Quantitative Fluorescence Resonance Energy Transfer Measurements Using Fluorescence Microscopy

Abstract

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

“…It has been previously concluded that calculation of the apparent E values (Ed in this paper, equation 2), which roughly represent FRET C normalized by the donor concentration, results in near-optimal measurement of FRET, and that its subsequent normalization by the acceptor concentration increases the variability in the resulting FRETN values 23 . Moreover, comparison of various methods of calculation of FRET efficiencies in the experimental system of multiple donor-acceptor interactions demonstrated that the method of E calculation 23 is the most reliable 32 .…”

“…The method of sensitized two-chromophore FRET measurement has been described previously 7,23 . For 3-FRET, images were acquired sequentially through FRET CY , FRET CR , FRET YR , CFP, YFP and mRFP filter channels (see Supplementary Table 2 online) using 2Â2 binning mode and 100-ms to 500-ms integration times.…”

“…FRET C was calculated for each of three two-chromophore FRET pairs in the presence of three fluorescent proteins on a pixel-by-pixel basis for the entire image as described for twochromophore FRET 7 using the formula shown in equation 1 in a modified notation 23 :…”

“…In experiments with concatemeric constructs (donor-acceptor 1:1), FRET efficiency E was calculated according to ref. 23 as:…”

“…The issue of quantification of intermolecular FRET data has been under much debate 23,31,32 . It has been previously concluded that calculation of the apparent E values (Ed in this paper, equation 2), which roughly represent FRET C normalized by the donor concentration, results in near-optimal measurement of FRET, and that its subsequent normalization by the acceptor concentration increases the variability in the resulting FRETN values 23 .…”