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Super-resolution Fluorescence Microscopy

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.

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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

Brightness demixing for simultaneous multi-target imaging in 3D single-molecule localization microscopy.

Laurent Le¹, Surabhi K Sreenivas^1,2, Emmanuel Fort²

¹Université Paris Saclay, CNRS, Institut des Sciences Moléculaires d'Orsay, Orsay, France.

|June 5, 2026

Summary

Brightness demixing allows super-resolution microscopy to image multiple targets simultaneously by distinguishing fluorophores based on their brightness, not spectral properties. This simplifies multi-target imaging without needing extra filters or cameras.

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Area of Science:

Biophysics
Optical Microscopy
Molecular Imaging

Background:

Single-molecule localization microscopy (SMLM) achieves high resolution but struggles with simultaneous multi-fluorophore detection due to spectral overlap.
Current methods rely on spectral separation, limiting multiplexing capabilities.

Purpose of the Study:

To introduce a novel method, brightness demixing, for discriminating fluorophores in SMLM.
To enable robust multi-target imaging independent of spectral properties.

Main Methods:

Brightness demixing quantifies photon flux from blinking events as a proxy for fluorophore brightness (extinction coefficient and quantum yield).
This method operates within a single detection channel, avoiding spectral filters and cameras.
Oversampling blinking events allows precise brightness quantification.

Main Results:

Demonstrated simultaneous two- and three-target imaging in 2D and 3D configurations.
Successfully differentiated fluorophores based on brightness, independent of spectral overlap.
Maintained single-wavelength excitation and minimized chromatic aberrations.

Conclusions:

Brightness demixing offers a simple, powerful method to enhance multiplexing in SMLM.
The approach is compatible with existing SMLM setups, expanding multi-target imaging capabilities.
Eliminates the need for spectral separation, overcoming a key limitation in SMLM.