Pulsed-Interleaved-Excitation Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Abstract

We report on pulsed-interleaved-excitation two-dimensional fluorescence lifetime correlation spectroscopy (PIE 2D FLCS) to study biomolecular structural dynamics with high sensitivity and high time resolution using Förster resonance energy transfer (FRET). PIE 2D FLCS is an extension of 2D FLCS, which is a unique single-molecule fluorescence method that uses fluorescence lifetime information to distinguish different fluorescence species in equilibrium and resolves their interconversion dynamics with a submicrosecond time resolution. Because 2D FLCS has used only a single-color excitation so far, it was difficult to distinguish a very low-FRET (or zero-FRET) species from only donor-labeled species. We overcome this difficulty by implementing the PIE scheme (i.e., alternate excitation of the donor and acceptor dyes using two temporally interleaved excitations with different colors) to 2D FLCS, realizing two-color excitation and two-color fluorescence detection in 2D FLCS. After proof-of-principle PIE 2D FLCS analysis on the photon data synthesized with Monte Carlo simulation, we apply PIE 2D FLCS to a DNA-hairpin sample and show that this method readily distinguishes four fluorescent species, i.e., high-FRET, low-FRET, and two single-dye-labeled species. In addition, we show that PIE 2D FLCS can also quantitatively evaluate the contributions of the donor-acceptor spectral crosstalk, which often appears as artifacts in FRET studies and degrades the information obtained.

Figure 1

Schematic of PIE 2D FLCS setup. DM: dichroic mirror, exF: optical filter for preparing excitation pulses, emF: optical filter to select the emission detected, smOF: single-mode optical fiber, OBJ: objective lens, PH: confocal pinhole, SPAD: single-photon avalanche photodiode.

Figure 2

Principle of PIE 2D FLCS. (A) Schematic illustration of photon data obtained with the PIE 2D FLCS setup. T, macrotime; t, microtime; d, donor excitation pulse; a, acceptor excitation pulse; D, fluorescence photon detected by the donor detector; A, fluorescence photon detected by the acceptor detector. dD, dA, aD, aA: excitation and detection color (c) of individual photons. (B) Representative dD, dA, aD, and aA fluorescence decays obtained with PIE 2D FLCS setup (left), and corresponding dD fluorescence decay with the integrated aA and dA intensities (right) that are used for constructing PIE-2D emission-delay correlation map. (C) PIE-2D emission-delay correlation map (leftmost panel, top) and corresponding PIE-2D lifetime correlation map (leftmost panel, bottom) obtained with inverse Laplace transform of the dD fluorescence decay. The panels on the right are those of individual fluorescence species contributing to the 2D maps on the leftmost panels.

Figure 3

PIE 2D FLCS of a simulated three-species static system. (A) Schematic of the system. (B, C) Results for an ideal case of no spectral crosstalk, i.e., no donor fluorescence leak into the acceptor detector and no direct acceptor excitation by the donor excitation pulse. (D, E) Results for a nonideal system with 10% donor fluorescence leak into the acceptor detector and 20% direct acceptor excitation by the donor excitation pulse. (B, D) Independent fluorescence components obtained with global 2D MEM analysis. Green: D-only, red: A-only, and blue: FRET-labeled species. (C, E) PIE-2D lifetime correlation maps. The τ_dD – τ_dD 2D maps are normalized using the maximum peak intensity of each map. Corresponding τ_dD – I_dA, I_dA – τ_dD, τ_dD – I_aA, and I_aA – τ_dD 1D maps are normalized using the intensity of the highest peak of these four maps.

Figure 4

PIE 2D FLCS of a simulated four-species dynamic system. (A) Schematic representation of the system. (B) Independent fluorescence components obtained with global 2D MEM analysis. Green: D-only, red: A-only, orange: low-FRET species, and blue: FRET-labeled species. (C) PIE-2D lifetime correlation maps. Black arrows indicate cross-peaks appearing with interconversion between the low-FRET and high-FRET species. The τ_dD – τ_dD 2D maps are normalized using the maximum peak intensity of each map. Corresponding τ_dD – I_dA, I_dA – τ_dD, τ_dD – I_aA, and I_aA – τ_dD 1D maps are normalized using the intensity of the highest peak of these four maps.

Figure 5

PIE 2D FLCS of the DNA hairpin. (A) Schematic of the DNA hairpin. (B) Independent fluorescence components obtained with global 2D MEM analysis. Green: D-only, red: A-only, orange: open form, and blue: closed form. (C) PIE-2D lifetime correlation maps. Green, orange, and blue dashed squares indicate the τ_dD peaks belonging to D-only, open form, and closed form, respectively. Green dashed ovals indicate I_aA and I_dA positions corresponding to the major τ_dD peak of D-only. Black arrows indicate cross-peaks between the open and closed forms. The τ_dD – τ_dD 2D maps are normalized using the maximum peak intensity of each map. Corresponding τ_dD – I_dA, I_dA – τ_dD, τ_dD – I_aA, and I_aA – τ_dD 1D maps are normalized using the intensity of the highest peak of these four maps.