Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Abstract

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease Eco RI.

Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution

“…Excitation power was monitored and kept well below the qdots' saturation power level (9). These qdots and dyes can both be excited at 900 nm by two-photon excitation; thus, the observation volume is the same for both f luorophores [critical for dual-color crosscorrelation FCS (26)] and gives an accurate measure of bright fraction. Methods of fluorescence lifetime measurements are described in ref.…”

“…The average number of fluorescent particles inside the focal volume is equal to the reciprocal of the zero-time autocorrelation amplitude G(0) (30); therefore, the bright qdot number (green particles) is found by G(0) in the green channel (G(0) green ), and the total qdot number (red particles) is found by G(0) in the red channel (G(0) red ). The fluorescence crosscorrelation function measures coincident fluctuations in both channels in case of binding of the two species with distinct colors (26,31). The cross-correlation amplitude (G(0) cross ) is the time-averaged intensity fluctuation product ͗I(t) green I(t) red ͘ divided by the product of the average intensities in the two channels, ͗I(t) green ͗͘I(t) red ͘.…”

Global analysis of fluorescence fluctuation data

“…In the last few years an increasing number of applications of dual-colour FCCS have been reported. These vary from enzyme kinetics Koltermann et al 1998, Rarbach et al 2001, nucleotide hybridisation (Schwille et al 1997;Rigler et al 1999b) and protein-DNA interactions (Rippe 2000) to the application of two-photon excitation (Heinze et al 2000). Dual-colour FCCS applications in live cells have been reported recently (Bacia et al 2002;Hink et al 2003).…”

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

“…[6][7][8] Due to the stringent alignment requirements in combining two excitation sources, the use of two-photon excitation overcame these technical disadvantages and facilitated the excitation of multicolor fluorescent mixtures. 9,10 For example, two-photon excitation of up to three dyes has been implemented to simultaneously resolve molecular concentrations and kinetics of three distinctly labeled, interacting species. 8,11 More recently, one-photon excitation has been implemented for FCCS studies, whereby a single laser line has been used to excite two fluorophores with similar excitation but different emission characteristics.…”

“…12 Complexes formed from the reactants labeled with different color fluorophores results in a new spectrally distinct species with a correlation trace that can be fit to quantify the kinetics, dynamics, and concentration of this complex. 6,9,13 Although the cross-correlation signal is specific to doubly labeled (or triply labeled) species, the accurate recovery of the dynamics and concentrations of the complex is critically dependent on the emission spectra of the labels. 5,14 For fluorescent labels that do not have cleanly separated spectra, the reliability of multicolor FCCS methodologies becomes limited as a result of spectral cross talk.…”