Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

Adrien C Descloux,Aleksandra Radenovic,Kristin S Grußmayer

doi:10.1038/s42003-021-02086-1

Abstract

Localization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

Highlights

Localization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters
We present a modified histogram method for SMLM dataset rendering that is compatible with resolution estimation using decorrelation analysis
ÄÅ xn denotes the x position of the nth localization event floored to the nearest integer multiple of the chosen pixel size. This rounding operation is problematic as it introduces a roundoff error, which can be detrimental to the resolution estimation, especially if the pixel size is on the order of the localization uncertainty

Summary

Introduction

Localization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. We presented decorrelation analysis on the single-molecule localization microscopy symposium (SMLMS), looking for feedback from this community that relies heavily on image processing. Unlike other super-resolution methods, most SMLM software does not directly output an image, but a set of localizations that need to be rendered for visualization. This adds another level of complexity to the interpretation of the images. We present a modified histogram method for SMLM dataset rendering that is compatible with resolution estimation using decorrelation analysis. We find that our method expects a localization density of about 1–4×104 loc. per μm[2] to work reliably with experimental data

Methods

Results

Conclusion