Methods are provided to identify spatially and spectrally multiplexed probes in a biological environment. Such probes are identified by the ordering and color of fluorophores of the probes. The devices and methods provided facilitate determination of the locations and colors of such fluorophores, such that a probe can be identified. In some embodiments, probes are identified by applying light from a target environment to a spatial light modulator that can be used to control the direction and magnitude of chromatic dispersion of the detected light; multiple images of the target, corresponding to multiple different spatial light modulator settings, can be deconvolved and used to determine the colors and locations of fluorophores. In some embodiments, light from a region of the target can be simultaneously imaged spatially and spectrally. Correlations between the spatial and spectral images over time can be used to determine the color of fluorophores in the target.