As a second messenger in signal transduction, calcium ion plays a very important role in neuronal information processing and integrating. Limited by the imaging technique, it is difficult to simultaneously perform deep tissue imaging and measure intracellular free calcium concentration ([Ca²+]_i) in different compartments of neurons in brain slice noncollinearly. By means of random access two-photon microscopy, which provides high optical penetration into tissues and low photo damage, we successfully measured [Ca²+]_i of different parts of pyramidal neurons in neocortical layer V in rat brain slices with high spatial and temporal resolution. Combining the patch clamp technique, we stimulated the soma with depolarizing current and explored the dynamics of calcium in pyramidal neurons.