Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy.

Fabian Hinterer,Ronny Ramlau,Gerhard J Schütz,Magdalena C Schneider,Montserrat López-Martinez,Alexander Jesacher,Simon Hubmer,Philipp Zelger,Marek Cebecauer

doi:10.1371/journal.pone.0263500

Abstract

Single molecule localization microscopy (SMLM) has the potential to resolve structural details of biological samples at the nanometer length scale. Compared to room temperature experiments, SMLM performed under cryogenic temperature achieves higher photon yields and, hence, higher localization precision. However, to fully exploit the resolution it is crucial to account for the anisotropic emission characteristics of fluorescence dipole emitters with fixed orientation. In case of slight residual defocus, localization estimates may well be biased by tens of nanometers. We show here that astigmatic imaging in combination with information about the dipole orientation allows to extract the position of the dipole emitters without localization bias and down to a precision of 1 nm, thereby reaching the corresponding Cramér Rao bound. The approach is showcased with simulated data for various dipole orientations, and parameter settings realistic for real life experiments.

Highlights

The rapid progress of superresolution microscopy in the last 15 years has enabled fluorescence microscopy well below the diffraction limit of light
We developed a workflow to localize single dye molecules characterized by a fixed transition dipole, as occurring in cryo-single molecule localization microscopy (SMLM)
The method yields a precise determination of single dye positions in x, y, and z

Summary

Introduction

The rapid progress of superresolution microscopy in the last 15 years has enabled fluorescence microscopy well below the diffraction limit of light. A resolution of around 20 nm can be routinely achieved in biological samples [1]. One prominent group of methods, termed single molecule localization microscopy (SMLM), exploits the stochastic blinking of single dye molecules to circumvent the diffraction barrier [2]. By determining the positions of all dye molecules to a precision far below the wavelength of light, localization maps can be obtained, which represent a superresolved image of the biological sample. Since the image acquisition requires the recording of ten thousands of frames, lasting tens of minutes, appropriate sample fixation has become inevitable.

Methods

Results

Conclusion