Single-molecule localization microscopy has become a reliable and go to technique for super-resolution imaging in biological samples. Biological issues owing to their complexity, demand for techniques that allow multiple localizations of several types of biomolecules simultaneously. The use of single excitation wavelength, allows one to observe dye with spectral overlap, but which minimize chromatic aberrations. We will show during the workshop that we can also use the flux analysis that is based on alternative intrinsic properties that can be used to differentiate fluorophores, especially with overlapping absorption-emission spectra. This presents the major advantage of being directly compatible with a single-camera configuration. We will show the benefits and constraints of this approach on DNA-PAINT imaging, and evaluate its performances compare to traditional spectral demixing strategy for multiple targets imaging. As an example, we will do demixing on COS-7 fixed cells with alpha-tubulin and clathrin-coated pits labelling. Furthermore, as this photon flux technique doesn’t require any modification of the optical implementation, its combination with 3D super-resolution localization microscopy is straightforward. In particular, we will present the combination of the photon flux demixing with supercritical angle fluorescence to achieve axial super-resolution, known as DONALD.