Accurate single-pair Förster resonant energy transfer through combination of pulsed interleaved excitation, time correlated single-photon counting, and fluorescence correlation spectroscopy

Abstract

Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem for the interpretation of the Forster resonant energy transfer (FRET) efficiency is the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescent acceptors. Other problems occurring are direct excitation of the acceptor, spectral crosstalk, and the determination of the quantum efficiency of the dyes as well as the detector sensitivity. Our approach to overcome these limitations is based on the pulsed-interleaved excitation (PIE) of both the acceptor and the donor molecule. PIE is used to excite the acceptor dye independently of the FRET process and to prove its existence via fluorescence. This technique enables us to differentiate a FRET molecule, even with a very low FRET efficiency, from a molecule with an absent or non-fluorescent acceptor. Crosstalk, direct acceptor excitation, and molecular brightness of acceptor and donor molecules are determined by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET efficiencies of the same data set are also determined by analyzing the lifetimes of the donor fluorophores. The advantages of the PIE-FRET approach are demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as donor and acceptor, respectively.

Periodic acceptor excitation spectroscopy of single molecules

“…Fluorescence emission from spectrally distinct fluorophores was recorded at two distinct wavelength ranges and further distinguished by the excitation period (donor or acceptor) resulting in four separate photonstreams, which were used for calculations of FRET efficiency E and stoichiometry S for each detected fluorescence burst (due to the transit of a single molecule). A similar approach uses pulsed lasers with an interleaved pulse sequence, giving access to the fluorescence lifetime of each fluorophore (Kukolka et al 2006;Laurence et al 2005;Muller et al 2005;Ruttinger et al 2006). ALEX has been implemented at different time scales (nanosecond: ns-ALEX, microsecond: μs-ALEX, and millisecond: ms-ALEX), in wide-field and pointdetection schemes, and on diffusing and immobilized molecules (Kapanidis et al 2005a), and has been combined with fluorescence correlation spectroscopy (Doose et al 2005;Muller et al 2005).…”

Mechanistic principles and applications of resonance energy transfer

“…When the same two chromophores feature, in spatially different configurations, in the chemical composition of two different systems (again, a common feature in biology), then the relative displacements of the chromophores can be quantitatively assessed on the basis of comparisons between the corresponding RET efficiencies (23)(24)(25)(26)(27)(28)47). Such a technique is popularly known as a "spectroscopic ruler".…”

“…2 exhibit typical implications for the spectral overlaps in the numerator and denominator of eq. [25]. For each chromophore, the fluorescence peak is Stokes-shifted to a lower frequency with respect to its absorption counterpart.…”

“…[10] and [11], and considering their counterparts for the inverse transfer process, it is in fact clear that the key to imposing directedness principally lies in exploiting differences in the relative spectral profiles. To quantify the relative rates or propensities for forward and backward transfer, it has been found convenient to introduce a dimensionless relative directional efficiency, ε, defined by [25] ε τ ωσ ωω ω τ ωσ ωω ω…”

“…In the fields of optical communications and computation, a number of optical-switching and logicgate devices are founded on the same principle (20)(21)(22). In the realm of molecular biology, the determination of protein structures and the characterization of dynamical processes is furthered by studies of the transfer of energy between intrinsic or "tag" chromophores (23)(24)(25)(26)(27)(28); other ultra-sensitive molecular-imaging applications are again based on the same underlying principle (29)(30)(31). Further applications include the study of polymer interfaces (32), energy-transfer systems designed to act as sensitizers for photodynamic therapy (33), and as analyte-specific sensors (34)(35)(36).…”