The mammalian brain is a complex system containing billions of neurons, and every neuron forms thousands of connections with other neurons, with information flowing from one neuron to another. Thus the neuronal connection map, a kind of “information highway,” plays a key role in understanding how the brain works. However, with hundreds years of development, investigators cannot draw out the complete structure of complex neurons, let alone the full connection map. Knowledge deficiency of brain connectivity is actually a result of lacking proper tools. In recent years, techniques have emerged that raise the potential to address such questions. Diffusion tension imaging–magnetic resonance imaging has provided a coarse map of different brain areas, the so-called “connectome,” and launched the Human Connectome Project. Serial electron microscopy has dissected brain tissue to as fine as a nanometer, and thus is able to map each synapse, the connection site between two neurons, and have been extending to large tissue blocks. With the help of molecular labeling, optical microscopic imaging fills the gap between electron microscopy and magnetic resonance imaging, and has been playing an important role in exploring the organization and function of the brain. The Special Section on Light Microscopy of Connectivity shows recent efforts to image the fine organization and function using light microscopy.