This work was supported by the Austrian Science Fund (FWF) projects P30214-N36, P32307-B, and by the Vienna Science and Technology Fund (WWTF) LS13-030.

<ol>
	<li>McKinney, S. A., D&#233;clais, A. -. C., Lilley, D. M. J., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+individual+holliday+junctions.">Structural dynamics of individual holliday junctions.</a> Nature Structural Biology. 10 (2), 93-97 (2002).</li><li>Wang, S., Vafabakhsh, R., Borschel, W. F., Ha, T., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+potassium-channel+gating+revealed+by+single-molecule+FRET.">Structural dynamics of potassium-channel gating revealed by single-molecule FRET.</a> Nature Structural &amp; Molecular Biology. 23 (1), 31-36 (2015).</li><li>Hellenkamp, B., Wortmann, P., Kandzia, F., Zacharias, M., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidomain+structure+and+correlated+dynamics+determined+by+self-consistent+FRET+networks.">Multidomain structure and correlated dynamics determined by self-consistent FRET networks.</a> Nature Methods. 14 (2), 174-180 (2016).</li><li>Kilic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+FRET+reveals+multiscale+chromatin+dynamics+modulated+by+HP1&#945;.">Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1&#945;.</a> Nature Communications. 9 (1), 235 (2018).</li><li>Stryer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+energy+transfer+as+a+spectroscopic+ruler.">Fluorescence energy transfer as a spectroscopic ruler.</a> Annual Review of Biochemistry. 47 (1), 819-846 (1978).</li><li>Wu, P. G., Brand, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonance+energy+transfer:+Methods+and+applications.">Resonance energy transfer: Methods and applications.</a> Analytical Biochemistry. 218 (1), 1-13 (1994).</li><li>Qiao, Y., Luo, Y., Long, N., Xing, Y., Tu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecular+f&#246;rster+resonance+energy+transfer+measurement+on+structures+and+interactions+of+biomolecules.">Single-molecular f&#246;rster resonance energy transfer measurement on structures and interactions of biomolecules.</a> Micromachines. 12 (5), 492 (2021).</li><li>Malkusch, N., D&#246;rfler, T., Nagy, J., Eilert, T., Michaelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=smFRET+experiments+of+the+RNA+polymerase+II+transcription+initiation+complex.">smFRET experiments of the RNA polymerase II transcription initiation complex.</a> Methods. 120, 115-124 (2017).</li><li>Lee, J. -. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+views+of+MutS+on+mismatched+DNA.">Single-molecule views of MutS on mismatched DNA.</a> DNA repair. 20, 82-93 (2014).</li><li>Phelps, C., Israels, B., Jose, D., Marsh, M. C., von Hippel, P. H., Marcus, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microsecond+single-molecule+FRET+to+determine+the+assembly+pathways+of+T4+ssDNA+binding+protein+onto+model+DNA+replication+forks.">Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (18), E3612-E3621 (2017).</li><li>Deindl, S., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+conformational+dynamics+with+single-molecule+fluorescence+energy+transfer:+Applications+in+nucleosome+remodeling.">Monitoring conformational dynamics with single-molecule fluorescence energy transfer: Applications in nucleosome remodeling.</a> Methods in Enzymology. 513, 59-86 (2012).</li><li>Crawford, D. J., Hoskins, A. A., Friedman, L. J., Gelles, J., Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+colocalization+FRET+evidence+that+spliceosome+activation+precedes+stable+approach+of+5'+splice+site+and+branch+site.">Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (17), 6783-6788 (2013).</li><li>Wang, Y., Xiao, M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+single+molecule+FRET+signals+reveals+multiple+active+ribosome+subpopulations.">Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations.</a> Proteins. 82 (1), 1-9 (2014).</li><li>Mori, T., Vale, R. D., Tomishige, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+kinesin+waits+between+steps.">How kinesin waits between steps.</a> Nature. 450 (7170), 750-754 (2007).</li><li>Huppa, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463 (7283), 963-967 (2010).</li><li>G&#246;hring, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analysis+of+T-cell+receptor-imposed+forces+via+quantitative+single+molecule+FRET+measurements.">Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements.</a> Nature Communications. 12 (1), 2502 (2021).</li><li>Hellenkamp, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+and+accuracy+of+single-molecule+FRET+measurements-a+multi-laboratory+benchmark+study.">Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.</a> Nature Methods. 15 (9), 669 (2018).</li><li>Kapanidis, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-aided+molecule+sorting:+Analysis+of+structure+and+interactions+by+alternating-laser+excitation+of+single+molecules.">Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (24), 8936-8941 (2004).</li><li>Sakon, J. J., Weninger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+the+conformation+of+individual+proteins+in+live+cells.">Detecting the conformation of individual proteins in live cells.</a> Nature Methods. 7 (3), 203-205 (2010).</li><li>McCann, J. J., Choi, U. B., Zheng, L., Weninger, K., Bowen, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+methods+to+recover+absolute+FRET+efficiency+from+immobilized+single+molecules.">Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.</a> Biophysical Journal. 99 (3), 961-970 (2010).</li><li>Lee, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+FRET+measurements+within+single+diffusing+biomolecules+using+alternating-laser+excitation.">Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.</a> Biophysical Journal. 88 (4), 2939-2953 (2005).</li><li>Asher, W. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+FRET+imaging+of+GPCR+dimers+in+living+cells.">Single-molecule FRET imaging of GPCR dimers in living cells.</a> Nature Methods. 18 (4), 397-405 (2021).</li><li>Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-molecule FRET with total internal reflection microscopy. (12), 1223-1237 (2012).</li><li>Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective-type+total+internal+reflection+microscopy+(excitation)+for+single-molecule+FRET.">Objective-type total internal reflection microscopy (excitation) for single-molecule FRET.</a> Cold Spring Harbor Protocols. 11, 1189-1191 (2012).</li><li>Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective-type+total+internal+reflection+microscopy+(emission)+for+single-molecule+FRET.">Objective-type total internal reflection microscopy (emission) for single-molecule FRET.</a> Cold Spring Harbor Protocols. 11, 1192-1194 (2012).</li><li>Allan, D. B., Caswell, T., van der Wel, C. M., Dimiduk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soft-matter/pims: PIMS v0.5. , (2020).</li><li>Anaconda Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Miniconda. , (2021).</li><li>conda-forge community. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The conda-forge project: community-based software distribution built on the conda package format and ecosystem. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> JupyterLab Contributors Notebooks - JupyterLab documentation. , (2021).</li><li>Babcock, H., Sigal, Y. M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-density+3D+localization+algorithm+for+stochastic+optical+reconstruction+microscopy.">A high-density 3D localization algorithm for stochastic optical reconstruction microscopy.</a> Optical Nanoscopy. 1 (6), (2012).</li><li>Gao, Y., Kilfoil, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+detection+and+complete+tracking+of+large+populations+of+features+in+three+dimensions.">Accurate detection and complete tracking of large populations of features in three dimensions.</a> Optics Express. 17 (6), 4685 (2009).</li><li>Schrangl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> sdt-python: Python library for fluorescence microscopy data analysis (v17.1). , (2021).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+digital+video+microscopy+for+colloidal+studies.">Methods of digital video microscopy for colloidal studies.</a> Journal of Colloid and Interface Science. 179 (1), 298-310 (1996).</li><li>Preibisch, S., Saalfeld, S., Schindelin, J., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Software+for+bead-based+registration+of+selective+plane+illumination+microscopy+data.">Software for bead-based registration of selective plane illumination microscopy data.</a> Nature Methods. 7 (6), 418-419 (2010).</li><li>Bradski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OpenCV+library.">The OpenCV library.</a> Dr. Dobb's Journal: Software Tools for the Professional Programmer. 25 (11), 120-123 (2000).</li><li>Allan, D. B., Caswell, T., Keim, N. C., van der Wel, C. M., Verweij, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft-matter/trackpy:+Trackpy+v0.5.0.">Soft-matter/trackpy: Trackpy v0.5.0.</a> Zenodo. , 4682814 (2021).</li><li>Killick, R., Fearnhead, P., Eckley, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+detection+of+changepoints+with+a+linear+computational+cost.">Optimal detection of changepoints with a linear computational cost.</a> Journal of the American Statistical Association. 107 (500), 1590-1598 (2012).</li><li>Schrangl, L., G&#246;hring, J., Sch&#252;tz, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+single+molecule+FRET+transitions+without+trajectories.">Kinetic analysis of single molecule FRET transitions without trajectories.</a> The Journal of Chemical Physics. 148 (12), 123328 (2018).</li><li>Santoso, Y., Torella, J. P., Kapanidis, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+single-molecule+FRET+dynamics+with+probability+distribution+analysis.">Characterizing single-molecule FRET dynamics with probability distribution analysis.</a> ChemPhysChem. 11 (10), 2209-2219 (2010).</li><li>Schrangl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+FRET+analysis+software+(3.0).">Single-molecule FRET analysis software (3.0).</a> Zenodo. , (2021).</li><li>Nye, J. A., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+control+of+histidine-tagged+protein+surface+density+on+supported+lipid+bilayers.">Kinetic control of histidine-tagged protein surface density on supported lipid bilayers.</a> Langmuir. 24 (8), 4145-4149 (2008).</li><li>Platzer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unscrambling+fluorophore+blinking+for+comprehensive+cluster+detection+via+photoactivated+localization+microscopy.">Unscrambling fluorophore blinking for comprehensive cluster detection via photoactivated localization microscopy.</a> Nature Communications. 11 (1), 4993 (2020).</li><li>Johnson, I., Spence, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The molecular probes handbook: A guide to fluorescent probes and labeling technologies. , (2010).</li><li>Szalai, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+of+energy+transfer+by+intensity-based+STED-FRET.">Super-resolution imaging of energy transfer by intensity-based STED-FRET.</a> Nano Letters. 21 (5), 2296-2303 (2021).</li><li>Miyawaki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388 (6645), 882-887 (1997).</li><li>Zhai, B., Zhai, S., Hao, R., Xu, J., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FRET-based+two-photon+probe+for+in+vivo+tracking+of+pH+during+a+traumatic+brain+injury+process.">A FRET-based two-photon probe for in vivo tracking of pH during a traumatic brain injury process.</a> New Journal of Chemistry. 43 (43), 17018-17022 (2019).</li></ol>

The authors declare no conflicts of interest.

This article details a pipeline for the automated recordings and quantitative analysis of smFRET data originating from mobile yet surface-tethered probe molecules. It complements the two predominant approaches to smFRET experiments, involving either surface-immobilized probes or probes diffusing in solution into and out of a confocal excitation volume17. It provides the correct FRET efficiency and the molecular positions as a function of time. It can therefore be used as input for specialized analysis programs, e.g., to quantify transition kinetics1, FRET histograms39, or two-dimensional diffusion22.
The software is released under a free and open-source license approved by the Open Source Initiative that grants the user the perpetual right to free usage, modification, and redistribution. Github was chosen as a development and distribution platform to make it as easy as possible to obtain the software and participate in the development process by reporting bugs or contributing code40. Written in Python, the software does not depend on proprietary components. The choice of Jupyter notebooks as user interfaces facilitates the inspection of data at every analysis step and allows for tailoring and extending the pipeline specifically for the experimental system at hand. The sdt-python library32 serves as the foundation and implements functionality to evaluate fluorescence microscopy data, such as single-molecule localization, diffusion analysis, fluorescence intensity analysis, color channel registration, colocalization analysis, and ROI handling.
In principle, single-particle tracking can be performed in one-, two- or three-dimensional systems. Here, the single-molecule analysis pipeline was tailored to the study of 2D mobile systems. This choice mirrors the availability of simple systems, such as planar-supported lipid bilayers (SLBs), to present mobile fluorescent probes. Such lipid bilayer systems are typically composed of two or more phospholipids moieties, where the bulk fraction determines the key physicochemical parameters of the SLB (such as phase and viscosity), and the minor fraction provides attachment sites for biomolecules. These attachment sites can be biotinylated phospholipids for avidin- or streptavidin-based protein platforms or nickel-NTA conjugated phospholipids for protein platforms with histidine tags41. The choice of the appropriate platform for linking proteins to the SLB depends on the scientific question. Readers can refer to the literature16,38,42 for examples of successfully employed strategies. The density of probes in the sample should be sufficiently low to avoid overlapping point spread functions; typically, less than 0.1 molecules per &#181;m2 are recommended. See the representative results section (in particular, Figure 6) for an example showing a suitable probe density. The analysis method is also applicable to single fluorescently labeled protein molecules diffusing in the plasma membrane of live cells.
One critical aspect of smFRET experiments is the production and characterization of the FRET probes themselves. When choosing fluorophores for a FRET pair, their F&#246;rster radius should match the expected inter-dye distances43. Dyes resistant to photobleaching are preferred as they yield long time traces. However, for elevated bleaching rates, one fluorophore species can be utilized to recognize multiemitter events originating from colocalized molecules via stepwise photobleaching analysis; see step 8.1.4 in the protocol section. Fluorophore pairs should be site-specifically and covalently attached to the molecules of interest, forming intra- or intermolecular FRET pairs.
Combining smFRET with other readily available techniques can increase its spatial resolution beyond the diffraction limit (via STED44). The smFRET tracking algorithm presented here widens the approach&#39;s applicability to new experimental settings and model systems. This includes studies of (i) kinetic changes in the stoichiometry of mobile biomolecules, (ii) dynamic association of mobile biomolecules, (iii) the rate of enzymatic reactions of freely diffusing reactants, and (iv) the kinetics of conformational changes of mobile biomolecules. The first two examples require model systems that show intermolecular FRET, i.e., donor and acceptor are conjugated to separate biomolecular entities of interest. The latter examples may make use of biosensors carrying donor and acceptor within the same molecular entity (intramolecular FRET).
Intramolecular FRET-based sensors can provide insight into intrinsic conformational changes of biomolecules1,2,3,4, conformational changes caused by endogenous or external force load (molecular force sensors16), or ion concentrations in the nano-environment such as calcium45 and pH46. Depending on the model system and the preferred anchoring platform, such smFRET events can either be tracked in 2D or 3D: (i) planar tracking of smFRET events can be employed for the quantification of receptor-ligand interaction times within a plasma membrane, the association of membrane-anchored signal amplification cascades, and the stoichiometry changes of surface receptors; (ii) volume tracking of smFRET events can be used for any intra- or intermolecular FRET probes in living cells or in in vitro reconstituted systems.
The smFRET tracking method was developed mainly with intramolecular FRET probes in mind. These probes feature a fixed and well-known number of fluorescent labels, a fact that was exploited to reject data from agglomerated and incorrectly synthesized (e.g., incompletely labeled) molecules, as well as from probes where one of the fluorophores has been photobleached. However, by adjusting the filtering steps, the method can also be applied to intermolecular FRET probes. For instance, instead of accepting only molecules featuring a single donor and a single acceptor fluorophore, one could examine the spatial trajectories of donor and acceptor dyes and select, for example, for co-diffusing donor-acceptor trajectories.
As the 3D-DAOSTORM algorithm has support for determining a signal&#39;s position along the optical axis via the astigmatism due to a cylindrical lens in the emission beam path, 3D experiments could be easily integrated into the analysis pipeline. In this case, the acceptor signal upon acceptor excitation would serve to determine the stoichiometry and the axial position. The analysis software can also be employed to evaluate data from experiments featuring immobilized probes by utilizing its large degree of automation and filtering schemes. In fact, smFRET efficiency datasets from Holliday junctions immobilized on gel-phase bilayers38 were analyzed using an early version of the software.

F&#246;rster resonance energy transfer (FRET) has been a major driver in molecular biological and biophysical research, as it allows the investigation of processes at sub-nanometer resolution. As the efficiency of energy transfer between donor and acceptor fluorophores strongly depends on the inter-dye distance in the sub-nanometer to nanometer range, it has been effectively used as a spectroscopic ruler to explore static and dynamic conformation of biomolecules1,2,3,4. Additionally, the FRET phenomenon has been widely used for colocalization studies of membrane-associated and intracellular proteins on a bulk level5,6. In the last two decades, the method was adapted for monitoring smFRET events7, which helped to substantially increase temporal and spatial resolution and resolved even rare subpopulations in heterogeneous samples. Equipped with these techniques, unique insights were gained into the dynamics of molecular machinery such as the transcript processing rate of RNA polymerase II8, replication speed of DNA polymerases9,10, nucleosome translocation rate11, transcript splicing and stalling rate of assembled spliceosomes12, the activity of ribosomal subpopulations13, and the walking speed of kinesin motors14, to name a few. Receptor-ligand interaction durations15 and molecular forces16 have been quantified.
Intensity-based smFRET studies typically rely on sensitized emission to measure FRET efficiency: a beam splitter in the emission path spatially separates light originating from donor and acceptor fluorophores upon donor excitation, allowing for the quantification of individual fluorescence intensities. The efficiency can subsequently be calculated as the fraction of photons emitted by the acceptor with respect to the total photon count17. In addition, acceptor excitation following donor excitation (ALEX) permits measurement of the stoichiometry of the FRET events, aiding in the discrimination between true low FRET signals from signals arising, e.g., from probes featuring a photobleached acceptor fluorophore18.
Single-molecule FRET experiments are commonly carried out in one of two ways. First, a small region in the sample volume is illuminated using a confocal microscope. Single probe molecules in solution are excited when they happen to diffuse within the focal volume. With this technique, fast photon-counting detectors can be used, enabling sub-microsecond time resolution. Second, probes are specifically immobilized on surfaces and monitored via widefield microscopy, often using total internal reflection (TIR) configuration to minimize background fluorescence. Probe immobilization allows for much longer recording times than using the first approach. In addition, the larger field of view permits the monitoring of multiple probes in parallel. The need for a camera makes this method slow compared to the one described above. Time resolution is limited to the millisecond to second range.
If long time traces are required, e.g., for studying dynamic processes on a millisecond to second time scale, the first method is not applicable, as the fluorescence bursts are typically too short. The second approach fails whenever immobilization is not feasible, e.g., in live-cell experiments featuring probes diffusing within the cell membrane. Furthermore, it has been observed that biological model systems can vary their response dramatically depending on the mobility of the contacted surface16.
While combined smFRET and single-particle tracking experiments recording mobile FRET probes have been performed in the past19, there is no publicly available software for the evaluation of the data. This prompted the development of a new analysis platform, which allows for the determination of multiple properties of mobile fluorescent probes, including smFRET efficiency and stoichiometry, positions with sub-pixel accuracy, and fluorescence intensities as functions of time. Methods for filtering the resulting traces by examining stepwise bleaching behavior, nearest-neighbor distances, emission intensities, and other traits were established to exclusively choose correctly synthesized and functional single-probe molecules. The software also supports experimental and analytical techniques recently agreed upon in a multilaboratory study to produce reliable, quantitative smFRET data17. In particular, the implementation adheres to the validated procedures for the calculation of FRET efficiency and stoichiometry. Fluorescence intensities upon donor excitation in the donor emission channel IDD and acceptor emission channel IDA are used for the calculation of the apparent FRET efficiency Eapp using Eq (1).
<img alt="Equation 1" src="/files/ftp_upload/63124/63124eq01.jpg" /> (1)
With the help of the fluorescence intensity in the acceptor emission channel upon acceptor excitation IAA, the apparent stoichiometry is calculated using Eq (2).
<img alt="Equation 2" src="/files/ftp_upload/63124/63124eq02.jpg" /> (2)
The FRET efficiency E and the stoichiometry S can be derived from Eapp and Sapp by considering four correction factors.
<img alt="Equation 3" src="/files/ftp_upload/63124/63124eq03.jpg" />
&#945; describes the leakage of donor fluorescence into the acceptor emission channel and can be determined using a sample containing only donor fluorophores or by analyzing parts of trajectories where the acceptor has been bleached.&#160;&#948; corrects for the direct excitation of the acceptor by the donor excitation light source and can be measured using a sample with only acceptor fluorophores or by analyzing parts of trajectories where the donor has been bleached.
<img alt="Equation 4" src="/files/ftp_upload/63124/63124eq04.jpg" />.
&#947; scales IDD to rectify diverging detection efficiencies in donor and acceptor emission channels and different quantum efficiencies of the fluorophores. The factor can be computed by analyzing the increase in donor intensity upon acceptor bleaching in trajectories with high FRET efficiencies20 or by studying a sample featuring multiple discrete FRET states.
<img alt="Equation 5" src="/files/ftp_upload/63124/63124eq05.jpg" />
&#946; scales IAA to correct for disparate efficiencies of donor and acceptor excitation. If &#947; was determined via acceptor bleaching analysis, &#946; could be calculated from a sample of known donor-to-acceptor ratio21. Otherwise, the multi-state FRET sample also yields &#946;.
Together, the corrections allow the calculation of the corrected FRET efficiency using Eq (3).
<img alt="Equation 6" src="/files/ftp_upload/63124/63124eq06.jpg" /> (3)
and the corrected stoichiometry using Eq (4).
<img alt="Equation 7" src="/files/ftp_upload/63124/63124eq07.jpg" /> (4)
Ideally, the corrected stoichiometry for a 1:1 donor-to-acceptor ratio gives S = 0.5. In practice, a reduced signal-to-noise ratio produces a spread of the measured values of S, hampering the discrimination from donor-only signals (S = 1) and acceptor-only signals (S = 0). The resulting time traces can be used as input for a more detailed analysis of the single-molecule trajectories to obtain information such as spatiotemporal force profiles16, the mobility of the single-molecule events22, or transition kinetics between different states1.
The following protocol describes experimental parameters and procedures for smFRET tracking experiments, as well as the working principle behind data analysis using the newly developed software suite. For the acquisition of experimental data, it is recommended to use a microscopy setup meeting the following requirements: i) capability of detecting the emission of single dye molecules; ii) widefield illumination: in particular for live-cell experiments, total internal reflection (TIR23,24,25) configuration is recommended; iii) spatial separation of emission light according to wavelength such that donor and acceptor fluorescence is projected onto different regions of the same camera chip25 or different cameras; iv) modulation of light sources for donor and acceptor excitation with millisecond precision, e.g., using directly modulatable lasers or modulation via acousto-optic modulators. This permits stroboscopic illumination to minimize photobleaching of fluorophores as well as alternating excitation to determine stoichiometries; v) output of one file per recorded image sequence in a format that can be read by the PIMS Python package26. In particular, multipage TIFF files are supported.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (Ni-NTA-DOGS)</td><td>Avanti Polar Lipids</td><td>790404P</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375P</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td><td>Avanti Polar Lipids</td><td>850457P</td><td></td></tr><tr><td>&amp;alpha; Plan-FLUAR 100x/1.45 oil objective</td><td>Zeiss</td><td>000000-1084-514</td><td></td></tr><tr><td>Axio Observer microscope body</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Bandpass filter</td><td>Chroma Technology Corp</td><td>ET570/60m</td><td>donor emission filter</td></tr><tr><td>Bandpass filter</td><td>Chroma Technology Corp</td><td>ET675/50m</td><td>acceptor emission filter</td></tr><tr><td>conda-forge</td><td>conda-forge community</td><td></td><td>community-maintaned Python package repository for Anaconda/miniconda</td></tr><tr><td>Coverslips 60 mm x 24 mm #1.5</td><td>MENZEL</td><td></td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock Inc</td><td>FF640-FDi01-25&amp;times;36</td><td>separation of donor and acceptor emission</td></tr><tr><td>Dichroic mirror (quad band)</td><td>Semrock Inc</td><td>Di01-R405/488/532/635-25&amp;times;36</td><td>separation of excitation and emission light</td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>FCS</td><td>Sigma-Aldrich</td><td>F7524</td><td>for imaging buffer</td></tr><tr><td>fret-analysis</td><td>Sch&amp;uuml;tz group at TU Wien</td><td></td><td>Python package for smFRET data analysis; version 3</td></tr><tr><td>Fura-2 AM</td><td>Thermo Fisher Scientific</td><td>11524766</td><td></td></tr><tr><td>HBSS</td><td>Sigma-Aldrich</td><td>H8264</td><td>for imaging buffer</td></tr><tr><td>iBeam Smart 405-S 405 nm laser</td><td>Toptica Photonics AG</td><td></td><td></td></tr><tr><td>iXon Ultra 897 EMCCD camera</td><td>Andor Technology Ltd</td><td></td><td></td></tr><tr><td>Lab-Tek chambers (8 wells)</td><td>Thermo Fisher Scientific</td><td>177402PK</td><td>for sample preparation and imaging</td></tr><tr><td>Millenia Prime 532 nm laser</td><td>Spectra Physics</td><td></td><td></td></tr><tr><td>miniconda</td><td>Anaconda Inc.</td><td></td><td>Python 3 distribution. Min. version: 3.7</td></tr><tr><td>Monovalent streptavidin (plasmids for bacterial expression)</td><td>Addgene</td><td>20860 &amp;amp; 20859</td><td></td></tr><tr><td>OBIS 640 nm laser</td><td>Coherent Inc</td><td>1185055</td><td></td></tr><tr><td>Optosplit II</td><td>Cairn Research</td><td></td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma-Aldrich</td><td>A5253</td><td>for imaging buffer</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td></td></tr><tr><td>sdt-python</td><td>Sch&amp;uuml;tz group at TU Wien</td><td></td><td>Python library for data analysis; version 17</td></tr><tr><td>TetraSpek bead size kit</td><td>Thermo Fisher Scientific</td><td>T14792</td><td>Randomly distributed, immobilized fiducial markers for image registration</td></tr><tr><td>USC500TH Ultrasound bath</td><td>VWR</td><td></td><td>for SUV formation</td></tr></tbody></table>

automated two-dimensional spatiotemporal analysis of mobile single-molecule fret probes

Single-molecule F&#246;rster 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.

A variety of low- and high-level information can be extracted from smFRET tracks depending on the scientific question of the experiment. Here, examples of analysis pipelines with analog and digital probes are presented: a peptide-based molecular force sensor16 and a DNA probe with stochastic switching of its conformation38, respectively. Refer to Figure 5 for the design and working principle of these probes.
After the localization and tracking algorithms have been executed as described in the protocol, the package offers multiple data visualization tools to optimize the initial parameters and subsequent filter steps: (i) visualization of individual smFRET events, (ii) optional image segmentation to analyze data in certain regions of interests, (iii) monitoring of filter steps via FRET efficiency vs. stoichiometry (E-S) plots. The visualization of the single-molecule data is presented in Figure 6.
Finally, the filtered FRET events are represented by an E-S plot and a FRET efficiency histogram (Figure 4). The E-S plot is a useful tool for optimizing the aforementioned filtering steps and investigating the final result. Partially bleached or incompletely labeled FRET sensors can be excluded by their stoichiometry value. Mobility parameters can be investigated by plotting an individual trajectory path in an x-y plot (Figure 6) or a mean square displacement (MSD) plot (Figure 4). The first method is especially useful for discriminating mobile from immobilized events, while the latter is used to calculate the diffusion coefficient.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63124/63124fig04.jpg" /> 
Figure 4: Exemplary output. (A) The FRET efficiency is plotted versus stoichiometry (E-S plot) for a population of the molecular force sensor (left panel) decorating a glass-supported lipid bilayer and strained by a T cell. Only one population cloud is visible. The respective histogram of FRET efficiencies exemplifies the difference between a force sensor population in presence and absence of cells (middle panel). No shift to lower FRET efficiencies of the sensor population in presence of T cells can be observed, indicating little to no force-dependent stretching of the sensor module. The MSD plot of these experimental conditions confirms that the force sensor population beneath a T cell moves considerably slower than their unbound counterparts (right panel). (B) The same analysis was performed with Holliday junction DNA sensor decorating a glass-supported fluid lipid bilayer. The E-S plot clearly shows two populations, which are also apparent in the FRET efficiency histogram. The MSD plot indicates the presence of one fast-moving sensor population. Abbreviations: FRET = F&#246;rster resonance energy transfer; MSD = mean square displacement. <a href="https://www.jove.com/files/ftp_upload/63124/63124fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63124/63124fig05.jpg" /> 
Figure 5: Design and working principle of intramolecular FRET probes. (A) Analog peptide sensor for quantification of mechanical molecular forces. The donor and acceptor fluorophores are covalently attached to either end of the peptide backbone. The sensor module is site-specifically attached to a specific ligand, which in turn binds a cell-resident surface receptor of interest (here, an antibody fragment specifically recognizing the beta chain of the T cell receptor). Upon receptor-ligand binding, force is exerted, and the sensor module extends and eventually recoils after bond cleavage. This panel is modified from 16. (B) Digital DNA sensor for quantification of FRET transitions. The FRET sensor is composed of four DNA strands forming a Holliday junction. The donor and acceptor fluorophore are covalently attached to two strands. Holliday junctions frequently switch their conformation depending on the surrounding buffer conditions. The stochastic switching of these conformations can be monitored by quantifying the FRET efficiency of individual probes. Abbreviations: TCR = T cell receptor; FRET = F&#246;rster resonance energy transfer. <a href="https://www.jove.com/files/ftp_upload/63124/63124fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63124/63124fig06.jpg" /> 
Figure 6: Examples of localization and tracking of FRET probes. (A) The FRET efficiency and stoichiometry of individual events are calculated by quantifying the intensity of the donor fluorophore upon donor excitation (D &#8594; D), the acceptor fluorophore upon donor excitation (D &#8594; A), and the acceptor fluorophore upon acceptor excitation (A &#8594; A). Nearest neighbor filtering prevents bias by overlapping point spread functions of close emitters. Image segmentation allows the user to choose certain smFRET events localized within an area of interest (e.g., a cell or a micropattern). As an example of image segmentation, T cells were stained with Fura-2 (displayed on the left) and subjected to adaptive thresholding to identify the cell edges (orange dotted line). Scale bars = 5 &#181;m. (B) smFRET trajectories using the molecular force sensor. Individual trajectories can be plotted in the x-y plane, visualizing their diffusion behavior and localization (left panel). Furthermore, each trajectory&#39;s intensities can be plotted over time to identify FRET transitions or bleaching steps (middle panel shows the red trajectory from the left panel). The resulting FRET efficiency and stoichiometry can be visualized similarly (right panel). (C) smFRET trajectories using the Holliday junction DNA sensor. HBSS + 12 mM MgCl2 was used as a buffer during the measurements. Apart from the apparent acceptor bleaching step near the sequence end of these examples, the frequency of FRET transitions for each sensor can be determined. The Holliday junctions switch their conformation with a high frequency, whereas the molecular force sensor does not exhibit FRET transitions. This information makes it possible to adjust the experimental conditions, such as the delay between the frames, to increase or reduce the number of observed transitions. Abbreviations: FRET = F&#246;rster resonance energy transfer; smFRET = single-molecule FRET; HBSS = Hank&#39;s balanced salt solution. <a href="https://www.jove.com/files/ftp_upload/63124/63124fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Information: Localization and tracking of single molecules (01. Tracking.ipynb). <a href="https://www.jove.com/files/ftp_upload/63124/si.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes at JoVE.com

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

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

automated-two-dimensional-spatiotemporal-analysis-mobile-single

Research

JoVE Journal

Bioengineering

2.5K Views.  TU Wien. Single-molecule FRET-experimenten met behulp van oppervlaktegebonden sondes zijn in het verleden bijna uitsluitend uitgevoerd op geïmmobiliseerde moleculen. Veel biomoleculen diffunderen echter en kunnen met onze methode worden geanalyseerd. Het combineren van single-molecule FRET met tracking maakt het mogelijk om niet alleen FRET-deficiëntietijdsporen van bewegende probes te analyseren, maar ook om spatiotemporale aspecten zoals het diffusiegedrag te onderzoeken.Onze methode is com...

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biology

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video