Easy-DHPSF 2.0: open-source software for three-dimensional localization and two-color registration of single molecules with nanoscale accuracy

Abstract

Abstract Automated processing of double-helix (DH) microscope images of fluorescent single molecules (SMs) streamlines the protocol required to obtain super-resolved three-dimensional (3D) reconstructions of ultrastructures in biological samples by single-molecule active control microscopy (SMACM). Here, we present a suite of MATLAB subroutines, bundled with an easy-to-use graphical user interface (GUI), that facilitates 3D localization of single emitters (e.g. SMs, fluorescent beads, or quantum dots) with typical precisions of tens of nanometers in multi-frame movies acquired using a wide-field DH epifluorescence microscope. The algorithmic approach is based upon template matching for SM recognition and least‑squares fitting for 3D position measurement, both of which are computationally expedient and precise. Overlapping images of SMs are ignored, and the precision of least-squares fitting is not as high as maximum likelihood-based methods. Version 2.0 of Easy-DHPSF augments the approach of Version 1.0 by incorporating code that facilitates combining localization data from two spectral channels using a locally-weighted quadratic 3D registration function.