Snapshot multicolor fluorescence imaging using double multiplexing of excitation and emission on a single detector

Fluorescence-based multispectral imaging of rapidly moving or dynamic samples requires both fast two-dimensional data acquisition as well as sufficient spectral sensitivity for species separation. As the number of fluorophores in the experiment increases, meeting both these requirements becomes technically challenging. Although several solutions for fast imaging of multiple fluorophores exist, they all have one main restriction; they rely solely on spectrally resolving either the excitation- or the emission characteristics of the fluorophores. This inability directly limits how many fluorophores existing methods can simultaneously distinguish. Here we present a snapshot multispectral imaging approach that not only senses the excitation and emission characteristics of the probed fluorophores but also all cross term combinations of excitation and emission. To the best of the authors’ knowledge, this is the only snapshot multispectral imaging method that has this ability, allowing us to even sense and differentiate between light of equal wavelengths emitted from the same fluorescing species but where the signal components stem from different excitation sources. The current implementation of the technique allows us to simultaneously gather 24 different spectral images on a single detector, from which we demonstrate the ability to visualize and distinguish up to nine fluorophores within the visible wavelength range.