Fluorescence (or Förster) resonance energy transfer (FRET) describes the flow of energy from a donor fluorophore to an acceptor chromophore. Among the parameters in determining FRET efficiency is the donor-acceptor energy matching, given by the overlap of the donor fluorescence spectrum and the acceptor absorption spectrum (J value). Calculations of the J value typically rely on experimentally acquired spectra. When a fluorescence spectrum is converted from the wavelength (nm) scale to the wavenumber (cm -1 ) scale, the Y-axis intensity needs to be corrected by the square of the wavelength (termed the l 2 correction), because fluorescence spectra (but not absorption spectra) are collected with a fixed wavelength bandpass. The l 2 correction causes the peak intensity of the short-wavelength (long-wavenumber) side of the fluorescence spectrum to decrease, or the peak intensity of the long-wavelength (short-wavenumber) side to increase. This issue has been known for >60 years but the impact remains little appreciated. The relatively new availability of libraries of spectral data enabled assessment here of the l 2 correction for various donor-acceptor pairs (i.e., donor fluorescence spectra and acceptor absorption spectra). The magnitude of error introduced upon omitting the l 2 correction increases with the width of the spectra. A meta-analysis of recent literature reveals trends in usage of wavenumber or wavelength scales. While either scale can be used, errors in calculating the ostensibly simple J term are not uncommon. Best methods are articulated here. The software program PhotochemCAD 3, which incorporates the l 2 correction for FRET calculations and also contains diverse spectral libraries, is freely downloadable at www.photochemcad.com.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.