Quantitative FRET analysis

Padilla-Parra, S, Auduge, N, Coppey-Moisan, M and Tramier, M. (2008). Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells. Biophys. J DOI 10.1529/bio-... 

Wallrabe, H., Chen, Y., Periasamy, A. and Barroso, M. (2006). Issues in confocal microscopy for quantitative FRET analysis. Microsc. Res. Tech. 69, 196-206. 

Because FRET-FLIM is so widely applied in biology, it has also received special attention with respect to quantitative data analysis. Usually, the biologist is not per se interested in the actual lifetime values, but he/she is interested in the degree of complex formation and the proximity/orientation of the donor/accep-tor pair in the complex. FLIM enables not just a contrast in these parameters but can provide quantitative estimation of the degree of complex formation and inter molecular distances in situ. To this end, special analysis routines have been developed based on (global) analysis of multiple lifetimes in images using a... 

Shi X, Basran J, Seward FIE, Childs W, Bagshaw CR, Boxer SG (2007) Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C) quantitative analysis of FRET in YFP dimers. Biochemistry 46 14403-14417... 

We can apply the quantitative analysis of two-component mixtures to the FRET data from Fig. 2.4 [20]. In this case, the two lifetimes are equal to the lifetimes of the donor in the absence and presence of acceptor. Figure 2.5a shows the results for EGFR-GFP. It can be seen that in the control sample, the relative concentrations of the short-lifetime component are not equal to zero, as would be expected for a monoexponential donor in the absence of FRET. Indeed, if we look at the plot of Nt versus D, for a subset of the pixels of the EGFR-GFP data, we see that the data for the control are not centered on a point on half-circle of monoexponential decays. In contrast, if we look at the results of EGFR-YFP (Fig. 2.5b), we find that the relative concentrations of the short-lifetime component are indeed much closer to zero. Indeed, the plot of Ni versus Dt shows that the data for the control are centered on a... 

FRET applications employing CFP and YFP are complicated due to considerable bleed-through between CFP and YFP fluorescence (Figs. 5.5B and 5.6B). Direct excitation of YFP and bleed-through of CFP fluorescence into the YFP detection channel have to be corrected for as shown in Chapters 7 and 8. The multiexponential fluorescence decay of all CFP variants complicates the quantification of FRET by donor lifetime methods. Altogether these factors make quantitative analysis of the FRET efficiency relatively difficult. 

The introduction of fluorescent proteins have been a great addition to the repertoire of FRET measurements of protein systems in solution and especially in fluorescence microscopy (138-141). Indeed, the number of applications of FRET in fluorescence microscopy have exploded in recent years, and novel analysis methods have also been developed in order to extract quantitative parameters from image data (142-146). 

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