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Related Experiment Video

Updated: Jun 13, 2026

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Published on: October 29, 2019

How accurately can a single molecule be localized in three dimensions using a fluorescence microscope?

Sripad Ram¹, E Sally Ward, Raimund J Ober

¹Center for Immunology NB9.106, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75235-8576, USA.

Proceedings of Spie--The International Society for Optical Engineering

|May 8, 2010

Summary

This study establishes a fundamental limit for the accuracy of single-molecule localization using fluorescence microscopy. Understanding these limits is crucial for optimizing 3D tracking of biomolecules in cellular research.

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Last Updated: Jun 13, 2026

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

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Published on: October 29, 2019

Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy

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Published on: November 11, 2025

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Published on: September 5, 2019

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Single molecule fluorescence microscopy enables the study of individual biomolecules within cells.
Three-dimensional tracking of single molecules offers valuable insights into cellular processes.
Accurate localization is essential for understanding biomolecular behavior.

Purpose of the Study:

To determine the fundamental limits of localization accuracy in fluorescence microscopy.
To provide a statistical framework for evaluating single-molecule localization performance.
To guide the optimization of experimental setups for 3D single-molecule tracking.

Main Methods:

Statistical analysis of the Fisher information matrix for single-molecule localization.
Derivation of a lower bound on the standard deviation for location estimation.
Evaluation of factors affecting localization accuracy, including noise and detector properties.

Main Results:

A theoretical lower bound for single-molecule localization accuracy was derived.
The bound depends on parameters like photon detection rate, acquisition time, and objective numerical aperture.
Noise, detector size, and pixelation were shown to degrade localization accuracy.

Conclusions:

The derived lower bound provides a benchmark for assessing localization accuracy in fluorescence microscopy.
Results offer guidelines for designing and optimizing experiments for 3D single-molecule tracking.
This work is critical for advancing the study of cellular dynamics at the single-molecule level.