![[feed]](/style/images/feed-icon-14x14.png){kind=icon}
Tools
Tools
Preview |
PDF (Original Article)
- Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
1MB |
| Item Type: | Article |
|---|---|
| Title: | Automated two-dimensional spatiotemporal analysis of mobile single-molecule FRET probes |
| Creators Name: | Schrangl, L., Göhring, J., Kellner, F., Huppa, J.B. and Schütz, G.J. |
| 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. |
| Keywords: | Fluorescence Resonance Energy Transfer, Nanotechnology, Single Molecule Imaging, Software, Spatio-Temporal Analysis |
| Source: | Journal of Visualized Experiments |
| ISSN: | 1940-087X |
| Publisher: | JoVE |
| Volume: | 177 |
| Page Range: | e63124 |
| Date: | 23 November 2021 |
| Official Publication: | https://doi.org/10.3791/63124 |
| PubMed: | View item in PubMed |
| Related to: |
Repository Staff Only: item control page
