Figure 3

The influence of free donor or free acceptor in the sample. (A) A theoretical S-E histogram with trajectories corresponding to the addition of free donor or free acceptor to a construct with 1:1 donor to acceptor ratio. The blue disk represents the area where pure donor samples would appear, whereas the green ellipse is where free acceptor samples would appear. (B) Experimental histogram of S versus E for constructs showing different FRET values (C32V and C5V) or different stoichiometries (CVC and VCV) as well as pure donor (Cerulean) and pure acceptor (Venus). This histogram was calculated using only the crosstalk correction factors and concatenating results from different experiments. (C). The same experimental E-S histogram with the complete calibration including \({\gamma }^{M}\) and \({\beta }^{X}\). In the completely corrected 2D histogram, the stoichiometry and FRET probability are uncorrelated (\(\rho =\mathrm{0.02,}\,N=5\cdot {10}^{6}\)). (D) An exemplary triplet of images showing a cell expressing C32V with a low signal-to-noise ratio, Scale bar 15 μm. (E) The corresponding RAW E and S maps and the FRET map for the images in panel D after filtering with a weigthed gaussian filter. (F) The corresponding stoichiometry histogram and the weights (W) as a function of the stoichiometry (line). For the weighting function, we used a Gaussian with a mean stoichiometry of \(S=0.5\) and a variance \({\sigma }_{S}=0.1\) (Eq. 16). The corresponding map of weights W is shown in (G). (H) Line profiles corresponding to the three maps shown in panel (E). Due to high intensity background in an endosome, the FRET efficiency drops (thin grey line). This anomaly is also observable in the stoichiometry (blue). By weighting the image with the measured stoichiometry, such artifacts can be recognized (magenta).