Appropriate handling of fluorescence spectra for accurate spectral overlap (J) values in Förster energy transfer (FRET) calculations

Masahiko Taniguchi; Hai Du; Jonathan S. Lindsey

doi:10.1117/12.3025086

28 May 2024 Appropriate handling of fluorescence spectra for accurate spectral overlap (J) values in Förster energy transfer (FRET) calculations

Masahiko Taniguchi, Hai Du, Jonathan S. Lindsey

Author Affiliations +

Proceedings Volume 13083, SPIE Future Sensing Technologies 2024; 130831J (2024) https://doi.org/10.1117/12.3025086
Event: SPIE Future Sensing Technologies, 2024, Yokohama, Japan

Conference Poster

Abstract

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 λ² correction), because fluorescence spectra (but not absorption spectra) are collected with a fixed wavelength bandpass. The λ² 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 λ² correction for various donor–acceptor pairs (i.e., donor fluorescence spectra and acceptor absorption spectra). The magnitude of error introduced upon omitting the λ² 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 λ² correction for FRET calculations and also contains diverse spectral libraries, is freely downloadable at www.photochemcad.com.

(2024) Published by SPIE. Downloading of the abstract is permitted for personal use only.

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Masahiko Taniguchi, Hai Du, and Jonathan S. Lindsey "Appropriate handling of fluorescence spectra for accurate spectral overlap (J) values in Förster energy transfer (FRET) calculations", Proc. SPIE 13083, SPIE Future Sensing Technologies 2024, 130831J (28 May 2024); https://doi.org/10.1117/12.3025086

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