Fluorescent proteins are increasingly being applied as non-invasive probes in living cells due to their ability to be genetically fused to proteins of interest for investigations of localization, transport, and dynamics. In addition, the spectral properties of fluorescent proteins are ideal for measuring the potential for intracellular molecular interactions using the technique of Förster (or fluorescence) resonance energy transfer (FRET) microscopy. Because energy transfer is limited to distances of less than 10 nanometers, the detection of FRET provides valuable information about the spatial relationships of fusion proteins on a sub-resolution scale. This interactive tutorial explores various combinations of fluorescent proteins as potential FRET partners and provides information about critical resonance energy transfer parameters, as well as suggestions for microscope optical filter and light source configuration.
The tutorial initializes in Widefield mode with the absorption and fluorescence emission spectra of the popular FRET pair, enhanced cyan and yellow fluorescent proteins (ECFP, labeled the Donor; and EYFP, labeled the Acceptor) appearing in the Spectral Profiles window superimposed over the emission spectrum of a mercury arc-discharge lamp (blue line spectrum). The Spectral Overlap region between the donor emission spectrum and acceptor absorption spectrum is illustrated with vertical lines and is displayed as a percentage in the yellow box beneath the window. Likewise, the absorption spectral overlap (AbOL) and emission spectral overlap (EmOL) regions are depicted in red and blue, respectively, and also displayed as percentages. The Förster Distance (calculated as described below) is presented in nanometers for each FRET pair and displayed above the Apply Presets button. In this tutorial, a variety of donor proteins can be selected to appear in Channel 1 and the acceptor proteins appear in Channel 2. The absorption and emission maxima are listed for each channel, and individual spectra or the entire channel can be toggled on or off using the checkboxes.
In order to operate the tutorial, select an imaging mode (Widefield or Confocal) and use the Choose A Light Source pull-down menu to select the appropriate illumination source (arc lamp or laser), which is then superimposed over the donor and acceptor spectra. Next, click on the Apply Presets button to activate the suggested excitation and emission bandpass filters as well as the dichromatic mirror for the selected FRET pair (Note: these values are intended only as a starting point in determining a useful filter combination). The filter passband regions can be adjusted by translating the sliders or clicking on the arrow button pairs at each end of the sliders. The left-hand arrow button pair moves the entire passband, while the right-hand arrow button pair adjusts the width. Filters can be temporarily removed from the window by deselecting the appropriate checkbox(es). The Dichromatic Mirror slider can be used to adjust the cut-on wavelength of this optical element. The Absorption Spectra, Emission Spectra, Excitation Source, and/or Dichromatic Mirror can be toggled on and off with the checkboxes on the right-hand side of the tutorial. Use the Maintain State checkbox to freeze all settings when toggling through donors and acceptors using the Choose A Donor and Choose An Acceptor pull-down menus. The fluorescent protein spectral class (blue, cyan, green, yellow and orange/red) listed in these menus can be selected using the radio buttons in the upper right-hand corner of the tutorial.
The spectral profiles illustrated in this tutorial were either gathered from the literature or carefully recorded under controlled conditions using purified fluorescent proteins. They are normalized for comparison and display purposes. In actual FRET investigations, spectral profiles may differ due to fluctuations in the extinction coefficient, quantum yield, probe concentration, and the maximum peak values for the localized environment within the various organelles and cytoplasm of living cells. As such, the information presented in this tutorial should be considered as being designed for instructional purposes only. In addition, the Förster distances calculated by the tutorial are also based on published spectral parameters of fluorescent proteins (extinction coefficient, quantum yield, wavelength distribution) and are in general agreement with other literature values. However, depending on the variables employed to calculate FRET efficiencies, these values may differ from those observed in an experimental setting.
One of the target purposes of this tutorial is to enable visitors to explore the optimum illumination sources and filter combinations for specific FRET pairs. For example, when investigating new cyan fluorescent proteins, such as Midori-ishi Cyan (MiCy), the tutorial is useful for examining lasers having lines in the violet and blue-violet spectral regions (405 to 473 nanometers) to determine the maximum excitation efficiency for this probe. Likewise, the various arc-discharge lamp (mercury, xenon, and metal-halide) spectra can be superimposed over the donor absorption spectrum to match excitation filter bandwidths and wavelength distributions that might be readily available in the laboratory. The tutorial is also efficacious for determining approximate excitation and emission spectral bleed-through levels for new probe combinations, which if excessive, could present problems in quantitative analysis.
When a fluorescent protein enters the excited state due to illumination by a laser or an intense arc lamp plasma source, the molecule behaves as an oscillating dipole and creates a characteristic electric field. In cases where the close proximity of a suitable acceptor fluorescent protein (or other fluorophore) places it within the boundaries of the electric field generated by the excited protein (the donor), energy can be transferred directly from the donor by electrodynamic interactions that induce transitions in the acceptor. An intermediate photon is not involved. The efficiency of energy transfer is dependent on the square of the donor electric field magnitude, and this field decays as the inverse sixth power of the distance between the donor and acceptor. The Förster distance represents the molecular separation at which energy transfer is 50-percent efficient. For measurable FRET to occur, several requirements must be met. Among these is a strong dependence on the physical distance between the donor and acceptor fluorophores, which is limited to only a few nanometers. In addition, there must be significant spectral overlap of the donor emission spectrum with the absorption spectrum of the acceptor. Finally, both the donor and acceptor molecules must be in a favorable mutual orientation.
A critical step in the practical application of resonance energy transfer to experimental biology is in the calculation of the Förster distance (Ro) value for the target donor-acceptor pair. The most important factors in this calculation are the overlap integral (J(λ)), the quantum yield of the donor in the absence of the acceptor (Q(d)), and the orientation factor between the two molecules (κ; this variable is squared). In this tutorial, the overlap integral is calculated according to the equation:
where the extinction coefficient (ε(λ)) is expressed in units of reciprocal moles per centimeter, wavelength in nanometers, and the normalized fluorescence intensity of the donor as a function of wavelength is dimensionless. The overlap integral calculation is dependent upon numerous variables, including environmental effects on the fluorescence emission spectrum profile and quantum yield of the donor. For donors having a high quantum yield and acceptors with large extinction coefficients, the spectral overlap integral will be greater, leading to more efficient FRET partners with a higher Ro value. Although the refractive index does not vary to a great degree between typical aqueous solvents or proteins, in the Förster distance calculation this parameter appears as the inverse of the fourth power and even a relatively minute change can render a much larger effect than quantum yields or the overlap integral. In addition, the efficiency of the dipole-dipole interaction between the donor and acceptor depends on the alignment of the two dipoles. The orientation factor, which describes this proximity, ranges from 0 (both dipoles perpendicular) to 4 (dipoles parallel). In general, the dipoles are assumed to be rapidly moving on timescales similar to the donor excited-state lifetime and the orientations are thus described as random, with an orientation factor of 0.67 (applied in this tutorial). Note that the orientation factor approximation can lead to uncertainty in the estimates of the distance separating two fluorophores based on FRET results in actual experiments.
Richard N. Day - Department of Endocrinology and Metabolism, Box 800578, Health System, University of Virginia, Charlottesville, Virginia, 22908.
Ammasi Periasamy - Keck Center for Cellular Imaging, Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903.
Matthew J. Parry-Hill, Adam Rainey, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.