Optimized measurements of separations and angles between intra-molecular fluorescent markers.
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.
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.
Kim I
KI
Mortensen
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.|Department of Applied Physics, Stanford University, Stanford, California 94305, USA.
Jongmin
J
Sung
Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby DK-2800, Denmark.
Henrik
H
Flyvbjerg
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
James A
JA
Spudich
D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation
D005456:Fluorescent Dyes; D004247:DNA