Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy: improved accuracy for samples with spatially varied autofluorescence to signal ratio

Session

Poster Session

Authors

István Rebenku (1), Cameron B. Lloyd (1, 2), János Szöllősi (1), György Vereb (1)

Affiliations

1. Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen
2. Department of Oncology and The Milner Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge

Keywords

Förster resonance energy transfer, autofluorescence correction, 

Abstract text

The importance of interaction between signaling species in cellular processes often precedes that of expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions since it is highly sensitive to proximity in the range of 1-10 nm. Intensity-based, spectral spillover-corrected FRET is a cost effective and versatile approach which can be applied in flow cytometry and various modalities of fluorescence microscopy, but may be hampered by varying levels of cellular autofluorescence. Here, we have implemented pixel by pixel autofluorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofluorescence that allow the correct determination of all spectral spillover factors, and present an ImageJ/FIJI plugin for manual and automatic creation of quantitative FRET efficiency maps. For validation, we have used bead and cell-based FRET models covering a range of signal to autofluorescence ratios and FRET efficiencies and compared the approach with conventional average autofluorescence/background correction. The pixel-by-pixel autofluorescence correction is easy to implement and is superior in the reliability of results, particularly for samples with spatially varying autofluorescence and low fluorescence to autofluorescence ratios, the latter often being the case for physiological expression levels.