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Updated: Nov 6, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Published on: June 30, 2018

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

Adrien C Descloux¹, Kristin S Grußmayer^2,3, Aleksandra Radenovic⁴

¹École Polytechnique Fédérale de Lausanne, Laboratory of Nanoscale Biology, Lausanne, Switzerland. adrien.descloux@epfl.ch.

Communications Biology

|May 12, 2021

Summary

Localization microscopy super-resolution imaging relies on separating blinking emitters. A new bilinear histogram rendering method improves resolution estimation accuracy in super-resolution microscopy.

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Area of Science:

Biophysics
Optical Microscopy
Super-resolution Imaging

Background:

Localization microscopy achieves super-resolution by spatially and temporally separating blinking fluorescent emitters.
Localization precision, below the diffraction limit, is influenced by photon count and sensor noise.
Estimating resolution from rendered localization data can be biased by rendering methods.

Purpose of the Study:

To investigate the impact of rendering techniques on resolution estimation in localization microscopy.
To introduce a novel rendering method that minimizes bias in resolution estimation.
To establish a parameter-free pipeline for accurate resolution assessment.

Main Methods:

Analysis of localization datasets using standard Gaussian and histogram rendering methods.
Development and application of a modified histogram rendering technique, termed bilinear histogram.
Validation using simulated datasets and state-of-the-art experimental data.

Main Results:

Standard rendering methods introduce biases in resolution estimation based on decorrelation analysis.
The proposed bilinear histogram rendering method effectively circumvents these biases.
Resolution estimation becomes dependent on localization density and precision with the new pipeline.

Conclusions:

Rendering methods significantly affect resolution estimation in localization microscopy.
Bilinear histogram rendering offers a more accurate approach to resolution assessment.
A parameter-free pipeline based on localization density and precision provides reliable resolution estimation.