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Register a new userLong-range Single Molecule Forster Resonance Energy Transfer Between Alexa Dyes in Zero-Mode Waveguides
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Zero-mode waveguides (ZMW) nanoapertures milled in metal films were proposed to improve the FRET efficiency and enable single molecule FRET detection beyond the 10 nm barrier, overcoming the restrictions of diffraction-limited detection in a homogeneous medium. However, the earlier ZMW demonstrations were limited to the Atto 550 - Atto 647N fluorophore pair, asking the question whether the FRET enhancement observation was an artefact related to this specific set of fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to investigate single molecule FRET at large donor-acceptor separations exceeding 10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different chemical structure, surface charge and hydrophobicity as compared to their Atto counterparts. Our single molecule data on Alexa 546 - Alexa 647 demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm, extending the spatial range available for FRET and confirming the earlier conclusions. By showing that the FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, this report is an important step to establish the relevance of ZMWs to extend the sensitivity and detection range of FRET, while preserving its ability to work on regular fluorescent dye pairs.
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Motivated by recent experiments on photon statistics from individual dye pairs planted on biomolecules and coupled by fluorescence resonance energy transfer (FRET), we show here that the FRET dynamics can be modelled by Gaussian random processes with colored noise. Using Monte-Carlo numerical simulations, the photon intensity correlations from the FRET pairs are calculated, and are turned out to be very close to those observed in experiment. The proposed stochastic description of FRET is consistent with existing theories for microscopic dynamics of the biomolecule that carries the FRET coupled dye pairs.
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