In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi-like staining of small numbers of cells and processes that can be resolved clearly using confocal microscopy; viability of stained cells is established by exclusion of the fluorescent DNA-binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high-resolution images are combined in an automated procedure to generate three-dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations.