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

Single-molecule localization microscopy has enabled 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 method for fluorophore discrimination that exploits brightness, which directly depends on the fluorophore 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 two- and three-dimensional configurations. By maintaining single-wavelength excitation and minimizing chromatic aberrations, this method notably enhances multiplexing in single-molecule localization microscopy 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.