Single-molecule brightness, a new dimension for super-resolution imaging in biology
In super-resolution imaging, a new method exploits the brightness of individual fluorophores, rather than their spectrum, to distinguish several proteins simultaneously with no notable loss of resolution. It opens the way to richer nanoscale mapping of biological systems.
References :
Brightness demixing for simultaneous multi-target imaging in 3D single-molecule localization microscopy. Laurent Le, Surabhi K. Sreenivas, Emmanuel Fort, Sandrine Lévêque-Fort, Nature Methods - Published: 05 June 2026.
DOI: 10.1038/s41592-026-03118-6
Open access: HAL
Understanding how biological systems function requires observing their organization at the nanoscale. Super-resolution fluorescence microscopy makes this observation possible by overcoming the diffraction limit of light: fluorescent molecules are activated in sparse subsets, localized one by one, and their positions are then accumulated to reconstruct a nanoscale image. However, a major challenge remains: distinguishing several different proteins simultaneously while preserving this very high localization precision. Current approaches rely mainly on the spectral separation of fluorescence emission, a strategy limited by the overlap between fluorophore emission spectra and by the experimental constraints required to separate them optically.
A highly interdisciplinary collaboration at the physics-biology interface has led to the development of a new approach for distinguishing several molecular targets labelled with fluorophores, no longer solely on the basis of their spectrum, but directly through their brightness, that is, the amount of light emitted by each individual fluorescent molecule.
This research was carried out in the following CNRS laboratories:
- Institut des Sciences Moléculaires d’Orsay (ISMO, CNRS/Université Paris-Saclay)
- Institut Langevin (CNRS/ESPCI Paris - PSL)
This work was supported in particular by the AXA Research Fund, Labex WIFI, ANR TimeLoc and the European ERC TimeNanolive funding.
This original strategy, called brightness demixing, exploits the fact that in single-molecule localization microscopy each fluorophore can be observed individually and characterized by the photon flux it emits. This property can thus be used as a genuine signature to separate different proteins associated with different fluorophores within the same biological sample. This new way of exploiting single-molecule signals builds on the two teams' work on the temporal control of their illumination. Initially developed to improve their localization, it now opens an additional information dimension for super-resolution imaging.
Unlike other methods for distinguishing several proteins in a single experiment, this approach does not require multiplying optical detection paths. It can be integrated into standard localization microscopes, provided that uniform illumination and an appropriate acquisition rate are available. The researchers validated it on several complex biological objects, including nuclear pore complexes, as well as on cellular structures in which three different proteins were labelled simultaneously. They also show that the method remains compatible with 3D imaging (Figure 1). These results indicate that more proteins can be distinguished simultaneously without notable degradation of spatial resolution. Moreover, by preserving a large field of view, this work could contribute to studying the nanoscale architecture of complex biological systems, notably in disease-related contexts.
