Maximizing the quantitative accuracy and reproducibility of Förster resonance energy transfer measurement for screening by high throughput widefield microscopy.

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) provides insights into the proximities and orientations of FPs as surrogates of the biochemical interactions and structures of the factors to which the FPs are genetically fused. As powerful as FRET methods are, technical issues have impeded their broad adoption in the biologic sciences. One hurdle to accurate and reproducible FRET microscopy measurement stems from variable fluorescence backgrounds both within a field and between different fields. Those variations introduce errors into the precise quantification of fluorescence levels on which the quantitative accuracy of FRET measurement is highly dependent. This measurement error is particularly problematic for screening campaigns since minimal well-to-well variation is necessary to faithfully identify wells with altered values. High content screening depends also upon maximizing the numbers of cells imaged, which is best achieved by low magnification high throughput microscopy. But, low magnification introduces flat-field correction issues that degrade the accuracy of background correction to cause poor reproducibility in FRET measurement. For live cell imaging, fluorescence of cell culture media in the fluorescence collection channels for the FPs commonly used for FRET analysis is a high source of background error. These signal-to-noise problems are compounded by the desire to express proteins at biologically meaningful levels that may only be marginally above the strong fluorescence background. Here, techniques are presented that correct for background fluctuations. Accurate calculation of FRET is realized even from images in which a non-flat background is 10-fold higher than the signal.