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Nat Commun. 2015 Oct 16;6:8621. doi: 10.1038/ncomms9621.

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

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Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby DK-2800, Denmark.
Department of Applied Physics, Stanford University, Stanford, California 94305, USA.


We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

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