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How to Measure Separations and Angles Between Intramolecular Fluorescent Markers
K I Mortensen1, J Sung2, J A Spudich3
1Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
Methods in Enzymology
|October 30, 2016
Summary
This study introduces a novel two-color colocalization microscopy method for precise biomolecule analysis. It enables simultaneous determination of fluorophore separation and 3D orientation, advancing molecular imaging capabilities.
Area of Science:
- Biophysics
- Molecular Imaging
- Microscopy
Background:
- Studying biomolecule structure and function often requires multiple fluorescent markers.
- Simultaneous imaging and spectral separation of different colored markers allow for colocalization analysis.
Purpose of the Study:
- To describe a two-color colocalization microscopy method.
- To extend this method for fluorescent markers with fixed orientations and intramolecular proximity.
- To establish DNA molecules as probes for 3D orientation and develop super-resolution mapping between color channels.
Main Methods:
- Simultaneous determination of relative (x,y)-separation and individual spatial orientations of fluorophores from color-separated microscope images in time-lapse movies.
- Utilizing short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores as a benchmark system.
- Developing super-resolution mapping between color-separated channels.
Main Results:
- Demonstrated accuracy and precision of localization and mapping methods using dsDNA structure as a benchmark.
- Resolved 10 base pair differences in fluorophore separations.
- Determined the unique 3D orientation of each DNA molecule.
Conclusions:
- The developed method allows for time-resolved analysis of relative positions and orientations of molecular domains.
- Short, double-labeled DNA molecules serve as effective probes for determining 3D orientation.
- Super-resolution mapping between color channels is beneficial for dual-color colocalization measurements.
