Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between two fluorescent molecules in which excitation is transferred from the donor to the acceptor without radiation. During the relaxation the excited acceptor molecule can emit photons that can be detected by flow cytometry.
Our goal was to upgrade and further develop an already existing simpler Matlab based program to analyze FCS format flow cytometry files. The original program offered only the basic processing options for users (1D and 2D histogram visualization, mean and standard deviation calculation).
Its improvement expanded to the enhancement of the 1D and 2D „region of interest” (ROI) drawing, designing an interface to modify the plotting options, and connecting to Microsoft Excel spreadsheet, so data can be read or exported to xls format easily. For the sake of flawless running the new functions required continuous testing.
Furthermore, as a result of our work we can define and generate new parameters from the data of the FCS format files. A list of mathematical expressions was created containing the necessary formulas for FRET calculations. Using it – and with the implemented formula-interpreter - we can determine the value of the energy transfer, and all necessary related data (S factors, acceptor and donor labeling values). The list can be arbitrarily expanded by available operations and functions in Matlab, making any complex fluorescence spectroscopy evaluation possible. The counting accuracy of the program was compared to the Reflex flow cytometry analysis software, which is frequently used. The results support the reliability of our program.
The developed program already allows users to process the data from any kind of FRET measurement, which can give accurate information about molecular distances and proximity-relationships of ligands on cell systems. This type of evaluations provides essential information in the field of biomedical research and diagnostics.