Brain metabolism is essential for the function of organisms. While established imaging methods provide valuable insights into brain metabolic function, they lack the resolution to capture important metabolic interactions and heterogeneity at the cellular level. Label-free, two-photon excited fluorescence imaging addresses this issue by enabling dynamic metabolic assessments at the single-cell level without manipulations. In this study, we demonstrate the impact of spectral imaging on the development of rigorous intensity and lifetime label-free imaging protocols to assess dynamically over time metabolic function in 3D engineered brain tissue models comprising human induced neural stem cells, astrocytes, and microglia. Specifically, we rely on multi-wavelength spectral imaging to identify the excitation/emission profiles of key cellular fluorophores within human brain cells, including NAD(P)H, LipDH, FAD, and lipofuscin. These enable development of methods to mitigate lipofuscin's overlap with NAD(P)H and flavin autofluorescence to extract reliable optical metabolic function metrics from images acquired at two excitation wavelengths over two emission bands. We present fluorescence intensity and lifetime metrics reporting on redox state, mitochondrial fragmentation, and NAD(P)H binding status in neuronal monoculture and triculture systems, to highlight the functional impact of metabolic interactions between different cell types. Our findings reveal significant metabolic differences between neurons and glial cells, shedding light on metabolic pathway utilization, including the glutathione pathway, OXPHOS, glycolysis, and fatty acid oxidation. Collectively, our studies establish a label-free, non-destructive approach to assess the metabolic function and interactions among different brain cell types relying on endogenous fluorescence and illustrate the complementary nature of information that is gained by combining intensity and lifetime-based images. Such methods can improve understanding of physiological brain function and dysfunction that occurs at the onset of cancers, traumatic injuries and neurodegenerative diseases.