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L. Schrangl, J. Göhring, F. Kellner, J. Huppa, G. Schütz:
"Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes";
JoVE, 177 (2021), e6312401 - e6312423.

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jove.com November 2021 . 177 . e63124 . Page 1 of 23
Automated Two-dimensional Spatiotemporal Analysis of
Mobile Single-molecule FRET Probes
Lukas Schrangl1, Janett Göhring2, Florian Kellner2, Johannes B. Huppa2, Gerhard J. Schütz1
1 Institute of Applied Physics, TU Wien 2 Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical
University of Vienna
Corresponding Author
Gerhard J. Schütz
schuetz@iap.tuwien.ac.at
Citation
Schrangl, L., Göhring, J., Kellner, F.,
Huppa, J.B., Schütz, G.J. Automated
Two-dimensional Spatiotemporal
Analysis of Mobile Single-molecule
FRET Probes. J. Vis. Exp. (177),
e63124, doi:10.3791/63124 (2021).
Date Published
November 23, 2021
DOI
10.3791/63124
URL
jove.com/video/63124
Abstract
Single-molecule Förster resonance energy transfer (smFRET) is a versatile technique
reporting on distances in the sub-nanometer to nanometer range. It has been
used in a wide range of biophysical and molecular biological experiments,
including the measurement of molecular forces, characterization of conformational
dynamics of biomolecules, observation of intracellular colocalization of proteins,
and determination of receptor-ligand interaction times. In a widefield microscopy
configuration, experiments are typically performed using surface-immobilized probes.
Here, a method combining single-molecule tracking with alternating excitation (ALEX)
smFRET experiments is presented, permitting the acquisition of smFRET time traces
of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid
bilayers. For the analysis of recorded data, an automated, open-source software
collection was developed supporting (i) the localization of fluorescent signals,
(ii) single-particle tracking, (iii) determination of FRET-related quantities including
correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive
presentation of the results. The generated data can conveniently be used as input for
further exploration via specialized software, e.g., for the assessment of the diffusional
behavior of probes or the investigation of FRET transitions.

https://publik.tuwien.ac.at/files/publik_299685.pdf