Despite considerable advances in confocal microscopy and related technologies, advanced light microscopy techniques have been rarely used to study the microstructure of cheese matrices. Here, we demonstrate the potential of advanced microscopic techniques, namely fluorescence lifetime imaging (FLIM), confocal Raman spectroscopy and non-linear microscopy (two-photon excitation and second harmonic generation microscopy), to study localized physicochemical properties of cheese matrices. These methods allow for precise localization of individual chemical components and help to determine their spatial organization in three dimensions. We discuss both fluorescent labelling and label-free methods that provide valuable information about the localized environment. Overall, these new technologies will enable a greater understanding of the influence of manufacturing processes on cheese quality and consistency.