Abstract
Accurate image alignment is critical in multicolor single-molecule fluorescence microscopy. Global alignment using affine transformations leaves residual errors due to the nonlinearity of the distortions, which decreases the effective field of view. Subsequent local refinement demands either large amounts of reference data and processing time or specialized imaging techniques like active stabilization. Here, we present a global alignment method, S/T polynomial decomposition, that uses sums of Zernike polynomial gradients to decompose the distortion between two images, correcting both linear and nonlinear distortions simultaneously. With minimal reference data, we gain diagnostic information about the distortion and achieve a colocalization accuracy comparable to local registration methods across the entire field of view.
Highlights
Modern biology has advanced through an ever-deeper understanding of the workings of cellular processes
Since the reference sample has a finite number of points, there will be an optimal number of S/T polynomials to include in the polynomial decomposition of the distortion field
We found that even with a comparatively sparse grid of 78 reference points, S/T polynomial decomposition (STPD) correction increased the accuracy of colocalization measurements on our instrument to ∼17 nm residual separation, which exceeds the accuracy of local affine correction despite using an order of magnitude less reference data
Summary
Modern biology has advanced through an ever-deeper understanding of the workings of cellular processes. To obtain how the dynamics of such cellular processes are governed at the molecular scale, the focus of study moves to smaller (sub-)systems, including individual biological molecules Such individual molecules are tagged with fluorescent markers, making it possible to e.g. visualise their localization and/or dynamics. CoSMoS (Colocalization Single-Molecule Spectroscopy) and SHREC (Single-molecule High-Resolution Colocalization) rely on the coincidence of components labeled in distinct colors to deduce the composition and accompanying kinetics of macromolecular complexes [5,6,7,8]. Both approaches rely on accurate registration of images in multiple wavelengths (Fig. 1(a)). In CoSMoS, an accuracy in the 10s of nm is necessary to reliably distinguish true complexes from mere binding at proximal sites
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