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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
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Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS
Charles Limouse1, Jason C Bell2, Colin J Fuller2
1Department of Applied Physics.
Biophysical Journal
|April 12, 2018
Summary
We developed tracking fluorescence correlation spectroscopy (tFCS) to study large molecules. This single-molecule method reveals conformational changes and interactions in mesoscale assemblies.
Area of Science:
- Biophysics
- Single-molecule biophysics
- Molecular dynamics
Background:
- Probing mesoscale molecular complexes (100-1000 nm) is challenging.
- Existing techniques lack resolution for dynamics and conformation at this scale.
Purpose of the Study:
- To develop a novel single-molecule technique for analyzing mesoscale molecular assemblies.
- To probe conformation and dynamics of biomolecules at the mesoscale.
Main Methods:
- Developed tracking fluorescence correlation spectroscopy (tFCS).
- Utilized a feedback scheme for subdiffraction spatial resolution in fluorescence correlation spectroscopy.
- Measured distance fluctuations between two fluorescently labeled sites in freely diffusing biomolecules.
Main Results:
- Characterized tFCS sensitivity using DNA molecule dynamics (1000 bp).
- Demonstrated detection of nucleosome array compaction changes.
- Showcased assaying transient protein-mediated interactions in individual DNA molecules.
Conclusions:
- tFCS is a powerful tool for studying mesoscale conformational dynamics.
- The technique is applicable to diverse biochemical processes.
- Enables detailed analysis of molecular complex behavior at the mesoscale.
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