Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation

Valentina Curcio,Luis A Alemán-Castañeda,Thomas G Brown,Sophie Brasselet,Miguel A Alonso

doi:10.1038/s41467-020-19064-6

Abstract

Super-resolution imaging based on single molecule localization allows accessing nanometric-scale information in biological samples with high precision. However, complete measurements including molecule orientation are still challenging. Orientation is intrinsically coupled to position in microscopy imaging, and molecular wobbling during the image integration time can bias orientation measurements. Providing 3D molecular orientation and orientational fluctuations would offer new ways to assess the degree of alignment of protein structures, which cannot be monitored by pure localization. Here we demonstrate that by adding polarization control to phase control in the Fourier plane of the imaging path, all parameters can be determined unambiguously from single molecules: 3D spatial position, 3D orientation and wobbling or dithering angle. The method, applied to fluorescent labels attached to single actin filaments, provides precisions within tens of nanometers in position and few degrees in orientation.

Highlights

Super-resolution imaging based on single molecule localization allows accessing nanometricscale information in biological samples with high precision
It is possible to preserve less-altered point spread function (PSF) images and restrict the measurements to 2D in-plane orientations by working under relatively low numerical aperture conditions and splitting polarization components[2], or using sequential polarization illumination[14,15,16,17]. None of these techniques have allowed the simultaneous measurement of 3D orientational properties and 3D spatial position of single molecules, in a single-shot image scheme compatible with super-resolution localization
The basis of the proposed technique is the placement at the pupil plane of an element referred to as a stressed-engineered optic (SEO), which is a BK7 glass window subjected to forces with trigonal symmetry at its edges[18,19,23]

Summary

Introduction

Super-resolution imaging based on single molecule localization allows accessing nanometricscale information in biological samples with high precision. Several methods have capitalized on this property by using Fourier-plane phase modification of the PSF7–10, or imaging finely sampled PSFs11 These approaches apply only to molecules with fixed position. None of these techniques have allowed the simultaneous measurement of 3D orientational properties (including both orientational fluctuations and mean orientation) and 3D spatial position of single molecules, in a single-shot image scheme compatible with super-resolution localization. The method is based on Fourier-plane filtering in phase and in polarization by using spatially varying birefringence It builds upon a prior technique for single-shot imaging polarimetry[18,19,20], where polarization is encoded in the shape of the PSF. We refer to the method as coordinate and height super-resolution imaging with dithering and orientation (CHIDO)

Methods

Results

Conclusion