Förster resonance energy transfer (FRET) from a fluorophore in the excited state (donor, D) to a fluorophore in the ground state (acceptor, A) provides a signal sensitive to D−A separation distances in the 0−10 nm range, and to various environmental parameters. A vast number of FRET imaging protocols and sensors have been developed and applied to reveal the spatial distribution and states of association and function of biomolecules in the cellular context, and the end is not in sight. A limitation of conventional FRET imaging is that it provides average information for an ensemble of molecules or nanostructures included in a diffraction-limited volume.
The advantages of a super-resolved FRET imaging method were clear since the advent of super-resolution microscopy, many years ago. However, obtaining a robust method of general applicability to obtain super-resolved FRET images has remained elusive until now.
In our recent paper in Nano Letters, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides more accurate quantification of interactions and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging.
STED-FRET can be applied on any two-color STED nanoscope, provides single-molecule sensitivity (Figure above), and enables the localization of biomolecular interactions on sub-diffraction structures, such as the example of actin-aducin in the MPS of neurons (Figure below).
This work was a collaboration with the group of Philip Tinnefeld, who (once again!) prepared wonderful DNA-origami systems to test the performance of STED-FRET to detect single-molecule FRET events.
There is an extra reason for us to be happy and proud of this work, that involves our long-time friend and supporter, the great Tom Jovin. It was in 2011 when we first talked about super-resolved FRET imaging. Since then, we had several ideas, some of which we tried in the lab with no success. This approach of intensity-based STED-FRET was clear to us that would work already in 2017, but for different reasons, we could not carry out the experiments properly. Finally, we made it! And we are already applying it to address biological questions.
Nano Letters 21 (2021) 2296-2303
“Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET”
Alan M. Szalai, Bruno Siarry, Jerónimo Lukin, Sebastián Giusti, Nicolás Unsain, Alfredo Cáceres, Florian Steiner, Philip Tinnefeld, Damián Refojo, Thomas M. Jovin, and Fernando D. Stefani
