Abstract: A system and method for determining fluorescence decay in a biological sample is provided. The method includes acquiring optical signal data from at least a part of a biological sample undergoing fluorescence, assembling the optical signal data into a set of spatial time-series data, and converting the set of spatial time-series data into a set of spatial frequency time-series data. The method also includes applying a spatial filter to the set of spatial frequency time-series data to yield a set of filtered frequency-series data, the spatial filter configured to separate, from the set of frequency-series data, fluorescence signals consistent with a non-diffuse component and converting the set of filtered frequency-series data into a set of filtered time-series data.