Tional changes take spot in nano- to microseconds (ns-ms), as in chain dynamics of disordered proteins, and protein folding in microseconds to minutes. Transitions along energetically unfavorable pathways can take as much as hours or longer, as in protein misfolding (Borgia et al., 2011; Tosatto et al., 2015). (2013 Elsevier Ltd. All rights reserved. The figure was originally published as Figure 1 in Schuler and Hofmann, 2013. Current Opinion in Structural Biology, 23(1): 367. Additional reproduction of this panel would require permission from the IL-17 Gene ID copyright holder.) Bottom: (A) Picosecond (ps) to millisecond (ms) processes are commonly examined with confocal strategies for instance polarization-resolved fluorescence lifetime measurements and Fluorescence Correlation Spectroscopy (FCS). Example shown: chain dynamics of an IDP from nsFCS. (B) Conformational states are identified by individual populations with characteristic positions inside the FRET efficiency – lifetime diagrams as discussed inside the sections Detection and characterization of intra-state dynamics and Future of smFRET (adapted from Soranno et al., 2012). (C) Rapid transitions measured employing confocal microscopy is usually analyzed applying the photon trajectory and applying a photon-by-photon maximum likelihood approach (2018 Elsevier Ltd. All rights reserved. The figure was initially published as Figures 2 and 3 in Chung and Eaton, 2018. Current Opinion in Structural Biology, 48: 309. Further adaptation of this panel would need to have permission in the copyright holder.) The timescale over which kinetics may be measured could be extended for diffusing molecules at low concentrations by utilizing a Adenosine A2A receptor (A2AR) medchemexpress recurrence evaluation of single particles (RASP, Hoffmann et al., 2011). (D) Non-equilibrium experiments over extended periods of time could be performed with microfluidic mixing devices. (Copyright 2011, Nature Publishing Group, a division of Macmillan Publishers Restricted. All Rights Reserved. Reproduced from Gambin et al., 2011, with permission. Nature Methods eight:23941. Further reproduction of this panel would require permission in the copyright holder.) (E) Slow alterations in conformations over a broad array of timescales can be followed in smFRET efficiency trajectories registered by single-photon counting (SPC) or cameras more than minutes to a lot of hours when the sample is immobilized (adapted from Figure 1 of Zosel et al., 2018). 2013, Elsevier Ltd. All rights reserved. Figure three (best) and panel A was initially published as Figure 1 in Schuler and Hofmann, 2013. Additional reproduction of this panel would have to have permission in the copyright holder. 2018, Elsevier Ltd. All rights reserved. Panel C was originally published as Figures two and 3 in Chung and Eaton, 2018. Additional adaptation of this panel would need to have permission from the copyright holder. 2011, Nature Publishing Group, a division of Macmillan Publishers Restricted. All Rights Reserved. Panel D was initially published as Figure 1f in Gambin et al., 2011. Further reproduction of this panel would have to have permission in the copyright holder.Lerner, Barth, Hendrix, et al. eLife 2021;10:e60416. DOI: https://doi.org/10.7554/eLife.10 ofReview ArticleBiochemistry and Chemical Biology Structural Biology and Molecular BiophysicsSample preparation DyesFor studying biomolecular conformations and their dynamics with smFRET, the biomolecules of interest have to be labeled with organic dyes that are appropriate for single-molecule fluorescence detection (intrinsically fluorescent aromatic amino acids.