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bioRxiv · 10.1101/2025.03.02.639924

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

Le, L.·Sreenivas, S. K.·Fort, E.·Leveque-Fort, S.
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Abstract

Single-Molecule Localization Microscopy (SMLM) has revolutionized high-resolution imaging, but the simultaneous detection of multiple fluorophores traditionally relies on spectral-based separation, which is inherently constrained by spectral overlap. Here, we introduce Brightness Demixing, a novel method for fluorophore discrimination that exploits brightness, which directly depends on the fluorophores extinction coefficient and quantum yield. By oversampling blinking events, we precisely quantify photon flux as a proxy for brightness, enabling robust differentiation of fluorophores independent of their spectral properties, without requiring additional spectral separation. Brightness Demixing operates within a single detection channel, eliminating the need for additional spectral filters or cameras. We demonstrate this approach with simultaneous two- and three-target imaging in both 2D and 3D configurations. By maintaining single-wavelength excitation and minimizing chromatic aberrations, this method significantly enhances multiplexing in SMLM while remaining fully compatible with existing setups. Brightness Demixing thus offers a simple yet powerful approach for expanding multi-target imaging capabilities in super-resolution microscopy.

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Le, L., Sreenivas, S. K., Fort, E., Leveque-Fort, S.. 2025-03-03. Brightness demixing for simultaneous multi-target imaging in 3D single-molecule localization microscopy. https://doi.org/10.1101/2025.03.02.639924

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