Epilepsy is a chronic disorder characterized by abnormal spatiotemporal neural activities. To clarify its physiological mechanisms and associated morphological features, we investigated neuronal activities using the flavoprotein fluorescence imaging technique and histopathological changes in epileptogenic tissue resected from patients with epilepsy. We applied an imaging technique suitable for examining human brain slices, and as a consequence achieved sufficient responses with high reproducibility. Moreover, we detected significant alterations in neuronal morphology associated with the acquired responses. Therefore, this strategy is useful for gaining a better understanding of the pathomechanisms underlying intractable epilepsy.