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

ABSTRACT: 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-p

SUBMITTER: Mortensen KI 

PROVIDER: S-EPMC4634324 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature