Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the useable spectral window. Here, we present a new method, exNEEMO, that enables perpixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use the approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging.