficiency, and analyzing the Net FRET and FRET Efficiency statistics. Use the FRET analysis tool and the following process to analyze the FRET data:
1. Calculate Donor and Acceptor Crosstalk involves creating Region of Interest (ROI) polygons on Donor and Acceptor Control images and determining representative background values that are subtracted from intensities in ROIs.
2. Calculate Net FRET and FRET Efficiency uses a FRET Experiment image as input along with Donor and Acceptor Crosstalk factors and background values for each channel and generates an image with 2 channels: Net FRET and FRET Efficiency (%E).
3. Analyze results Analyzing Net FRET and FRET Efficiency statistics involves specifying a FRET Results Image and one or more ROI polygons to generate a table and graph of statistics.
Acquiring and Preparing FRET Data