Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Abstract

Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License 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.