Multi-modal label-free imaging of cellular metabolism and oxidative stress in 3D brain tissue models

Label-free, two-photon fluorescence imaging enables non-destructive, single-cell metabolic assessments that capture metabolic reprogramming and heterogeneity. Here, we use intensity and lifetime imaging to report on cell metabolic function and dynamic interactions in 3D engineered brain tissue models, comprising human induced neural stem cells, astrocytes and microglia. We rely on spectral imaging to define excitation/emission profiles of key fluorophores, including NAD(P)H, lipoamide dehydrogenase, FAD, and lipofuscin. These profiles inform a multi-wavelength imaging strategy that accounts for lipofuscin and spectral overlap, ensuring accurate redox state, mitochondrial fragmentation, and NAD(P)H bound state analysis. Intensity imaging over 11 weeks reveals metabolic differences between neurons and astrocytes, highlighting glial support for neuronal function and oxidative stress mitigation. Lifetime measurements provide insights into NADPH utilization and antioxidant pathways. Collectively, our results support multi-modal, label-free optical imaging for assessing dynamic brain cell metabolism and interactions, laying the foundation for studying metabolic and mitochondrial dysfunction in brain disorders.