Spines are tiny protrusions that densely stud the dendrites of neurons in the brain. Individual spines are the primary recipients of synaptic inputs from single axons, which emanate from other neurons in the central nervous system. A dendritic tree on one neuron may have hundreds of thousands of spines, making connections to a corresponding number of axons. The morphology of the spine typically consists of an ∼1-μm-diameter bulbous head connected to the dendritic shaft by a 50–150-nm-diameter × 500-nm-long cylindrical neck (Fig. 1). This morphology can be quite variable even on a single dendrite and has been characterized primarily by electron microscopy, because standard light microscopy approaches have insufficient resolution for these tiny structures. But, of course, electron microscopy precludes the analysis of spines in living tissue. To remedy this, a pair of articles in this issue of Biophysical Journal (1,2) combines two-photon and stimulated emission depletion (STED) microscopy to characterize individual spine morphologies on neuronal dendrites within freshly harvested live brain tissue (acute brain slices). They increase the resolution of two-photon excitation fluorescence microscopy from ∼350 nm to 60–80 nm, which is in the same range as the size of the spine necks (Fig. 1). Determining the size of the spine neck in living tissue will allow neuroscientists to explore how spines can behave as biochemical or electrical compartments to individually process synaptic inputs. Combining the virtues of two-photon microscopy with those of STED holds great promise for unraveling dynamic morphological changes of neuronal spines and their physiological role in the brain.