Förster Resonance Energy Transfer (FRET) governs energy exchanges at the nanoscale and can be used to accurately measure intra- and inter-molecular distances with sub-nm accuracy. While photonic nanoantennas have been shown to modify FRET, most of the earlier experiments lacked the ability to manipulate the distance between the antenna and the emitters.
In a collaboration with ICFO and INSP, scanning optical antenna probes are used to directly modulate the FRET efficiency in the near-field, and image the FRET efficiencies distributions at the nanoscale. The results, published in Nanophotonics, show that the antenna creates a local perturbation competing with the FRET acceptor, which is imaged for the first time at the single molecule level as a function of the relative position between the antenna and the FRET donor-acceptor pair.
Still not as famous on youtube as our esteemed colleague from Marseille, but going on the dark path
Soon our flag will flutter again over the Sea of Nanos.
Wed. May 13th 2pm CEST: Ebbesen's Blade is on fly again.
Thu. May 14th 3pm CEST: Shadow Princess took off and is fully operational.
Fri. May 15th 12am CEST: Arcadia hashin!
Thu. May 28th 4pm CEST: Queen Emeraldas Revenge is on fly. All our microscope setups are back and operational.
Link to some well-documented articles about Captain Harlock
Long-Range Single-Molecule Förster Resonance Energy Transfer between Alexa Dyes in Zero-Mode Waveguides
Förster resonance energy transfer (FRET) is widely used as a molecular ruler to monitor biomolecular conformations and interactions dynamics, but FRET is generally limited to distances below 10 nm.
Last year, we showed that zero-mode waveguides (ZMW) nanoapertures can enable single molecule FRET detection at spatial distances exceeding 10 nm with higher FRET efficiencies. However, this earlier work was limited to a specific Atto 550 – Atto 647N fluorescent dye pair, rising the issue that observations of FRET enhancement could be an artefact related to this specific set of fluorescent dyes.
In a recent ACS Omega paper, we use a markedly different set of fluorescent dyes (Alexa Fluor 546 and Alexa Fluor 647). Our new single molecule FRET data quantitatively demonstrate enhanced FRET efficiencies at large separations exceeding 10 nm confirming our earlier conclusions.
- The FRET enhancement inside a ZMW does not depend on the set of fluorescent dyes, validating the ZMW approach.
- Nanoapertures and nanophotonics are demonstrated to extend the spatial range of FRET to distances where dipole-dipole interactions would otherwise be too weak to produce detectable FRET signals.
Also available on ArXiv 2004.04513
Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins
The natural autofluorescence of proteins in the UV is appealing to get the detailed information from single molecule data without requiring a potentially disturbing external fluorescent label. However, proteins feature significantly lower autofluorescence brightness and photostabilities than conventional fluorescent dyes. This issue has largely prevented so far the detection of label-free proteins in the ultraviolet.
In a recent article in the Journal of Physical Chemistry Letters, we use a dedicated combination of oxygen scavengers and reducing agents to promote the protein photostability, reduce the photobleaching probability and improve the net UV autofluorescence signal.
- This is the first time that different photostability improvement strategies are reported and quantitatively assessed for label-free proteins in the ultraviolet range.
- We show that the underlying photochemical concepts initially derived for organic fluorescent dyes are still valid for protein autofluorescence in the UV, and therefore appear to be quite general.
- Fluorescence correlation spectroscopy (FCS) is demonstrated on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5× less than the current state-of-the-art.
Also freely available on ArXiv 2002.09761