Abstract
Two-photon imaging is a powerful tool used to examine molecular and cellular functions in living
tissues. In particular, calcium imaging can quantitatively measure neuronal activity i.e. action potential
fi ring. Two-photon calcium imaging can detect the multicellular activity of neuronal circuits in the brain
at the single cell level while animals perform behavioral tasks. Normal two-photon microscopy can be
applied to head-restrained mice, while fi ber-optic, head-mounted miniaturized two-photon microscopes
can be used in unrestrained mice. Here, we review the general mechanisms and methodologies for twophoton
calcium imaging in awake behaving mice. In addition, we also briefl y discuss the manipulation
of photoactivatable proteins (optogenetics), which can be used to activate or inhibit specifi c types of
neurons and animal behaviors with millisecond precision.
