Label-free nonlinear microscopy probes cellular metabolism and myelin dynamics in live tissue.

Metabolic coupling between neurons and glial cells plays a critical role in brain activity and myelin plasticity. Understanding its role in physiological and pathological contexts requires advanced methods to map metabolism and myelin in live tissue with high spatiotemporal resolution. Here, we present a label-free, multimodal, nonlinear optical microscopy platform integrated with an advanced image processing framework that simultaneously maps cellular metabolism and myelin distribution in organotypic cerebellar cultures. We combine third-harmonic generation microscopy for high-resolution myelin imaging with single axon precision with two-photon fluorescence lifetime microscopy of NAD(P)H metabolic biomarker to assess redox states with single-cell resolution. We introduce automated image analysis methods for cell segmentation and myelinated axon detection, enabling quantitative metabolic and myelin assessment in intact tissue during experimental myelination, demyelination and remyelination. Using this framework, we map the 3D myelin distribution in cerebellar folia and identify distinct metabolic signatures in neurons, oligodendrocytes, and microglia. Furthermore, we measure a metabolic shift in microglia along with myelin distribution changes during experimental demyelination. In conclusion, we establish label-free optical imaging as a powerful tool for the non-invasive characterization of neuro-glial metabolic coupling and myelin organization in living brain tissue, opening new perspectives for research in neuroinflammation and neurodegeneration.