Quantitative Förster resonance energy transfer efficiency measurements using simultaneous spectral unmixing of excitation and emission spectra

Abstract

Abstract. Accurate quantification of Förster resonance energy transfer (FRET) using intensity-based methods is difficult due to the overlap of fluorophore excitation and emission spectra. Consequently, mechanisms are required to remove bleedthrough of the donor emission into the acceptor channel and direct excitation of the acceptor when aiming to excite only the donor fluorophores. Methods to circumvent donor bleedthrough using the unmixing of emission spectra have been reported, but these require additional corrections to account for direct excitation of the acceptor. Here we present an alternative method for robust quantification of FRET efficiencies based upon the simultaneous spectral unmixing of both excitation and emission spectra. This has the benefit over existing methodologies in circumventing the issue of donor bleedthrough and acceptor cross excitation without the need for additional corrections. Furthermore, we show that it is applicable with as few as two excitation wavelengths and so can be used for quantifying FRET efficiency in microscope images as easily as for data collected on a spectrofluorometer. We demonstrate the accuracy of the approach by reproducing efficiency values in well characterized FRET standards: HEK cells expressing a variety of linked cerulean and venus fluorescent proteins. Finally we describe simple ImageJ plugins that can be used to calculate and create images of FRET efficiencies from microscope images.

Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra

“…Since 405 nm and 436 nm excitations share the same D455, our system with the two excitations has the same emission-spectral response. Because ExEm method can be achieved with as few as two excitation wavelengths [18], 405 nm and 436 nm excitations may be enough for our EES-FRET method. In this case, we just need to measure the emission-spectral responses of our system with D455.…”

“…Where, E is FRET efficiency of the paired donor-acceptor in FRET sample; C d is the concentration of free donor, C da is the concentration of the paired donor-acceptor, and C t A is the concentration of total acceptor; K D and K A are the total donor absorption and total acceptor absorption respectively in all excitation wavelengths used; Q D and Q A are the quantum yield of donor and acceptor, respectively; S D , S A and S S are the unit-area-normalized excitationemission spectral signatures of donor, acceptor and donor-acceptor sensitization, respectively, and they are the outer product of the excitation spectra (S ex X, X = D and A) and the emission spectra (S em X, X = D and A) measured from donor-only and acceptor-only sample, respectively [5,18]:…”

“…In sharp contrast to the ExEm-FRET method proposed by Mustafa and associates [18], EES-FRET method adopts complete instrumental calibration including the excitation intensity spectrum (wavelength-dependent excitation intensity) and the emission-spectral response (optical transfer function of the emission light path) of system. In addition, we introduce a ratio factor (r K ) of total acceptor absorption (K A ) to total donor absorption (K D ) in all excitation wavelengths used for the determination of concentration ratio (R C ) of acceptor to donor.…”

“…Recently, Mustafa and associates demonstrated that ExEm unmixing with as few as two excitation wavelengths could be used for quantitative FRET measurement, and for the first time implemented ExEm unmixing-based quantitative FRET measurement on a laser scanning confocal microscope with two different excitations to image the FRET efficiency (E) of a genetically encoded FRET construct in living HEK cells [18]. Rigorous system calibration and signal correction are required for almost every quantitative FRET measurement on confocal microscope due to the mutable status of confocal microscope [20][21][22].…”

“…Therefore, donor spectral bleedthrough can be overcome by Em unmixing, but the acceptor excitation cross must be corrected using additional manner [13][14][15][16]. Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, FRET-sensitized acceptor emission and direct acceptor emission [5,6,17,18]. Thus ExEm unmixing is capable of simultaneously resolving the donor spectral bleedthrough and acceptor excitation cross without additional correction.…”