Pulsed Interleaved Excitation

Abstract

In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

“…Single-pair FRET experiments with pulsed interleaved excitation (PIE) [28,29] and multiparameter detection (MFD) [20] were performed on a home-build confocal dual-color dual polarization detection setup ( Figure 1). The system is built around a Nikon TE2000 microscope.…”

“…Classical spFRET experiments without MFD and PIE require determination of the relative quantum yield of the donor and acceptor fluorophores. In PIE or ALEX experiments, the relationship between the FRET efficiency and the stoichiometry can be used when multiple FRET species are present to determine g. [28,29] On the other hand, MFD experiments allow calculation of the FRET efficiency from both the fluorescence intensity and lifetime of the donor and thereby determination of g from a single species. Third, analysis of the time-resolved or steady state anisotropy is required to estimate the uncertainty in the orientation factor, k 2 .…”

“…Knowing the direct acceptor excitation coefficient, a, and the spectral crosstalk coefficient, b, the g factor [Equation (6)] can be calculated from a pair of double-labeled species [28,29] with different FRET efficiencies (DA1, DA2) by requiring S DA1 = S DA2 (Figure 3). According to Kapanidis et al, [28] g is determined from the best linear fit to the data plotted as 1/S PR versus E PR .…”

“…However, for MFD experiments, the lifetime information is lost due to the microsecond alternating timescale of msALEX. Pulsed interleaved excitation (PIE) [29] and nanosecond ALEX (nsALEX) [30] utilize pulsed lasers alternated on the ns timescale to probe the presence of the acceptor. The excitation source for each detected photon is distinguished using TCSPC.…”

Studying Ion Exchange in Solution and at Biological Membranes by FCS

“…Alternating excitation, introduced some years ago under the acronyms ALEX (Alternating Laser EXcitation) (Kapanidis et al, 2004) and PIE (Pulsed Interleaved Excitation) (Müller et al, 2005), can provide information about which excitation source has generated each detected fluorescence photon. Thereby, spectral cross-talk can be strongly suppressed, significantly improving the sensitivity by which the interaction between two spectrally distinct interaction partners can be characterized, on a single-molecule level or by FCCS.…”