Despite extraordinary progress in figuring out how our organs provide the functions necessary for life, an in-depth understanding of how the brain works remains elusive. Unlike the heart or kidneys, brain structures we can easily discern provide few clues. Further complicating matters, the brain resides in a largely inaccessible skull.

Methodological development in optical microscopy can now provide cellular-level information on neuronal signaling in behaving mice and rats.

Most investigation of the brain has involved examining the function of individual brain cells or examining the relationship between regional neuronal activity and organism function—studies dominated by electrophysiology and magnetic resonance imaging. Optical probes of activity provide a way of bridging these two scales of investigation, but until recently optical studies of the functioning intact brain were stymied by inadequate methods.

But new developments are redefining what is possible. Two-photon microscopy increases the penetration depth of light microscopy tenfold, making it possible to peer as much as a millimeter into the brain. Fluorescent sensors of neuronal activity are improving and report neuronal signaling with greater reliability. The coupling of these developments with preparative techniques that provide physical and optical access to the brain of mice and rats has made it possible to visualize neuronal signaling in the intact brain of living animals.

Early studies were limited to anesthetized mice, which limited the applications. Neuroscientists now have mice that walk on spinning Styrofoam balls in virtual-reality environments while a two-photon microscope records the firing of networks of neurons (Nature 461, 941–946, 2009). Miniaturized two-photon microscopes can be carried by freely moving rats while imaging takes place (Proc. Natl. Acad. Sci. USA 106, 19557–19562, 2009). Or fiber-based microscopes can peer into the deep recesses of the brain in freely moving mice (Nat. Methods 5, 935–938, 2008).

The spatial scales covered by these new methods—from subcellular to networks of hundreds of cells—promise to open new doors of understanding for this most impenetrable of organs.