High-resolution calcium imaging of neuronal activity. (A) Two-photon image of transverse section of mouse neocortex loaded with Fura-2 AM. Scale bar, 250 μm. Note that thousands of neurons can be adequately visualized. (B) Correspondence between action potentials and somatic fluorescence change. (i) Whole-cell recording of a burst of action potentials in response to a single intracellular depolarizing current step (150 pA, 200 msec). (ii) Normalized fluorescence change for the recorded neuron imaged with a cooled charge-coupled device (CCD) camera (Micromax, Princeton Instruments). The recording pipette contained 50 µm of Fura pentapotassium salt (comparable to the intracellular concentration of Fura following bulk loading). Although individual action potentials cannot be resolved, the onset of neuronal activity is accurately imaged.