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Updated: Mar 10, 2026

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

Orthogonally Dispersed Spectroscopic Single-Molecule Localization Microscopy.

Jun Lu¹, Lei Xu¹, Zhenyao Zhao¹

¹College of Biomedical Engineering Yiwu Research Institute Fudan University Shanghai China.

Nanophotonics (Berlin, Germany)

|March 9, 2026

Summary

This summary is machine-generated.

Orthogonally dispersed sSMLM (ODsSMLM) overcomes the photon budget trade-off in single-molecule microscopy. This new method enhances both spatial localization and spectral characterization for advanced molecular imaging.

Keywords:

orthogonal dispersion single‐molecule localization microscopy spectroscopy super‐resolution imaging

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

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Area of Science:

Biophysics
Optical Microscopy
Spectroscopy

Background:

Spectroscopic single-molecule localization microscopy (sSMLM) enables simultaneous spatial and spectral data acquisition.
A key challenge in sSMLM is the photon budget limitation, forcing a compromise between localization and spectral performance.
Existing methods often suffer from dispersion-dependent artifacts affecting localization precision.

Purpose of the Study:

To introduce orthogonally dispersed sSMLM (ODsSMLM) to overcome the photon budget dilemma in sSMLM.
To enable the use of all photons for both localization and spectral characterization.
To achieve isotropic lateral localization precision and reduce artifacts.

Main Methods:

Modulating single-molecule emission spectra using an orthogonal structure.
Implementing ODsSMLM for simultaneous spatial and spectral data acquisition.
Validating the method with simulated data and dual-color imaging experiments.

Main Results:

ODsSMLM utilizes all photons for both localization and spectral analysis.
Simulated data showed 10 nm localization and 1.1 nm spectral precision with a 3000-photon budget.
Dual-color imaging of microtubules and clathrin achieved 27 nm isotropic lateral resolution.

Conclusions:

ODsSMLM effectively resolves the trade-off between localization and spectral precision in sSMLM.
The method provides isotropic lateral localization, eliminating dispersion-dependent artifacts.
ODsSMLM offers a significant advancement for multicolor imaging and molecular characterization.