Deep Learning-Augmented Stimulated Raman Imaging for Cell-Type-Specific Metabolic Profiling in Live Neuronal Cocultures

Neuronal metabolism is fundamental to brain functions and diseases, yet its spatial and temporal dynamics and interactions remain poorly understood. Here, we introduce a tandem deep-learning approach integrated with bioorthogonal chemical imaging using stimulated Raman scattering (SRS) microscopy. This method achieves high-speed and quantitative metabolic profiling in live neuronal cocultures. Our deep-learning framework consists of a recurrent convolutional neural network (RCNN) that enables high-resolution 3D imaging with minimal photodamage and a U-Net segmentation model for cell-type-specific metabolic analysis. Using deuterium-labeled metabolites, we demonstrate the ability to trace lipid, protein, glucose, and D₂O metabolism in live neurons, astrocytes, and oligodendrocytes under physiological and pathological conditions, including NMDA receptor activation, proteasome inhibition, and Huntington's disease. Our findings reveal distinct metabolic adaptations among neuronal cell types and underscore the importance of noninvasive metabolic profiling for understanding neuronal interactions and disease mechanisms. This platform significantly advances live-cell dynamic imaging with broad applications in neuroscience, disease modeling, and therapeutic screening.