(489x) FRET Efficiencies in Varying Cellular Microenvironments and Equipment Configurations

Förster resonance energy transfer (FRET), also known as fluorescence resonance energy transfer, is a radiationless process through which energy is transferred from an excited fluorophore (the donor) to a different fluorophore (the acceptor) by means of intermolecular, long-range, dipole-dipole coupling. Four conditions must be satisfied for FRET to occur: 1) the donor emission spectrum must significantly overlap the absorption spectrum of the acceptor; 2) the distance between the donor and acceptor fluorophores must fall within the range of 10 to 100 angstroms; 3) the donor emission dipole moment, the acceptor absorption dipole moment, and their separation vectors must be in favorable mutual orientation; and 4) the donor should have a high quantum yield. In cellular imaging assays, fluorescent protein pairs can be used to measure the kinetics of protein-protein interactions or protein folding by measuring the FRET efficiency. Hence, FRET is an excellent technique for quantitatively assessing enzyme kinetics, protein-protein binding, and protein folding in living cells. However, fluorescent protein excitation and emission spectra (one common pair is cyan fluorescent protein ? yellow fluorescent protein) are sensitive to environmental conditions, such as pH and ionic gradients. These are conditions that commonly vary between compartments within the cell, establishing local cellular microenvironments.

Because of the dependence of fluorescent protein excitation and emission spectra on environmental conditions, the measured FRET efficiency will also be sensitive to changes in cellular microenvironment. These changes in the fluorophore excitation and emission spectra can be measured using spectrophotometry. However, most fluorescence imaging assays typically use two or three emission wavelengths, making them unable to accurately quantify FRET efficiency when the excitation or emission spectra of either the donor or acceptor fluorophore are altered. In addition, spectral variations in fluorescence microscopy equipment (light source fluctuations, filter damage, etc.) can also lead to incorrect FRET efficiency measurements. Hence, it is very important to fully understand the effects of varying cellular microenvironments and equipment operating conditions on measured FRET response.

We have developed a model for FRET response that takes into account the varying optical components of a fluorescence microscope. This allows the comparison of spectrophotometric measurements (taken on a spectrophotometer) and two- or three-wavelength measurements that are typically made using a fluorescence microscope. We will present the results from this model and the practical implications for calculating FRET efficiencies in living cells and different equipment configurations. In addition, we will present a hybrid model that combines experimental measurements of fluorophore excitation spectra, emission spectra, and fluorescence microscope parameters with theoretical modeling of perturbations introduced by cellular microenvironments. This model can be used for assessing the sensitivity of a specific assay or equipment configuration to environmental changes. Finally, we will present the practical improvements in sensitivity and statistical accuracy that can be achieved when this model is applied to the design and execution of FRET assays in living cells.