• Integration of genetically encoded fluorescent sensors enables real-time monitoring of ion fluxes, energy metabolites, and redox states during NECSO.
• NC1-induced potassium loss via TRPM4 is captured with high spatiotemporal resolution using K+ sensors.
• NECSO is associated with rapid NADH accumulation and ATP depletion in mitochondria and cytosol, alongside reduced mitochondrial oxidative stress.
• The study provides a methodological framework for applying biosensors to study cell death and metabolic disorders.