No abstract

Metallic nanoparticles were shown to affect Förster energy transfer between fluorophore pairs. However, to date, the net plasmonic effect on FRET is still under dispute, with experiments showing efficiency enhancement and reduction. This controversy is due to the challenges involved in the precise positioning of FRET pairs in the near field of a metallic nanostructure, as well as in the accurate characterization of the plasmonic impact on the FRET mechanism. Here, we use the DNA origami technique to place a FRET pair 10 nm away from the surface of gold nanoparticles with sizes ranging from 5 to 20 nm. In this configuration, the fluorophores experience only moderate plasmonic quenching. We use the acceptor bleaching approach to extract the FRET rate constant and efficiency on immobilized single FRET pairs based solely on the donor lifetime. This technique does not require a posteriori correction factors neither a priori knowledge of the acceptor quantum yield, and importantly, it is performed in a single spectral channel. Our results allow us to conclude that, despite the plasmon-assisted Purcell enhancement experienced by donor and acceptor partners, the gold nanoparticles in our samples have a negligible effect on the FRET rate, which in turns yields a reduction of the transfer efficiency.

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

We present a comprehensive theory of dead-time effects on Time-Correlated Single Photon Counting (TCSPC) as used for fluorescence lifetime measurements, and develop a correction algorithm to remove these artifacts. We apply this algorithm to fluorescence lifetime measurements as well as to Fluorescence Lifetime Imaging Microscopy (FLIM), where rapid data acquisition is necessarily connected with high count rates. There, dead-time effects cannot be neglected, and lead to distortions in the observed lifetime image. The algorithm is quite general and completely independent of the particular nature of the measured signal. It can also be applied to any other single-event counting measurement with detector and/or electronics dead-time.