Issues in confocal microscopy for quantitative FRET analysis

Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.