Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask

Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging. Until now, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push towards the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high-efficiency dielectric metasurface polarization/phase device composed of nanoposts with subwavelength spacing. The method is demonstrated both on fluorophores embedded in a polymer matrix and in dL5 protein complexes that bind malachite green

Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function

Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25-nm precision in the x and y dimensions and 50-nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe’s diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20–40 nm in 3D) over a large field of view (12 × 12 μm)