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-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.
Highlights
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
It is very sensitive to proper choice of theoretical point spread function (PSF), and the latter depends on experimental conditions
We determine the total number of source photons emitted by the probe, a constant background level, and the objective’s distance from its focus (Methods, Supplementary Fig. 1). The latter does not directly carry information about the molecule, its determination is paramount for accurate estimates of the molecule’s position and orientation, we show. This general procedure of maximum likelihood estimation (MLE) based on a theoretical PSF was dubbed MLEwT (Maximum Likelihood Estimation with the Theoretical PSF) in ref
Summary
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. One may fit the latter to the measured intensity distribution and localize the fluorophore with a precision that increases with the number of photons in the imaged spot With this method, fluorescent probes are routinely localized with nanometre precision[1,2,3,4,5,6], that is, nanometre reproducibility. The orientation of a fluorophore may be fixed deliberately to a molecule of interest[4,10,11,12,13] In both cases, the PSF is typically anisotropic, and if it is approximated by a 2D Gaussian, localization accuracy is compromised: Systematic errors may amount to tens of nanometres[14,15], irrespective of precision, which may still be nanometres. The true position of the fluorophore can be tens of nanometres away from the precisely estimated position
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