PFRET Data Processing Software

The precision FRET (PFRET) data processing software package of ImageJ plugins can be used to process Fluorescence Resonance Energy Transfer (FRET) microscopy data. The PFRET software package is designed for intensity-based quantitative FRET microscopy data analysis:

• Provides accurate spectral bleedthrough (SBT) corrections for varying intensity levels, FRET efficiency calculations, and distance estimations
• Works with TIFF images generated by any microscopy system (e.g. widefield, TIRF, confocal, spectral and multiphoton)
• Compatible with all computer operating systems – ImageJ plugin written in JAVA

One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor.  As a result of spectral overlap, the FRET signal is always contaminated by donor emission in the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength.  Both of these signals are termed spectral bleed-through (SBT) signal into the acceptor channel.  In addition to SBT, the FRET signals in the acceptor channel also require correction for spectral variations in donor and acceptor channels, autofluorescence, and detector and optical noise, which contaminate the FRET signal. Details of the PFRET algorithm to remove SBT and the relevant biological applications can be found in the literature:

• Elangovan et al (2003) Methods 29, 58-73
• Chen et al (2005) In Molecular imaging: FRET microscopy and spectroscopy. A. Periasamy, R. N. Day, editors. Oxford University Press, New York. 126-145
• Chen and Periasamy (2006) J Fluoresc 16, 95-104
• Chen et al (2007) J Microsc 228, 139-52
• Sun and Periasamy (2010) J Biomed Opt 15, 020513