Abstract
Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales (≲ 50nm). This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.
Competing Interest Statement
The authors have declared no competing interest.